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Medical Applications of Synthetic Biology - Samantha Simpson

2007-12-06T04:24:39Z

Sasimpson: /* Paper */

== Project Proposal ==

My project will be on medical applications of synthetic biology. I will reference Anderson's paper on utilizing quorum-sensing and hypoxia-responsive genes coupled with invasin from ''Yersinia tuberculosis'' to invade cancer-causing cells. Coupled with Critchley's work, which describes a bacterial system that invades eukaryotic cells and delivers proteins coded for in the bacteria's genome, one could possibly create 'search and destroy' ''E.coli'' that can locate, invade, and kill tumor cells. Garmory's paper also highlights the possibility of using bacteria as a drug-delivering system, specifically vaccine vectors. Kobayashi describes cells that produce a protective biofilm layer after exposure to DNA-damaging agents. Ro engineers yeast to create an anti-malarial drug that can be created at a lower cost than the previous standard production process. These papers, and others that have a focus on direct medical applications such as novel cancer therapies, vaccination technology, biological protection, and the creation of new medicines, will be referenced to create a cohesive overview of applications of synthetic biology to the medical field.

== Paper ==

Synthetic biology is a rapidly emerging field that strives to re-design existing biological systems and components or fabricate novel biological systems and components. The end result of experimentation in synthetic biology is to help scientists understand a naturally occurring genetic pathway more fully or to create a new, useful pathway. Recent research efforts have focused on engineering bacteria to create a new form of biofuel or to make promoters of varying efficiencies for more exact gene expression. Other research efforts focus on engineering yeast to make antimalarial drugs or engineering ''E. coli'' to recognize and invade tumor-like cells, both of which are medical applications of synthetic biology. Advancements such as novel cancer therapies, vaccination technology, biological protection, and the creation of new medicines highlight synthetic biology’s potential to create breakthroughs in both the prevention and treatment side of medical science. This paper will examine various medical applications of synthetic biology and further development that needs to be done before current research can be used in hospitals and doctors’ offices.

'''Prevention'''

Engineering live bacteria to act as a vaccine vector is an area of interest in synthetic biology. Bacteria was first thought to be a good vector because it would not degrade at mucosal surface and it would survive the low pH in the gastrointestinal tract like most other orally administered antigens (Garmory, 2003). A particular strain of ''Salmonella'' with one or more deletions in the [http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&uid=15012217&cmd=showdetailview&indexed=google shikimate pathway], which creates a precursor for phenylalanine and tyrosine, is a promising vector because it gets into the body easily but does not harmfully effect the tissue and is gone within a week or two of introduction. Scientists engineered this strain to carry the [http://en.wikipedia.org/wiki/Yersinia_pestis ''Yersinia pestis''] V antigen, and studied its ability to protect mice from ''Y. pestis''. The V antigen is generally injected via an intramuscular route, however, in this study, 20 mice were given the V antigen via the intragastric route so scientists could test the effectiveness of an oral administration. Unfortunately, the inoculation was not extremely successful and only 6 of 20 mice infected with ''Y. pestis'' showed a strong response (Garmory, 2003). Garmory and colleagues cited several reasons for this: the copy number of the plasmid containing the V antigen might not have been high enough; the attenuated ''Salmonella'' may not have been able to reach cells that would be targeted by ''Y. pestis''; and the inability of the cell to secrete the antigen. The authors remain hopeful that attenuated bacteria vectors may someday produce an orally administered long-lasting response for protection against bubonic and pneumonic plague (Garmory, 2003). Many changes need to be made to live vector vaccine design before it is an effective method of treating or preventing disease, such as solving the issue of specifity of a vaccine administered orally, and creating a bacterial chassey that can secrete an antigen at an appropriate time.

Another method of disease prevention with a synthetic biology approach is related to skin cancer. ''E. coli'' was engineered to sense single-stranded DNA, and this DNA damage was coupled with a mechanism to produce a biofilm around the cell. The biofilm output module was tested with DNA damage done by [http://en.wikipedia.org/wiki/Mitomycin_c mitomycin C], a DNA crosslinking agent, or UV irradiation, a known cause of skin cancer. Single-stranded DNA activates [http://en.wikipedia.org/wiki/RecA RecA], which in turn represses the C1 repressor protein, thus allowing the transcription of the PL promoter (Kobayashi, 2004). The PL promoter is on the biofilm-forming output plasmid (pBFR), which controls biofilm formation (Fig. 1). The biofilm protection, when part of a toggle switch mechanism, lasted indefinitely when pulsed with UV light at 8 J/m2 for only 2 seconds (Kobayashi, 2004) (Fig. 2). The biofilm-production mechanism has not been engineered in eukaryotic cells, but a prokaryote-based system that could aid with human skin disease is imaginable. For example, a sunscreen that changes colors when the DNA damage may be too intense for a sunbather as a warning, or a sunscreen that becomes more protective as the possibility of genetic damage increased. Further research should focus on how to best implement the biofilm producing output module to protect human DNA.

<center>

{|
|-
|[[Image:Toggleswitch.JPG]]
|[[Image:UVinducedbiofilm.JPG]]
|-
|'''Fig. 1.''' The RecA / traA toggleswitch mechanism. (Kobayashi, 2004 - Permission Pending)
|'''Fig. 2.''' Crystal violet absorbance measures biofilm formation. (Kobayashi, 2004 - Permission Pending)
|}
</center>

'''Treatment'''

Synthetic biologists consider finding a cure for cancer a reachable goal. Previous to the development of synthetic biology as a field, it was known that three types of bacteria, ''Bifodobacterium'', ''Clostridium'', and ''Salmonella'' all preferentially infect the dense cells of tumors (Pawelek, 2003). All three types of bacteria are associated with reducing tumor size in patients who are infected with them, and it was noted that all three bacteria act anaerobically. Capitalizing on the idea that tumors create an unusually dense, anaerobic mass of cells, Christopher Voigt’s lab engineered bacteria to identify and invade cancer cells. They utilized the ''lux'' quorum sensing from ''Vibrio fischeri'' to identify cells growing at high densities (Fig. 3) and the ''fdhF'' promoter to identify cells growing in anaerobic conditions ''in vitro'' (Fig. 4) (Anderson, 2006). They coupled this identification mechanism with the invasin output module from ''Yersinia pseudotuberculosis'' so the ''E. coli'' could invade specific cells.

<center>

{|
|-
|[[Image:InvasinDensity.JPG]]
|[[Image:InvasinAnaerobic2.JPG]]
|-
|'''Fig. 3.''' The red line represents when the invasin output module was paired with the inducible promoter ''lux'' which senses high cell density, the black line is constitutively on invasin. (Anderson, 2006 - Permission Pending)
|'''Fig. 4.''' The invasin output module was paired with the inducible promoter ''fdhF'' which senses anaerobic conditions. (Anderson, 2006 - Permission Pending)
|}

</center>

Next, scientists hope to insert a gene that would destroy the invaded cells, making the complete cycle a search, invade, and destroy loop that would only be sensitive to cancer cells. One promising destroy mechanism has been implemented in humans via intratumoral injection, based on ''Salmonella'' that contains the ''E. coli'' cytosine deaminase gene that converts 5-fluorocytosine to 5-fluorouracil, a chemotherapy drug (Nemunaitis, 2003). Unfortunately, while the tumors did produce [http://en.wikipedia.org/wiki/Fluorouracil 5-fluorouracil] (5-FU), they did not produce it in high enough yields to cause regression of the cancer. Another experiment flooded mouse cells via intratumoral injection with 6-methylpurine-2’deoxyribose (6-MPDR), which was converted to the toxin [http://jpet.aspetjournals.org/cgi/content/abstract/304/3/1280 6-methyl purine] (MeP) by the enzyme purine nucleoside phosphorylase (PNP), which is naturally found in ''E. coli'' (Critchley, 2004) (Fig. 5).

<center>

[[Image:Tumor.JPG]]

'''Fig. 5.''' Tumor size after injections. Cancer cells were injected into mice, and 5 days later injections with PBS, 6-MPDR, and invasin-enhanced ''E-coli'' began. Tumor cells recieving injections of invasin-enhanced ''E.coli'' and 6-MPDR grew the slowest, but did not get smaller. (Critchley, 2004 - Permission Pending)

</center>

The problem with this is that even though MeP was injected into the tumor, all cells in the body might be exposed to the toxin. In addition to the search and destroy mechanism, scientists hope to increase selectivity by combining the hypoxia-sensing and density-sensing units so that ''E. coli'' will only enter eukaryotic cells that exhibit both characteristics. This would make it more likely that invasin-enhanced ''E. coli'' would only invade cancer cells rather than muscle tissue that had low oxygen due to exercise (Anderson, 2006). To create a fully functional, synthetic biology-based approach to cancer therapy, scientists need to develop bacteria that discriminately invades cancer cells and destroys them. Most of the pieces are there – scientists have engineered ''E. coli'' that selectively invades hypoxic cells or dense cells and ''Salmonella'' that can destroy tumors in vivo – they just need to be put together effectively.

Finally, synthetic biology has made its mark on the field of medicine by creating drugs that are less expensive to produce than those made by pharmaceutical companies. Jay Keasling’s project involving the antimalarial drug artemisinin is the best example. Yeast was engineered to increase normal farnesyl pyrophosphate (FPP) production, convert FPP to amorphadiene, and oxidizing amorphadiene to artemisinic acid, which can then easily be separated from the yeast cells and converted to artemisinin in the laboratory (Fig. 6). This innovation allows the production of artemisinin at lower prices than those currently on the market, and without regard to environmental constraints (Ro, 2006). On a completely different tangent, Collins’s lab engineered T7 bacteriophages to deliver biofilm-degrading enzymes such as dispersin B after infecting and replicating in an ''E. coli'' biofilm. Dispersin B works to degrade an adhesin that is critical for biofilm formation. Phages that released dispersin B were found to be 99.997% effective at removing biofilm, which is 4.5 orders of magnitude higher than non-enzymatic phages, and caused a 3.65log10 reduction in bacterial cells recovered from biofilmwhen compared to untreated biofilms (Lu, 2007) (Fig. 7). The specificity of phages for a certain type of bacteria make them a viable vector to be used in human treatments, which may range from removing dental plaques to cleaning inserted medical devices. Before phages are ready for use in medicine, however, a well-characterized phage library must be created so that way any specific biofilm can be targeted using the dispersin B method. This also requires scientists being able to identify the type of bacteria in the biofilm, which may be growing inside the body. Using synthetic biology to engineer an antimalarial drug in yeast or a biofilm-destroying enzyme in bacteriophages greatly increases the possible contributions to the field of medicine.

<center>

{|
|-
|[[Image:Artemesinin.JPG]]
|[[Image:BiofilmDegradation.JPG]]
|-
|'''Fig. 6.''' All the steps in the box indicate steps that were optimized in yeast for a maximum artemisinic acid output. (Ro, 2006 - Permission Pending)
|'''Fig. 7.''' The amount of biofilm was decreased when the dispersin B enzymes were incorporated into T7 bacteriophages. (Lu, 2007 - Permission Pending)
|}
</center>

'''Conclusion'''

Medical applications of synthetic biology are wide-ranging and eminently applicable to daily life. Synthetic biology based cancer treatments that utilize ''E.coli'' engineered to sense and attack tissues with high cell density and low oxygen availability are promising and need to be tested in vivo. Designing vectors out of attenuated bacteria may be a safe, effective method for disease treatment, but need to be able to reach a target tissue if administered orally. Using biofilms to sense sun damage potential may decrease the likelihood of skin cancer. Finally, using engineered cells to produce medicines in a cost-effective, environmentally-friendly way may revolutionize the pharmaceutical industry. Investing more research efforts in the field of synthetic biology may turn out to be an investment in medical technology.

== Works Cited ==

-[http://www.bio.davidson.edu/courses/synthetic/papers/2006_anderson.pdf Anderson JC, Clarke EJ, Arkin, AP, and Voigt CA (2006). Environmentally controlled invasion of cancer cells by engineered bacteria. Journal of Molecular Biology 355:619-27.]

-[http://www.nature.com/gt/journal/v11/n15/abs/3302281a.html Critchley RJ, Jezzard S, Radford KJ, Goussard S, Lemoine NR, et al. (2004). Potential therapeutic applications of recombinant, invasive ''E. coli''. Gene Therapy 11:1224-33.]

-[http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&uid=15203915&cmd=showdetailview&indexed=google Garmory HS, Leary SEC, Griffin, KF, Williamson D, Brown, KA, and Titball RW (2003). The use of live attenuated bacteria as a delivery system for heterologous antigens. Journal of Drug Targeting 11:471-79.]

-[http://www.pnas.org/cgi/reprint/101/22/8414 Kobayashi H, et al. (2004). Programmable cells: Interfacing natural and engineered gene networks. PNAS 101: 8414-19.]

-[http://www.bio.davidson.edu/courses/synthetic/papers/Phage_BioFilms.pdf Lu, TK, and Collins JJ (2007). Dispersing biofilms with engineered enzymatic bacteriophage. PNAS 104: 11197-11202.]

-[http://www.nature.com/cgt/journal/v10/n10/abs/7700634a.html Nemunaitis J, Cunningham C, Senzer N, Kuhn J, Cramm J, Litz C, et al. (2003). Pilot trial of genetically modified, attenuated ''Salmonella'' expressing cytosine deaminase gene in refractory cancer patients. Cancer Gene Therapy 10:737-744.]

-[http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6W85-49FGKHC-N&_user=10&_coverDate=09%2F30%2F2003&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=d4529b45974bfe7d9ddab1fc865f088b Pawelek JM, Low KB, and Bermudes D (2003). Bacteria as tumour-targeting vectors. Lancet Oncology 4:548-56.]

-[http://www.bio.davidson.edu/courses/synthetic/papers/Keasling_malaria.pdf Ro D, et al. (2006). Production of the antimalarial drug precursor artemisinic acid in engineered yeast. Nature 440:940-43.]

Medical Applications of Synthetic Biology - Samantha Simpson

2007-12-06T04:13:09Z

Sasimpson: /* Paper */

== Project Proposal ==

My project will be on medical applications of synthetic biology. I will reference Anderson's paper on utilizing quorum-sensing and hypoxia-responsive genes coupled with invasin from ''Yersinia tuberculosis'' to invade cancer-causing cells. Coupled with Critchley's work, which describes a bacterial system that invades eukaryotic cells and delivers proteins coded for in the bacteria's genome, one could possibly create 'search and destroy' ''E.coli'' that can locate, invade, and kill tumor cells. Garmory's paper also highlights the possibility of using bacteria as a drug-delivering system, specifically vaccine vectors. Kobayashi describes cells that produce a protective biofilm layer after exposure to DNA-damaging agents. Ro engineers yeast to create an anti-malarial drug that can be created at a lower cost than the previous standard production process. These papers, and others that have a focus on direct medical applications such as novel cancer therapies, vaccination technology, biological protection, and the creation of new medicines, will be referenced to create a cohesive overview of applications of synthetic biology to the medical field.

== Paper ==

Synthetic biology is a rapidly emerging field that strives to re-design existing biological systems and components or fabricate novel biological systems and components. The end result of experimentation in synthetic biology is to help scientists understand a naturally occurring genetic pathway more fully or to create a new, useful pathway. Recent research efforts have focused on engineering bacteria to create a new form of biofuel or to make promoters of varying efficiencies for more exact gene expression. Other research efforts focus on engineering yeast to make antimalarial drugs or engineering ''E. coli'' to recognize and invade tumor-like cells, both of which are medical applications of synthetic biology. Advancements such as novel cancer therapies, vaccination technology, biological protection, and the creation of new medicines highlight synthetic biology’s potential to create breakthroughs in both the prevention and treatment side of medical science. This paper will examine various medical applications of synthetic biology and further development that needs to be done before current research can be used in hospitals and doctors’ offices.

'''Prevention'''

Engineering live bacteria to act as a vaccine vector is an area of interest in synthetic biology. Bacteria was first thought to be a good vector because it would not degrade at mucosal surface and it would survive the low pH in the gastrointestinal tract like most other orally administered antigens (Garmory, 2003). A particular strain of ''Salmonella'' with one or more deletions in the [http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&uid=15012217&cmd=showdetailview&indexed=google shikimate pathway], which creates a precursor for phenylalanine and tyrosine, is a promising vector because it gets into the body easily but does not harmfully effect the tissue and is gone within a week or two of introduction. Scientists engineered this strain to carry the [http://en.wikipedia.org/wiki/Yersinia_pestis ''Yersinia pestis''] V antigen, and studied its ability to protect mice from ''Y. pestis''. The V antigen is generally injected via an intramuscular route, however, in this study, 20 mice were given the V antigen via the intragastric route so scientists could test the effectiveness of an oral administration. Unfortunately, the inoculation was not extremely successful and only 6 of 20 mice infected with ''Y. pestis'' showed a strong response (Garmory, 2003). Garmory and colleagues cited several reasons for this: the copy number of the plasmid containing the V antigen might not have been high enough; the attenuated ''Salmonella'' may not have been able to reach cells that would be targeted by ''Y. pestis''; and the inability of the cell to secrete the antigen. The authors remain hopeful that attenuated bacteria vectors may someday produce an orally administered long-lasting response for protection against bubonic and pneumonic plague (Garmory, 2003). Many changes need to be made to live vector vaccine design before it is an effective method of treating or preventing disease, such as solving the issue of specifity of a vaccine administered orally, and creating a bacterial chassey that can secrete an antigen at an appropriate time.

Another method of disease prevention with a synthetic biology approach is related to skin cancer. ''E. coli'' was engineered to sense single-stranded DNA, and this DNA damage was coupled with a mechanism to produce a biofilm around the cell. The biofilm output module was tested with DNA damage done by [http://en.wikipedia.org/wiki/Mitomycin_c mitomycin C], a DNA crosslinking agent, or UV irradiation, a known cause of skin cancer. Single-stranded DNA activates [http://en.wikipedia.org/wiki/RecA RecA], which in turn represses the C1 repressor protein, thus allowing the transcription of the PL promoter (Kobayashi, 2004). The PL promoter is on the biofilm-forming output plasmid (pBFR), which controls biofilm formation (Fig. 1). The biofilm protection, when part of a toggle switch mechanism, lasted indefinitely when pulsed with UV light at 8 J/m2 for only 2 seconds (Kobayashi, 2004) (Fig. 2). The biofilm-production mechanism has not been engineered in eukaryotic cells, but a prokaryote-based system that could aid with human skin disease is imaginable. For example, a sunscreen that changes colors when the DNA damage may be too intense for a sunbather as a warning, or a sunscreen that becomes more protective as the possibility of genetic damage increased. Further research should focus on how to best implement the biofilm producing output module to protect human DNA.

<center>

{|
|-
|[[Image:Toggleswitch.JPG]]
|[[Image:UVinducedbiofilm.JPG]]
|-
|'''Fig. 1.''' The RecA / traA toggleswitch mechanism. (Kobayashi, 2004 - Permission Pending)
|'''Fig. 2.''' Crystal violet absorbance measures biofilm formation. (Kobayashi, 2004 - Permission Pending)
|}
</center>

'''Treatment'''

Synthetic biologists consider finding a cure for cancer a reachable goal. Previous to the development of synthetic biology as a field, it was known that three types of bacteria, ''Bifodobacterium'', ''Clostridium'', and ''Salmonella'' all preferentially infect the dense cells of tumors (Pawelek, 2003). All three types of bacteria are associated with reducing tumor size in patients who are infected with them, and it was noted that all three bacteria act anaerobically. Capitalizing on the idea that tumors create an unusually dense, anaerobic mass of cells, Christopher Voigt’s lab engineered bacteria to identify and invade cancer cells. They utilized the ''lux'' quorum sensing from ''Vibrio fischeri'' to identify cells growing at high densities (Fig. 3) and the ''fdhF'' promoter to identify cells growing in anaerobic conditions ''in vitro'' (Fig. 4) (Anderson, 2006). They coupled this identification mechanism with the invasin output module from ''Yersinia pseudotuberculosis'' so the ''E. coli'' could invade specific cells.

<center>

{|
|-
|[[Image:InvasinDensity.JPG]]
|[[Image:InvasinAnaerobic2.JPG]]
|-
|'''Fig. 3.''' The red line represents when the invasin output module was paired with the inducible promoter ''lux'' which senses high cell density, the black line is constitutively on invasin. (Anderson, 2006 - Permission Pending)
|'''Fig. 4.''' The invasin output module was paired with the inducible promoter ''fdhF'' which senses anaerobic conditions. (Anderson, 2006 - Permission Pending)
|}

</center>

Next, scientists hope to insert a gene that would destroy the invaded cells, making the complete cycle a search, invade, and destroy loop that would only be sensitive to cancer cells. One promising destroy mechanism has been implemented in humans via intratumoral injection, based on ''Salmonella'' that contains the ''E. coli'' cytosine deaminase gene that converts 5-fluorocytosine to 5-fluorouracil, a chemotherapy drug (Nemunaitis, 2003). Unfortunately, while the tumors did produce [http://en.wikipedia.org/wiki/Fluorouracil 5-fluorouracil] (5-FU), they did not produce it in high enough yields to cause regression of the cancer. Another experiment flooded mouse cells via intratumoral injection with 6-methylpurine-2’deoxyribose (6-MPDR), which was converted to the toxin [http://jpet.aspetjournals.org/cgi/content/abstract/304/3/1280 6-methyl purine] (MeP) by the enzyme purine nucleoside phosphorylase (PNP), which is naturally found in ''E. coli'' (Critchley, 2004) (Fig. 5).

<center>

[[Image:Tumor.JPG]]

'''Fig. 5.''' Tumor size after injections. Cancer cells were injected into mice, and 5 days later injections with PBS, 6-MPDR, and invasin-enhanced ''E-coli'' began. Tumor cells recieving injections of invasin-enhanced ''E.coli'' and 6-MPDR grew the slowest, but did not get smaller. (Critchley, 2004 - Permission Pending)

</center>

The problem with this is that even though MeP was injected into the tumor, all cells in the body might be exposed to the toxin. In addition to the search and destroy mechanism, scientists hope to increase selectivity by combining the hypoxia-sensing and density-sensing units so that ''E. coli'' will only enter eukaryotic cells that exhibit both characteristics. This would make it more likely that invasin-enhanced ''E. coli'' would only invade cancer cells rather than muscle tissue that had low oxygen due to exercise (Anderson, 2006). To create a fully functional, synthetic biology-based approach to cancer therapy, scientists need to develop bacteria that discriminately invades cancer cells and destroys them. Most of the pieces are there – scientists have engineered ''E. coli'' that selectively invades hypoxic cells or dense cells and ''Salmonella'' that can destroy tumors in vivo – they just need to be put together effectively.

Finally, synthetic biology has made its mark on the field of medicine by creating drugs that are less expensive to produce than those made by pharmaceutical companies. Jay Keasling’s project involving the antimalarial drug artemisinin is the best example. Yeast was engineered to increase normal farnesyl pyrophosphate (FPP) production, convert FPP to amorphadiene, and oxidizing amorphadiene to artemisinic acid, which can then easily be separated from the yeast cells and converted to artemisinin in the laboratory (Fig. 6). This innovation allows the production of artemisinin at lower prices than those currently on the market, and without regard to environmental constraints (Ro, 2006). On a completely different tangent, Collins’s lab engineered T7 bacteriophages to deliver biofilm-degrading enzymes such as dispersin B after infecting and replicating in an ''E. coli'' biofilm. Dispersin B works to degrade an adhesin that is critical for biofilm formation. Phages that released dispersin B were found to be 99.997% effective at removing biofilm, which is 4.5 orders of magnitude higher than non-enzymatic phages, and caused a 3.65log10 reduction in bacterial cells recovered from biofilmwhen compared to untreated biofilms (Lu, 2007) (Fig. 7). The specificity of phages for a certain type of bacteria make them a viable vector to be used in human treatments, which may range from removing dental plaques to cleaning inserted medical devices. Using synthetic biology to engineer an antimalarial drug in yeast or a biofilm-destroying enzyme in bacteriophages greatly increases the possible contributions to the field of medicine.

<center>

{|
|-
|[[Image:Artemesinin.JPG]]
|[[Image:BiofilmDegradation.JPG]]
|-
|'''Fig. 6.''' All the steps in the box indicate steps that were optimized in yeast for a maximum artemisinic acid output. (Ro, 2006 - Permission Pending)
|'''Fig. 7.''' The amount of biofilm was decreased when the dispersin B enzymes were incorporated into T7 bacteriophages. (Lu, 2007 - Permission Pending)
|}
</center>

'''Conclusion'''

Medical applications of synthetic biology are wide-ranging and eminently applicable to daily life. Synthetic biology based cancer treatments that utilize ''E.coli'' engineered to sense and attack tissues with high cell density and low oxygen availability are promising and need to be tested in vivo. Designing vectors out of attenuated bacteria may be a safe, effective method for disease treatment. Using biofilms to sense sun damage potential may decrease the likelihood of skin cancer. Finally, using engineered cells to produce medicines in a cost-effective, environmentally-friendly way may revolutionize the pharmaceutical industry. Investing more research efforts in the field of synthetic biology may turn out to be an investment in medical technology.

== Works Cited ==

-[http://www.bio.davidson.edu/courses/synthetic/papers/2006_anderson.pdf Anderson JC, Clarke EJ, Arkin, AP, and Voigt CA (2006). Environmentally controlled invasion of cancer cells by engineered bacteria. Journal of Molecular Biology 355:619-27.]

-[http://www.nature.com/gt/journal/v11/n15/abs/3302281a.html Critchley RJ, Jezzard S, Radford KJ, Goussard S, Lemoine NR, et al. (2004). Potential therapeutic applications of recombinant, invasive ''E. coli''. Gene Therapy 11:1224-33.]

-[http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&uid=15203915&cmd=showdetailview&indexed=google Garmory HS, Leary SEC, Griffin, KF, Williamson D, Brown, KA, and Titball RW (2003). The use of live attenuated bacteria as a delivery system for heterologous antigens. Journal of Drug Targeting 11:471-79.]

-[http://www.pnas.org/cgi/reprint/101/22/8414 Kobayashi H, et al. (2004). Programmable cells: Interfacing natural and engineered gene networks. PNAS 101: 8414-19.]

-[http://www.bio.davidson.edu/courses/synthetic/papers/Phage_BioFilms.pdf Lu, TK, and Collins JJ (2007). Dispersing biofilms with engineered enzymatic bacteriophage. PNAS 104: 11197-11202.]

-[http://www.nature.com/cgt/journal/v10/n10/abs/7700634a.html Nemunaitis J, Cunningham C, Senzer N, Kuhn J, Cramm J, Litz C, et al. (2003). Pilot trial of genetically modified, attenuated ''Salmonella'' expressing cytosine deaminase gene in refractory cancer patients. Cancer Gene Therapy 10:737-744.]

-[http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6W85-49FGKHC-N&_user=10&_coverDate=09%2F30%2F2003&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=d4529b45974bfe7d9ddab1fc865f088b Pawelek JM, Low KB, and Bermudes D (2003). Bacteria as tumour-targeting vectors. Lancet Oncology 4:548-56.]

-[http://www.bio.davidson.edu/courses/synthetic/papers/Keasling_malaria.pdf Ro D, et al. (2006). Production of the antimalarial drug precursor artemisinic acid in engineered yeast. Nature 440:940-43.]

Medical Applications of Synthetic Biology - Samantha Simpson

2007-12-06T04:09:04Z

Sasimpson: /* Paper */

== Project Proposal ==

My project will be on medical applications of synthetic biology. I will reference Anderson's paper on utilizing quorum-sensing and hypoxia-responsive genes coupled with invasin from ''Yersinia tuberculosis'' to invade cancer-causing cells. Coupled with Critchley's work, which describes a bacterial system that invades eukaryotic cells and delivers proteins coded for in the bacteria's genome, one could possibly create 'search and destroy' ''E.coli'' that can locate, invade, and kill tumor cells. Garmory's paper also highlights the possibility of using bacteria as a drug-delivering system, specifically vaccine vectors. Kobayashi describes cells that produce a protective biofilm layer after exposure to DNA-damaging agents. Ro engineers yeast to create an anti-malarial drug that can be created at a lower cost than the previous standard production process. These papers, and others that have a focus on direct medical applications such as novel cancer therapies, vaccination technology, biological protection, and the creation of new medicines, will be referenced to create a cohesive overview of applications of synthetic biology to the medical field.

== Paper ==

Synthetic biology is a rapidly emerging field that strives to re-design existing biological systems and components or fabricate novel biological systems and components. The end result of experimentation in synthetic biology is to help scientists understand a naturally occurring genetic pathway more fully or to create a new, useful pathway. Recent research efforts have focused on engineering bacteria to create a new form of biofuel or to make promoters of varying efficiencies for more exact gene expression. Other research efforts focus on engineering yeast to make antimalarial drugs or engineering ''E. coli'' to recognize and invade tumor-like cells, both of which are medical applications of synthetic biology. Advancements such as novel cancer therapies, vaccination technology, biological protection, and the creation of new medicines highlight synthetic biology’s potential to create breakthroughs in both the prevention and treatment side of medical science. This paper will examine various medical applications of synthetic biology and further development that needs to be done before current research can be used in hospitals and doctors’ offices.

'''Prevention'''

Engineering live bacteria to act as a vaccine vector is an area of interest in synthetic biology. Bacteria was first thought to be a good vector because it would not degrade at mucosal surface and it would survive the low pH in the gastrointestinal tract like most other orally administered antigens (Garmory, 2003). A particular strain of ''Salmonella'' with one or more deletions in the [http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&uid=15012217&cmd=showdetailview&indexed=google shikimate pathway], which creates a precursor for phenylalanine and tyrosine, is a promising vector because it gets into the body easily but does not harmfully effect the tissue and is gone within a week or two of introduction. Scientists engineered this strain to carry the [http://en.wikipedia.org/wiki/Yersinia_pestis ''Yersinia pestis''] V antigen, and studied its ability to protect mice from ''Y. pestis''. The V antigen is generally injected via an intramuscular route, however, in this study, 20 mice were given the V antigen via the intragastric route so scientists could test the effectiveness of an oral administration. Unfortunately, the inoculation was not extremely successful and only 6 of 20 mice infected with ''Y. pestis'' showed a strong response (Garmory, 2003). Garmory and colleagues cited several reasons for this: the copy number of the plasmid containing the V antigen might not have been high enough; the attenuated ''Salmonella'' may not have been able to reach cells that would be targeted by ''Y. pestis''; and the inability of the cell to secrete the antigen. The authors remain hopeful that attenuated bacteria vectors may someday produce an orally administered long-lasting response for protection against bubonic and pneumonic plague (Garmory, 2003). Many changes need to be made to live vector vaccine design before it is an effective method of treating or preventing disease, such as solving the issue of specifity of a vaccine administered orally, and creating a bacterial chassey that can secrete an antigen at an appropriate time.

Another method of disease prevention with a synthetic biology approach is related to skin cancer. ''E. coli'' was engineered to sense single-stranded DNA, and this DNA damage was coupled with a mechanism to produce a biofilm around the cell. The biofilm output module was tested with DNA damage done by [http://en.wikipedia.org/wiki/Mitomycin_c mitomycin C], a DNA crosslinking agent, or UV irradiation, a known cause of skin cancer. Single-stranded DNA activates [http://en.wikipedia.org/wiki/RecA RecA], which in turn represses the C1 repressor protein, thus allowing the transcription of the PL promoter (Kobayashi, 2004). The PL promoter is on the biofilm-forming output plasmid (pBFR), which controls biofilm formation (Fig. 1). The biofilm protection, when part of a toggle switch mechanism, lasted indefinitely when pulsed with UV light at 8 J/m2 for only 2 seconds (Kobayashi, 2004) (Fig. 2). The biofilm-production mechanism has not been engineered in eukaryotic cells, but a prokaryote-based system that could aid with human skin disease is imaginable. For example, a sunscreen that changes colors when the DNA damage may be too intense for a sunbather as a warning, or a sunscreen that becomes more protective as the possibility of genetic damage increased. Further research should focus on how to best implement the biofilm producing output module to protect human DNA.

<center>

{|
|-
|[[Image:Toggleswitch.JPG]]
|[[Image:UVinducedbiofilm.JPG]]
|-
|'''Fig. 1.''' The RecA / traA toggleswitch mechanism. (Kobayashi, 2004 - Permission Pending)
|'''Fig. 2.''' Crystal violet absorbance measures biofilm formation. (Kobayashi, 2004 - Permission Pending)
|}
</center>

'''Treatment'''

Synthetic biologists consider finding a cure for cancer a reachable goal. Previous to the development of synthetic biology as a field, it was known that three types of bacteria, ''Bifodobacterium'', ''Clostridium'', and ''Salmonella'' all preferentially infect the dense cells of tumors (Pawelek, 2003). All three types of bacteria are associated with reducing tumor size in patients who are infected with them, and it was noted that all three bacteria act anaerobically. Capitalizing on the idea that tumors create an unusually dense, anaerobic mass of cells, Christopher Voigt’s lab engineered bacteria to identify and invade cancer cells. They utilized the ''lux'' quorum sensing from ''Vibrio fischeri'' to identify cells growing at high densities (Fig. 3) and the ''fdhF'' promoter to identify cells growing in anaerobic conditions ''in vitro'' (Fig. 4) (Anderson, 2006). They coupled this identification mechanism with the invasin output module from ''Yersinia pseudotuberculosis'' so the ''E. coli'' could invade specific cells.

<center>

{|
|-
|[[Image:InvasinDensity.JPG]]
|[[Image:InvasinAnaerobic2.JPG]]
|-
|'''Fig. 3.''' The red line represents when the invasin output module was paired with the inducible promoter ''lux'' which senses high cell density, the black line is constitutively on invasin. (Anderson, 2006 - Permission Pending)
|'''Fig. 4.''' The invasin output module was paired with the inducible promoter ''fdhF'' which senses anaerobic conditions. (Anderson, 2006 - Permission Pending)
|}

</center>

Next, scientists hope to insert a gene that would destroy the invaded cells, making the complete cycle a search, invade, and destroy loop that would only be sensitive to cancer cells. One promising destroy mechanism has been implemented in humans via intratumoral injection, based on ''Salmonella'' that contains the ''E. coli'' cytosine deaminase gene that converts 5-fluorocytosine to 5-fluorouracil, a chemotherapy drug (Nemunaitis, 2003). Unfortunately, while the tumors did produce [http://en.wikipedia.org/wiki/Fluorouracil 5-fluorouracil] (5-FU), they did not produce it in high enough yields to cause regression of the cancer. Another experiment flooded mouse cells via intratumoral injection with 6-methylpurine-2’deoxyribose (6-MPDR), which was converted to the toxin [http://jpet.aspetjournals.org/cgi/content/abstract/304/3/1280 6-methyl purine] (MeP) by the enzyme purine nucleoside phosphorylase (PNP), which is naturally found in ''E. coli'' (Critchley, 2004) (Fig. 5).

<center>

[[Image:Tumor.JPG]]

'''Fig. 5.''' Tumor size after injections. Cancer cells were injected into mice, and 5 days later injections with PBS, 6-MPDR, and invasin-enhanced ''E-coli'' began. Tumor cells recieving injections of invasin-enhanced ''E.coli'' and 6-MPDR grew the slowest, but did not get smaller. (Critchley, 2004 - Permission Pending)

</center>

The problem with this is that even though MeP was injected into the tumor, all cells in the body might be exposed to the toxin. In addition to the search and destroy mechanism, scientists hope to increase selectivity by combining the hypoxia-sensing and density-sensing units so that ''E. coli'' will only enter eukaryotic cells that exhibit both characteristics. This would make it more likely that invasin-enhanced ''E. coli'' would only invade cancer cells rather than muscle tissue that had low oxygen due to exercise (Anderson, 2006). To create a fully functional, synthetic biology-based approach to cancer therapy, scientists need to develop bacteria that discriminately invades cancer cells and destroys them. Most of the pieces are there – scientists have engineered ''E. coli'' that selectively invades hypoxic cells or dense cells and ''Salmonella'' that can destroy tumors in vivo – they just need to be put together effectively.

Finally, synthetic biology has made its mark on the field of medicine by creating drugs that are less expensive to produce than those made by pharmaceutical companies. Jay Keasling’s project involving the antimalarial drug artemisinin is the best example. Yeast was engineered to increase normal farnesyl pyrophosphate (FPP) production, convert FPP to amorphadiene, and oxidizing amorphadiene to artemisinic acid, which can then easily be separated from the yeast cells and converted to artemisinin in the laboratory (Fig. 6). This innovation allows the production of artemisinin at lower prices than those currently on the market, and without regard to environmental constraints (Ro, 2006). On a completely different tangent, Collins’s lab engineered T7 bacteriophages to deliver biofilm-degrading enzymes such as dispersin B after infecting and replicating in an ''E. coli'' biofilm. Dispersin B works to degrade an adhesin that is critical for biofilm formation. Phages that released dispersin B were found to be 99.997% effective at removing biofilm, which is 4.5 orders of magnitude higher than non-enzymatic phages (Lu, 2007) (Fig. 7). The specificity of phages for a certain type of bacteria make them a viable vector to be used in human treatments, which may range from removing dental plaques to cleaning inserted medical devices. Using synthetic biology to engineer an antimalarial drug in yeast or a biofilm-destroying enzyme in bacteriophages greatly increases the possible contributions to the field of medicine.

<center>

{|
|-
|[[Image:Artemesinin.JPG]]
|[[Image:BiofilmDegradation.JPG]]
|-
|'''Fig. 6.''' All the steps in the box indicate steps that were optimized in yeast for a maximum artemisinic acid output. (Ro, 2006 - Permission Pending)
|'''Fig. 7.''' The amount of biofilm was decreased when the dispersin B enzymes were incorporated into T7 bacteriophages. (Lu, 2007 - Permission Pending)
|}
</center>

'''Conclusion'''

Medical applications of synthetic biology are wide-ranging and eminently applicable to daily life. Synthetic biology based cancer treatments that utilize ''E.coli'' engineered to sense and attack tissues with high cell density and low oxygen availability are promising and need to be tested in vivo. Designing vectors out of attenuated bacteria may be a safe, effective method for disease treatment. Using biofilms to sense sun damage potential may decrease the likelihood of skin cancer. Finally, using engineered cells to produce medicines in a cost-effective, environmentally-friendly way may revolutionize the pharmaceutical industry. Investing more research efforts in the field of synthetic biology may turn out to be an investment in medical technology.

== Works Cited ==

-[http://www.bio.davidson.edu/courses/synthetic/papers/2006_anderson.pdf Anderson JC, Clarke EJ, Arkin, AP, and Voigt CA (2006). Environmentally controlled invasion of cancer cells by engineered bacteria. Journal of Molecular Biology 355:619-27.]

-[http://www.nature.com/gt/journal/v11/n15/abs/3302281a.html Critchley RJ, Jezzard S, Radford KJ, Goussard S, Lemoine NR, et al. (2004). Potential therapeutic applications of recombinant, invasive ''E. coli''. Gene Therapy 11:1224-33.]

-[http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&uid=15203915&cmd=showdetailview&indexed=google Garmory HS, Leary SEC, Griffin, KF, Williamson D, Brown, KA, and Titball RW (2003). The use of live attenuated bacteria as a delivery system for heterologous antigens. Journal of Drug Targeting 11:471-79.]

-[http://www.pnas.org/cgi/reprint/101/22/8414 Kobayashi H, et al. (2004). Programmable cells: Interfacing natural and engineered gene networks. PNAS 101: 8414-19.]

-[http://www.bio.davidson.edu/courses/synthetic/papers/Phage_BioFilms.pdf Lu, TK, and Collins JJ (2007). Dispersing biofilms with engineered enzymatic bacteriophage. PNAS 104: 11197-11202.]

-[http://www.nature.com/cgt/journal/v10/n10/abs/7700634a.html Nemunaitis J, Cunningham C, Senzer N, Kuhn J, Cramm J, Litz C, et al. (2003). Pilot trial of genetically modified, attenuated ''Salmonella'' expressing cytosine deaminase gene in refractory cancer patients. Cancer Gene Therapy 10:737-744.]

-[http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6W85-49FGKHC-N&_user=10&_coverDate=09%2F30%2F2003&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=d4529b45974bfe7d9ddab1fc865f088b Pawelek JM, Low KB, and Bermudes D (2003). Bacteria as tumour-targeting vectors. Lancet Oncology 4:548-56.]

-[http://www.bio.davidson.edu/courses/synthetic/papers/Keasling_malaria.pdf Ro D, et al. (2006). Production of the antimalarial drug precursor artemisinic acid in engineered yeast. Nature 440:940-43.]

Medical Applications of Synthetic Biology - Samantha Simpson

2007-12-06T04:03:05Z

Sasimpson: /* Paper */

== Project Proposal ==

My project will be on medical applications of synthetic biology. I will reference Anderson's paper on utilizing quorum-sensing and hypoxia-responsive genes coupled with invasin from ''Yersinia tuberculosis'' to invade cancer-causing cells. Coupled with Critchley's work, which describes a bacterial system that invades eukaryotic cells and delivers proteins coded for in the bacteria's genome, one could possibly create 'search and destroy' ''E.coli'' that can locate, invade, and kill tumor cells. Garmory's paper also highlights the possibility of using bacteria as a drug-delivering system, specifically vaccine vectors. Kobayashi describes cells that produce a protective biofilm layer after exposure to DNA-damaging agents. Ro engineers yeast to create an anti-malarial drug that can be created at a lower cost than the previous standard production process. These papers, and others that have a focus on direct medical applications such as novel cancer therapies, vaccination technology, biological protection, and the creation of new medicines, will be referenced to create a cohesive overview of applications of synthetic biology to the medical field.

== Paper ==

Synthetic biology is a rapidly emerging field that strives to re-design existing biological systems and components or fabricate novel biological systems and components. The end result of experimentation in synthetic biology is to help scientists understand a naturally occurring genetic pathway more fully or to create a new, useful pathway. Recent research efforts have focused on engineering bacteria to create a new form of biofuel or to make promoters of varying efficiencies for more exact gene expression. Other research efforts focus on engineering yeast to make antimalarial drugs or engineering ''E. coli'' to recognize and invade tumor-like cells, both of which are medical applications of synthetic biology. Advancements such as novel cancer therapies, vaccination technology, biological protection, and the creation of new medicines highlight synthetic biology’s potential to create breakthroughs in both the prevention and treatment side of medical science. This paper will examine various medical applications of synthetic biology and further development that needs to be done before current research can be used in hospitals and doctors’ offices.

'''Prevention'''

Engineering live bacteria to act as a vaccine vector is an area of interest in synthetic biology. Bacteria was first thought to be a good vector because it would not degrade at mucosal surface and it would survive the low pH in the gastrointestinal tract like most other orally administered antigens (Garmory, 2003). A particular strain of ''Salmonella'' with one or more deletions in the [http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&uid=15012217&cmd=showdetailview&indexed=google shikimate pathway], which creates a precursor for phenylalanine and tyrosine, is a promising vector because it gets into the body easily but does not harmfully effect the tissue and is gone within a week or two of introduction. Scientists engineered this strain to carry the [http://en.wikipedia.org/wiki/Yersinia_pestis ''Yersinia pestis''] V antigen, and studied its ability to protect mice from ''Y. pestis''. The V antigen is generally injected via an intramuscular route, however, in this study, 20 mice were given the V antigen via the intragastric route so scientists could test the effectiveness of an oral administration. Unfortunately, the inoculation was not extremely successful and only 6 of 20 mice infected with ''Y. pestis'' showed a strong response (Garmory, 2003). Garmory and colleagues cited several reasons for this: the copy number of the plasmid containing the V antigen might not have been high enough; the attenuated ''Salmonella'' may not have been able to reach cells that would be targeted by ''Y. pestis''; and the inability of the cell to secrete the antigen. The authors remain hopeful that attenuated bacteria vectors may someday produce an orally administered long-lasting response for protection against bubonic and pneumonic plague (Garmory, 2003). Many changes need to be made to live vector vaccine design before it is an effective method of treating or preventing disease, such as solving the issue of specifity of a vaccine administered orally, and creating a bacterial chassey that can secrete an antigen at an appropriate time.

Another method of disease prevention with a synthetic biology approach is related to skin cancer. ''E. coli'' was engineered to sense single-stranded DNA, and this DNA damage was coupled with a mechanism to produce a biofilm around the cell. The biofilm output module was tested with DNA damage done by [http://en.wikipedia.org/wiki/Mitomycin_c mitomycin C], a DNA crosslinking agent, or UV irradiation, a known cause of skin cancer. Single-stranded DNA activates [http://en.wikipedia.org/wiki/RecA RecA], which in turn represses the C1 repressor protein, thus allowing the transcription of the PL promoter (Kobayashi, 2004). The PL promoter is on the biofilm-forming output plasmid (pBFR), which controls biofilm formation (Fig. 1). The biofilm protection, when part of a toggle switch mechanism, lasted indefinitely when pulsed with UV light at 8 J/m2 for only 2 seconds (Kobayashi, 2004) (Fig. 2). The biofilm-production mechanism has not been engineered in eukaryotic cells, but a prokaryote-based system that could aid with human skin disease is imaginable. For example, a sunscreen that changes colors when the DNA damage may be too intense for a sunbather as a warning, or a sunscreen that becomes more protective as the possibility of genetic damage increased. Further research should focus on how to best implement the biofilm producing output module to protect human DNA.

<center>

{|
|-
|[[Image:Toggleswitch.JPG]]
|[[Image:UVinducedbiofilm.JPG]]
|-
|'''Fig. 1.''' The RecA / traA toggleswitch mechanism. (Kobayashi, 2004 - Permission Pending)
|'''Fig. 2.''' Crystal violet absorbance measures biofilm formation. (Kobayashi, 2004 - Permission Pending)
|}
</center>

'''Treatment'''

Synthetic biologists consider finding a cure for cancer a reachable goal. Previous to the development of synthetic biology as a field, it was known that three types of bacteria, ''Bifodobacterium'', ''Clostridium'', and ''Salmonella'' all preferentially infect the dense cells of tumors (Pawelek, 2003). All three types of bacteria are associated with reducing tumor size in patients who are infected with them, and it was noted that all three bacteria act anaerobically. Capitalizing on the idea that tumors create an unusually dense, anaerobic mass of cells, Christopher Voigt’s lab engineered bacteria to identify and invade cancer cells. They utilized the ''lux'' quorum sensing from ''Vibrio fischeri'' to identify cells growing at high densities (Fig. 3) and the ''fdhF'' promoter to identify cells growing in anaerobic conditions ''in vitro'' (Fig. 4) (Anderson, 2006). They coupled this identification mechanism with the invasin output module from ''Yersinia pseudotuberculosis'' so the ''E. coli'' could invade specific cells.

<center>

{|
|-
|[[Image:InvasinDensity.JPG]]
|[[Image:InvasinAnaerobic2.JPG]]
|-
|'''Fig. 3.''' The red line represents when the invasin output module was paired with the inducible promoter ''lux'' which senses high cell density, the black line is constitutively on invasin. (Anderson, 2006 - Permission Pending)
|'''Fig. 4.''' The invasin output module was paired with the inducible promoter ''fdhF'' which senses anaerobic conditions. (Anderson, 2006 - Permission Pending)
|}

</center>

Next, scientists hope to insert a gene that would destroy the invaded cells, making the complete cycle a search, invade, and destroy loop that would only be sensitive to cancer cells. One promising destroy mechanism has been implemented in humans via intratumoral injection, based on ''Salmonella'' that contains the ''E. coli'' cytosine deaminase gene that converts 5-fluorocytosine to 5-fluorouracil, a chemotherapy drug (Nemunaitis, 2003). Unfortunately, while the tumors did produce [http://en.wikipedia.org/wiki/Fluorouracil 5-fluorouracil] (5-FU), they did not produce it in high enough yields to cause regression of the cancer. Another experiment flooded mouse cells via intratumoral injection with 6-methylpurine-2’deoxyribose (6-MPDR), which was converted to the toxin [http://jpet.aspetjournals.org/cgi/content/abstract/304/3/1280 6-methyl purine] (MeP) by the enzyme purine nucleoside phosphorylase (PNP), which is naturally found in ''E. coli'' (Critchley, 2004) (Fig. 5).

<center>

[[Image:Tumor.JPG]]

'''Fig. 5.''' Tumor size after injections. Cancer cells were injected into mice, and 5 days later injections with PBS, 6-MPDR, and invasin-enhanced ''E-coli'' began. Tumor cells recieving injections of invasin-enhanced ''E.coli'' and 6-MPDR grew the slowest, but did not get smaller. (Critchley, 2004 - Permission Pending)

</center>

The problem with this is that even though MeP was injected into the tumor, all cells in the body might be exposed to the toxin. In addition to the search and destroy mechanism, scientists hope to increase selectivity by combining the hypoxia-sensing and density-sensing units so that ''E. coli'' will only enter eukaryotic cells that exhibit both characteristics. This would make it more likely that invasin-enhanced ''E. coli'' would only invade cancer cells rather than muscle tissue that had low oxygen due to exercise (Anderson, 2006). To create a fully functional, synthetic biology-based approach to cancer therapy, scientists need to develop bacteria that discriminately invades cancer cells and destroys them. Most of the pieces are there – scientists have engineered ''E. coli'' that selectively invades hypoxic cells or dense cells and ''Salmonella'' that can destroy tumors in vivo – they just need to be put together effectively.

Finally, synthetic biology has made its mark on the field of medicine by creating drugs that are less expensive to produce than those made by pharmaceutical companies. Jay Keasling’s project involving the antimalarial drug artemisinin is the best example. Yeast was engineered to increase normal farnesyl pyrophosphate (FPP) production, convert FPP to amorphadiene, and oxidizing amorphadiene to artemisinic acid, which can then easily be separated from the yeast cells and converted to artemisinin in the laboratory (Fig. 6). This innovation allows the production of artemisinin at lower prices than those currently on the market, and without regard to environmental constraints (Ro, 2006). On a completely different tangent, Collins’s lab engineered T7 bacteriophages to deliver biofilm-degrading enzymes such as dispersin B after infecting and replicating in an ''E. coli'' biofilm. Dispersin B works to degrade an adhesin that is critical for biofilm formation. Phages that released dispersin B were found to be 99.997% effective at removing biofilm, which is 4.5 orders of magnitude higher than non-enzymatic phages (Lu, 2007) (Fig. 7). The specificity of phages for a certain type of bacteria make them a viable vector to be used in human treatments, which may range from removing dental plaques to cleaning inserted medical devices. Using synthetic biology to engineer an antimalarial drug in yeast or a biofilm-destroying enzyme in bacteriophages greatly increases the possible contributions to the field of medicine.

<center>

{|
|-
|[[Image:Artemesinin.JPG]]
|[[Image:BiofilmDegradation.JPG]]
|-
|'''Fig. 6.''' All the steps in the box indicate steps that were optimized in yeast for a maximum artemisinic acid output. (Ro, 2006 - Permission Pending)
|'''Fig. 7.''' The amount of biofilm was decreased when the dispersin B enzymes were incorporated into T7 bacteriophages. (Lu, 2007 - Permission Pending)
|}
</center>

'''Conclusion'''

Medical applications of synthetic biology are wide-ranging and eminently applicable to daily life. Synthetic biology based cancer treatments that utilize ''E.coli'' engineered to sense and attack tissues with high cell density and low oxygen availability are promising and need to be tested in vivo. Designing vectors out of attenuated bacteria may be a safe, effective method for disease treatment. Using biofilms to sense sun damage potential may decrease the likelihood of skin cancer. Finally, using engineered cells to produce medicines in a cost-effective, environmentally-friendly way may revolutionize the pharmaceutical industry. Investing more research efforts in the field of synthetic biology may turn out to be an investment in medical technology.

== Works Cited ==

-[http://www.bio.davidson.edu/courses/synthetic/papers/2006_anderson.pdf Anderson JC, Clarke EJ, Arkin, AP, and Voigt CA (2006). Environmentally controlled invasion of cancer cells by engineered bacteria. Journal of Molecular Biology 355:619-27.]

-[http://www.nature.com/gt/journal/v11/n15/abs/3302281a.html Critchley RJ, Jezzard S, Radford KJ, Goussard S, Lemoine NR, et al. (2004). Potential therapeutic applications of recombinant, invasive ''E. coli''. Gene Therapy 11:1224-33.]

-[http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&uid=15203915&cmd=showdetailview&indexed=google Garmory HS, Leary SEC, Griffin, KF, Williamson D, Brown, KA, and Titball RW (2003). The use of live attenuated bacteria as a delivery system for heterologous antigens. Journal of Drug Targeting 11:471-79.]

-[http://www.pnas.org/cgi/reprint/101/22/8414 Kobayashi H, et al. (2004). Programmable cells: Interfacing natural and engineered gene networks. PNAS 101: 8414-19.]

-[http://www.bio.davidson.edu/courses/synthetic/papers/Phage_BioFilms.pdf Lu, TK, and Collins JJ (2007). Dispersing biofilms with engineered enzymatic bacteriophage. PNAS 104: 11197-11202.]

-[http://www.nature.com/cgt/journal/v10/n10/abs/7700634a.html Nemunaitis J, Cunningham C, Senzer N, Kuhn J, Cramm J, Litz C, et al. (2003). Pilot trial of genetically modified, attenuated ''Salmonella'' expressing cytosine deaminase gene in refractory cancer patients. Cancer Gene Therapy 10:737-744.]

-[http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6W85-49FGKHC-N&_user=10&_coverDate=09%2F30%2F2003&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=d4529b45974bfe7d9ddab1fc865f088b Pawelek JM, Low KB, and Bermudes D (2003). Bacteria as tumour-targeting vectors. Lancet Oncology 4:548-56.]

-[http://www.bio.davidson.edu/courses/synthetic/papers/Keasling_malaria.pdf Ro D, et al. (2006). Production of the antimalarial drug precursor artemisinic acid in engineered yeast. Nature 440:940-43.]

Medical Applications of Synthetic Biology - Samantha Simpson

2007-12-06T03:39:48Z

Sasimpson: /* Paper */

== Project Proposal ==

My project will be on medical applications of synthetic biology. I will reference Anderson's paper on utilizing quorum-sensing and hypoxia-responsive genes coupled with invasin from ''Yersinia tuberculosis'' to invade cancer-causing cells. Coupled with Critchley's work, which describes a bacterial system that invades eukaryotic cells and delivers proteins coded for in the bacteria's genome, one could possibly create 'search and destroy' ''E.coli'' that can locate, invade, and kill tumor cells. Garmory's paper also highlights the possibility of using bacteria as a drug-delivering system, specifically vaccine vectors. Kobayashi describes cells that produce a protective biofilm layer after exposure to DNA-damaging agents. Ro engineers yeast to create an anti-malarial drug that can be created at a lower cost than the previous standard production process. These papers, and others that have a focus on direct medical applications such as novel cancer therapies, vaccination technology, biological protection, and the creation of new medicines, will be referenced to create a cohesive overview of applications of synthetic biology to the medical field.

== Paper ==

Synthetic biology is a rapidly emerging field that strives to re-design existing biological systems and components or fabricate novel biological systems and components. The end result of experimentation in synthetic biology is to help scientists understand a naturally occurring genetic pathway more fully or to create a new, useful pathway. Recent research efforts have focused on engineering bacteria to create a new form of biofuel or to make promoters of varying efficiencies for more exact gene expression. Other research efforts focus on engineering yeast to make antimalarial drugs or engineering ''E. coli'' to recognize and invade tumor-like cells, both of which are medical applications of synthetic biology. Advancements such as novel cancer therapies, vaccination technology, biological protection, and the creation of new medicines highlight synthetic biology’s potential to create breakthroughs in both the prevention and treatment side of medical science. This paper will examine various medical applications of synthetic biology and further development that needs to be done before current research can be used in hospitals and doctors’ offices.

'''Prevention'''

Engineering live bacteria to act as a vaccine vector is an area of interest in synthetic biology. Bacteria was first thought to be a good vector because it would not degrade at mucosal surface and it would survive the low pH in the gastrointestinal tract like most other orally administered antigens (Garmory, 2003). A particular strain of ''Salmonella'' with one or more deletions in the [http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&uid=15012217&cmd=showdetailview&indexed=google shikimate pathway], which creates a precursor for phenylalanine and tyrosine, is a promising vector because it gets into the body easily but does not harmfully effect the tissue and is gone within a week or two of introduction. Scientists engineered this strain to carry the [http://en.wikipedia.org/wiki/Yersinia_pestis ''Yersinia pestis''] V antigen, and studied its ability to protect mice from ''Y. pestis''. The V antigen is generally injected via an intramuscular route, however, in this study, 20 mice were given the V antigen via the intragastric route so scientists could test the effectiveness of an oral administration. Unfortunately, the inoculation was not extremely successful and only 6 of 20 mice infected with ''Y. pestis'' showed a strong response (Garmory, 2003). Garmory and colleagues cited several reasons for this: the copy number of the plasmid containing the V antigen might not have been high enough; the attenuated ''Salmonella'' may not have been able to reach cells that would be targeted by ''Y. pestis''; and the inability of the cell to secrete the antigen. The authors remain hopeful that attenuated bacteria vectors may someday produce an orally administered long-lasting response for protection against bubonic and pneumonic plague (Garmory, 2003). Many changes need to be made to live vector vaccine design before it is an effective method of treating or preventing disease, such as solving the issue of specifity of a vaccine administered orally, and creating a bacterial chassey that can secrete an antigen at an appropriate time.

Another method of disease prevention with a synthetic biology approach is related to skin cancer. ''E. coli'' was engineered to sense single-stranded DNA, and this DNA damage was coupled with a mechanism to produce a biofilm around the cell. The biofilm output module was tested with DNA damage done by [http://en.wikipedia.org/wiki/Mitomycin_c mitomycin C], a DNA crosslinking agent, or UV irradiation, a known cause of skin cancer. Single-stranded DNA activates [http://en.wikipedia.org/wiki/RecA RecA], which in turn represses the C1 repressor protein, thus allowing the transcription of the PL promoter (Kobayashi, 2004). The PL promoter is on the biofilm-forming output plasmid (pBFR), which controls biofilm formation (Fig. 1). The biofilm protection, when part of a toggle switch mechanism, lasted indefinitely when pulsed with UV light at 8 J/m2 for only 2 seconds (Kobayashi, 2004) (Fig. 2). The biofilm-production mechanism has not been engineered in eukaryotic cells, but a prokaryote-based system that could aid with human skin disease is imaginable. For example, a sunscreen that changes colors when the DNA damage may be too intense for a sunbather as a warning, or a sunscreen that becomes more protective as the possibility of genetic damage increased. Further research should focus on how to best implement the biofilm producing output module to protect human DNA.

<center>

{|
|-
|[[Image:Toggleswitch.JPG]]
|[[Image:UVinducedbiofilm.JPG]]
|-
|'''Fig. 1.''' The RecA / traA toggleswitch mechanism. (Kobayashi, 2004 - Permission Pending)
|'''Fig. 2.''' Crystal violet absorbance measures biofilm formation. (Kobayashi, 2004 - Permission Pending)
|}
</center>

'''Treatment'''

Synthetic biologists consider finding a cure for cancer a reachable goal. Previous to the development of synthetic biology as a field, it was known that three types of bacteria, ''Bifodobacterium'', ''Clostridium'', and ''Salmonella'' all preferentially infect the dense cells of tumors (Pawelek, 2003). All three types of bacteria are associated with reducing tumor size in patients who are infected with them, and it was noted that all three bacteria act anaerobically. Capitalizing on the idea that tumors create an unusually dense, anaerobic mass of cells, Christopher Voigt’s lab engineered bacteria to identify and invade cancer cells. They utilized the ''lux'' quorum sensing from ''Vibrio fischeri'' to identify cells growing at high densities (Fig. 3) and the ''fdhF'' promoter to identify cells growing in anaerobic conditions (Fig. 4) (Anderson, 2006). They coupled this identification mechanism with the invasin output module from ''Yersinia pseudotuberculosis'' so the ''E. coli'' could invade specific cells.

<center>

{|
|-
|[[Image:InvasinDensity.JPG]]
|[[Image:InvasinAnaerobic2.JPG]]
|-
|'''Fig. 3.''' The red line represents when the invasin output module was paired with the inducible promoter ''lux'' which senses high cell density, the black line is constitutively on invasin. (Anderson, 2006 - Permission Pending)
|'''Fig. 4.''' The invasin output module was paired with the inducible promoter ''fdhF'' which senses anaerobic conditions. (Anderson, 2006 - Permission Pending)
|}

</center>

Next, scientists hope to insert a gene that would destroy the invaded cells, making the complete cycle a search, invade, and destroy loop that would only be sensitive to cancer cells. One promising destroy mechanism has been implemented in humans via intratumoral injection, based on ''Salmonella'' that contains the ''E. coli'' cytosine deaminase gene that converts 5-fluorocytosine to 5-fluorouracil, a chemotherapy drug (Nemunaitis, 2003). Unfortunately, while the tumors did produce [http://en.wikipedia.org/wiki/Fluorouracil 5-fluorouracil] (5-FU), they did not produce it in high enough yields to cause regression of the cancer. Another experiment flooded mouse cells with 6-methylpurine-2’deoxyribose (6-MPDR), which was converted to the toxin [http://jpet.aspetjournals.org/cgi/content/abstract/304/3/1280 6-methyl purine] (MeP) by the enzyme purine nucleoside phosphorylase (PNP), which is naturally found in ''E. coli'' (Critchley, 2004) (Fig. 5).

<center>

[[Image:Tumor.JPG]]

'''Fig. 5.''' Tumor size after injections. Cancer cells were injected into mice, and 5 days later injections with PBS, 6-MPDR, and invasin-enhanced ''E-coli'' began. Tumor cells recieving injections of invasin-enhanced ''E.coli'' and 6-MPDR grew the slowest, but did not get smaller. (Critchley, 2004 - Permission Pending)

</center>

The problem with this is that all cells in the body might be exposed to the toxin MeP. In addition to the search and destroy mechanism, scientists hope to increase selectivity by combining the hypoxia-sensing and density-sensing units so that ''E. coli'' will only enter eukaryotic cells that exhibit both characteristics. This would make it more likely that invasin-enhanced ''E. coli'' would only invade cancer cells rather than muscle tissue that had low oxygen due to exercise (Anderson, 2006). To create a fully functional, synthetic biology-based approach to cancer therapy, scientists need to develop bacteria that discriminately invades cancer cells and destroys them. Most of the pieces are there – scientists have engineered ''E. coli'' that selectively invades hypoxic cells or dense cells and ''Salmonella'' that can destroy tumors in vivo – they just need to be put together effectively.

Finally, synthetic biology has made its mark on the field of medicine by creating drugs that are less expensive to produce than those made by pharmaceutical companies. Jay Keasling’s project involving the antimalarial drug artemisinin is the best example. Yeast was engineered to increase normal farnesyl pyrophosphate (FPP) production, convert FPP to amorphadiene, and oxidizing amorphadiene to artemisinic acid, which can then easily be separated from the yeast cells and converted to artemisinin in the laboratory (Fig. 6). This innovation allows the production of artemisinin at lower prices than those currently on the market, and without regard to environmental constraints (Ro, 2006). On a completely different tangent, Collins’s lab engineered T7 bacteriophages to deliver biofilm-degrading enzymes such as dispersin B after infecting and replicating in an ''E. coli'' biofilm. Dispersin B works to degrade an adhesin that is critical for biofilm formation. Phages that released dispersin B were found to be 99.997% effective at removing biofilm, which is 4.5 orders of magnitude higher than non-enzymatic phages (Lu, 2007) (Fig. 7). The specificity of phages for a certain type of bacteria make them a viable vector to be used in human treatments, which may range from removing dental plaques to cleaning inserted medical devices. Using synthetic biology to engineer an antimalarial drug in yeast or a biofilm-destroying enzyme in bacteriophages greatly increases the possible contributions to the field of medicine.

<center>

{|
|-
|[[Image:Artemesinin.JPG]]
|[[Image:BiofilmDegradation.JPG]]
|-
|'''Fig. 6.''' All the steps in the box indicate steps that were optimized in yeast for a maximum artemisinic acid output. (Ro, 2006 - Permission Pending)
|'''Fig. 7.''' The amount of biofilm was decreased when the dispersin B enzymes were incorporated into T7 bacteriophages. (Lu, 2007 - Permission Pending)
|}
</center>

'''Conclusion'''

Medical applications of synthetic biology are wide-ranging and eminently applicable to daily life. Synthetic biology based cancer treatments that utilize ''E.coli'' engineered to sense and attack tissues with high cell density and low oxygen availability are promising and need to be tested in vivo. Designing vectors out of attenuated bacteria may be a safe, effective method for disease treatment. Using biofilms to sense sun damage potential may decrease the likelihood of skin cancer. Finally, using engineered cells to produce medicines in a cost-effective, environmentally-friendly way may revolutionize the pharmaceutical industry. Investing more research efforts in the field of synthetic biology may turn out to be an investment in medical technology.

== Works Cited ==

-[http://www.bio.davidson.edu/courses/synthetic/papers/2006_anderson.pdf Anderson JC, Clarke EJ, Arkin, AP, and Voigt CA (2006). Environmentally controlled invasion of cancer cells by engineered bacteria. Journal of Molecular Biology 355:619-27.]

-[http://www.nature.com/gt/journal/v11/n15/abs/3302281a.html Critchley RJ, Jezzard S, Radford KJ, Goussard S, Lemoine NR, et al. (2004). Potential therapeutic applications of recombinant, invasive ''E. coli''. Gene Therapy 11:1224-33.]

-[http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&uid=15203915&cmd=showdetailview&indexed=google Garmory HS, Leary SEC, Griffin, KF, Williamson D, Brown, KA, and Titball RW (2003). The use of live attenuated bacteria as a delivery system for heterologous antigens. Journal of Drug Targeting 11:471-79.]

-[http://www.pnas.org/cgi/reprint/101/22/8414 Kobayashi H, et al. (2004). Programmable cells: Interfacing natural and engineered gene networks. PNAS 101: 8414-19.]

-[http://www.bio.davidson.edu/courses/synthetic/papers/Phage_BioFilms.pdf Lu, TK, and Collins JJ (2007). Dispersing biofilms with engineered enzymatic bacteriophage. PNAS 104: 11197-11202.]

-[http://www.nature.com/cgt/journal/v10/n10/abs/7700634a.html Nemunaitis J, Cunningham C, Senzer N, Kuhn J, Cramm J, Litz C, et al. (2003). Pilot trial of genetically modified, attenuated ''Salmonella'' expressing cytosine deaminase gene in refractory cancer patients. Cancer Gene Therapy 10:737-744.]

-[http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6W85-49FGKHC-N&_user=10&_coverDate=09%2F30%2F2003&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=d4529b45974bfe7d9ddab1fc865f088b Pawelek JM, Low KB, and Bermudes D (2003). Bacteria as tumour-targeting vectors. Lancet Oncology 4:548-56.]

-[http://www.bio.davidson.edu/courses/synthetic/papers/Keasling_malaria.pdf Ro D, et al. (2006). Production of the antimalarial drug precursor artemisinic acid in engineered yeast. Nature 440:940-43.]

Medical Applications of Synthetic Biology - Samantha Simpson

2007-12-06T03:33:05Z

Sasimpson: /* Paper */

== Project Proposal ==

My project will be on medical applications of synthetic biology. I will reference Anderson's paper on utilizing quorum-sensing and hypoxia-responsive genes coupled with invasin from ''Yersinia tuberculosis'' to invade cancer-causing cells. Coupled with Critchley's work, which describes a bacterial system that invades eukaryotic cells and delivers proteins coded for in the bacteria's genome, one could possibly create 'search and destroy' ''E.coli'' that can locate, invade, and kill tumor cells. Garmory's paper also highlights the possibility of using bacteria as a drug-delivering system, specifically vaccine vectors. Kobayashi describes cells that produce a protective biofilm layer after exposure to DNA-damaging agents. Ro engineers yeast to create an anti-malarial drug that can be created at a lower cost than the previous standard production process. These papers, and others that have a focus on direct medical applications such as novel cancer therapies, vaccination technology, biological protection, and the creation of new medicines, will be referenced to create a cohesive overview of applications of synthetic biology to the medical field.

== Paper ==

Synthetic biology is a rapidly emerging field that strives to re-design existing biological systems and components or fabricate novel biological systems and components. The end result of experimentation in synthetic biology is to help scientists understand a naturally occurring genetic pathway more fully or to create a new, useful pathway. Recent research efforts have focused on engineering bacteria to create a new form of biofuel or to make promoters of varying efficiencies for more exact gene expression. Other research efforts focus on engineering yeast to make antimalarial drugs or engineering ''E. coli'' to recognize and invade tumor-like cells, both of which are medical applications of synthetic biology. Advancements such as novel cancer therapies, vaccination technology, biological protection, and the creation of new medicines highlight synthetic biology’s potential to create breakthroughs in both the prevention and treatment side of medical science. This paper will examine various medical applications of synthetic biology and further development that needs to be done before current research can be used in hospitals and doctors’ offices.

'''Prevention'''

Engineering live bacteria to act as a vaccine vector is an area of interest in synthetic biology. Bacteria was first thought to be a good vector because it would not degrade at mucosal surface and it would survive the low pH in the gastrointestinal tract like most other orally administered antigens (Garmory, 2003). A particular strain of ''Salmonella'' with one or more deletions in the [http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&uid=15012217&cmd=showdetailview&indexed=google shikimate pathway], which creates a precursor for phenylalanine and tyrosine, is a promising vector because it gets into the body easily but does not harmfully effect the tissue and is gone within a week or two of introduction. Scientists engineered this strain to carry the [http://en.wikipedia.org/wiki/Yersinia_pestis ''Yersinia pestis''] V antigen, and studied its ability to protect mice from ''Y. pestis''. The V antigen is generally injected via an intramuscular route, however, in this study, 20 mice were given the V antigen via the intragastric route so scientists could test the effectiveness of an oral administration. Unfortunately, the inoculation was not extremely successful and only 6 of 20 mice infected with ''Y. pestis'' showed a strong response (Garmory, 2003). Garmory and colleagues cited several reasons for this: the copy number of the plasmid containing the V antigen might not have been high enough; the attenuated ''Salmonella'' may not have been able to reach cells that would be targeted by ''Y. pestis''; and the inability of the cell to secrete the antigen. The authors remain hopeful that attenuated bacteria vectors may someday produce an orally administered long-lasting response for protection against bubonic and pneumonic plague (Garmory, 2003). Many changes need to be made to live vector vaccine design before it is an effective method of treating or preventing disease, such as solving the issue of specifity of a vaccine administered orally, and creating a bacterial chassey that can secrete an antigen at an appropriate time.

Another method of disease prevention with a synthetic biology approach is related to skin cancer. ''E. coli'' was engineered to sense single-stranded DNA, and this DNA damage was coupled with a mechanism to produce a biofilm around the cell. The biofilm output module was tested with DNA damage done by [http://en.wikipedia.org/wiki/Mitomycin_c mitomycin C], a DNA crosslinking agent, or UV irradiation, a known cause of skin cancer. Single-stranded DNA activates [http://en.wikipedia.org/wiki/RecA RecA], which in turn represses the C1 repressor protein, thus allowing the transcription of the PL promoter (Kobayashi, 2004). The PL promoter is in charge of the ''traA'' gene, which controls biofilm formation (Fig. 1). The biofilm protection, when part of a toggle switch mechanism, lasted indefinitely when pulsed with UV light at 8 J/m2 for only 2 seconds (Kobayashi, 2004) (Fig. 2). The biofilm-production mechanism has not been engineered in eukaryotic cells, but a prokaryote-based system that could aid with human skin disease is imaginable. For example, a sunscreen that changes colors when the DNA damage may be too intense for a sunbather as a warning, or a sunscreen that becomes more protective as the possibility of genetic damage increased. Further research should focus on how to best implement the biofilm producing output module to protect human DNA.

<center>

{|
|-
|[[Image:Toggleswitch.JPG]]
|[[Image:UVinducedbiofilm.JPG]]
|-
|'''Fig. 1.''' The RecA / traA toggleswitch mechanism. (Kobayashi, 2004 - Permission Pending)
|'''Fig. 2.''' Crystal violet absorbance measures biofilm formation. (Kobayashi, 2004 - Permission Pending)
|}
</center>

'''Treatment'''

Synthetic biologists consider finding a cure for cancer a reachable goal. Previous to the development of synthetic biology as a field, it was known that three types of bacteria, ''Bifodobacterium'', ''Clostridium'', and ''Salmonella'' all preferentially infect the dense cells of tumors (Pawelek, 2003). All three types of bacteria are associated with reducing tumor size in patients who are infected with them, and it was noted that all three bacteria act anaerobically. Capitalizing on the idea that tumors create an unusually dense, anaerobic mass of cells, Christopher Voigt’s lab engineered bacteria to identify and invade cancer cells. They utilized the ''lux'' quorum sensing from ''Vibrio fischeri'' to identify cells growing at high densities (Fig. 3) and the ''fdhF'' promoter to identify cells growing in anaerobic conditions (Fig. 4) (Anderson, 2006). They coupled this identification mechanism with the invasin output module from ''Yersinia pseudotuberculosis'' so the ''E. coli'' could invade specific cells.

<center>

{|
|-
|[[Image:InvasinDensity.JPG]]
|[[Image:InvasinAnaerobic2.JPG]]
|-
|'''Fig. 3.''' The red line represents when the invasin output module was paired with the inducible promoter ''lux'' which senses high cell density, the black line is constitutively on invasin. (Anderson, 2006 - Permission Pending)
|'''Fig. 4.''' The invasin output module was paired with the inducible promoter ''fdhF'' which senses anaerobic conditions. (Anderson, 2006 - Permission Pending)
|}

</center>

Next, scientists hope to insert a gene that would destroy the invaded cells, making the complete cycle a search, invade, and destroy loop that would only be sensitive to cancer cells. One promising destroy mechanism has been implemented in humans via intratumoral injection, based on ''Salmonella'' that contains the ''E. coli'' cytosine deaminase gene that converts 5-fluorocytosine to 5-fluorouracil, a chemotherapy drug (Nemunaitis, 2003). Unfortunately, while the tumors did produce [http://en.wikipedia.org/wiki/Fluorouracil 5-fluorouracil] (5-FU), they did not produce it in high enough yields to cause regression of the cancer. Another experiment flooded mouse cells with 6-methylpurine-2’deoxyribose (6-MPDR), which was converted to the toxin [http://jpet.aspetjournals.org/cgi/content/abstract/304/3/1280 6-methyl purine] (MeP) by the enzyme purine nucleoside phosphorylase (PNP), which is naturally found in ''E. coli'' (Critchley, 2004) (Fig. 5).

<center>

[[Image:Tumor.JPG]]

'''Fig. 5.''' Tumor size after injections. Cancer cells were injected into mice, and 5 days later injections with PBS, 6-MPDR, and invasin-enhanced ''E-coli'' began. Tumor cells recieving injections of invasin-enhanced ''E.coli'' and 6-MPDR grew the slowest, but did not get smaller. (Critchley, 2004 - Permission Pending)

</center>

The problem with this is that all cells in the body might be exposed to the toxin MeP. In addition to the search and destroy mechanism, scientists hope to increase selectivity by combining the hypoxia-sensing and density-sensing units so that ''E. coli'' will only enter eukaryotic cells that exhibit both characteristics. This would make it more likely that invasin-enhanced ''E. coli'' would only invade cancer cells rather than muscle tissue that had low oxygen due to exercise (Anderson, 2006). To create a fully functional, synthetic biology-based approach to cancer therapy, scientists need to develop bacteria that discriminately invades cancer cells and destroys them. Most of the pieces are there – scientists have engineered ''E. coli'' that selectively invades hypoxic cells or dense cells and ''Salmonella'' that can destroy tumors in vivo – they just need to be put together effectively.

Finally, synthetic biology has made its mark on the field of medicine by creating drugs that are less expensive to produce than those made by pharmaceutical companies. Jay Keasling’s project involving the antimalarial drug artemisinin is the best example. Yeast was engineered to increase normal farnesyl pyrophosphate (FPP) production, convert FPP to amorphadiene, and oxidizing amorphadiene to artemisinic acid, which can then easily be separated from the yeast cells and converted to artemisinin in the laboratory (Fig. 6). This innovation allows the production of artemisinin at lower prices than those currently on the market, and without regard to environmental constraints (Ro, 2006). On a completely different tangent, Collins’s lab engineered T7 bacteriophages to deliver biofilm-degrading enzymes such as dispersin B after infecting and replicating in an ''E. coli'' biofilm. Dispersin B works to degrade an adhesin that is critical for biofilm formation. Phages that released dispersin B were found to be 99.997% effective at removing biofilm, which is 4.5 orders of magnitude higher than non-enzymatic phages (Lu, 2007) (Fig. 7). The specificity of phages for a certain type of bacteria make them a viable vector to be used in human treatments, which may range from removing dental plaques to cleaning inserted medical devices. Using synthetic biology to engineer an antimalarial drug in yeast or a biofilm-destroying enzyme in bacteriophages greatly increases the possible contributions to the field of medicine.

<center>

{|
|-
|[[Image:Artemesinin.JPG]]
|[[Image:BiofilmDegradation.JPG]]
|-
|'''Fig. 6.''' All the steps in the box indicate steps that were optimized in yeast for a maximum artemisinic acid output. (Ro, 2006 - Permission Pending)
|'''Fig. 7.''' The amount of biofilm was decreased when the dispersin B enzymes were incorporated into T7 bacteriophages. (Lu, 2007 - Permission Pending)
|}
</center>

'''Conclusion'''

Medical applications of synthetic biology are wide-ranging and eminently applicable to daily life. Synthetic biology based cancer treatments that utilize ''E.coli'' engineered to sense and attack tissues with high cell density and low oxygen availability are promising and need to be tested in vivo. Designing vectors out of attenuated bacteria may be a safe, effective method for disease treatment. Using biofilms to sense sun damage potential may decrease the likelihood of skin cancer. Finally, using engineered cells to produce medicines in a cost-effective, environmentally-friendly way may revolutionize the pharmaceutical industry. Investing more research efforts in the field of synthetic biology may turn out to be an investment in medical technology.

== Works Cited ==

-[http://www.bio.davidson.edu/courses/synthetic/papers/2006_anderson.pdf Anderson JC, Clarke EJ, Arkin, AP, and Voigt CA (2006). Environmentally controlled invasion of cancer cells by engineered bacteria. Journal of Molecular Biology 355:619-27.]

-[http://www.nature.com/gt/journal/v11/n15/abs/3302281a.html Critchley RJ, Jezzard S, Radford KJ, Goussard S, Lemoine NR, et al. (2004). Potential therapeutic applications of recombinant, invasive ''E. coli''. Gene Therapy 11:1224-33.]

-[http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&uid=15203915&cmd=showdetailview&indexed=google Garmory HS, Leary SEC, Griffin, KF, Williamson D, Brown, KA, and Titball RW (2003). The use of live attenuated bacteria as a delivery system for heterologous antigens. Journal of Drug Targeting 11:471-79.]

-[http://www.pnas.org/cgi/reprint/101/22/8414 Kobayashi H, et al. (2004). Programmable cells: Interfacing natural and engineered gene networks. PNAS 101: 8414-19.]

-[http://www.bio.davidson.edu/courses/synthetic/papers/Phage_BioFilms.pdf Lu, TK, and Collins JJ (2007). Dispersing biofilms with engineered enzymatic bacteriophage. PNAS 104: 11197-11202.]

-[http://www.nature.com/cgt/journal/v10/n10/abs/7700634a.html Nemunaitis J, Cunningham C, Senzer N, Kuhn J, Cramm J, Litz C, et al. (2003). Pilot trial of genetically modified, attenuated ''Salmonella'' expressing cytosine deaminase gene in refractory cancer patients. Cancer Gene Therapy 10:737-744.]

-[http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6W85-49FGKHC-N&_user=10&_coverDate=09%2F30%2F2003&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=d4529b45974bfe7d9ddab1fc865f088b Pawelek JM, Low KB, and Bermudes D (2003). Bacteria as tumour-targeting vectors. Lancet Oncology 4:548-56.]

-[http://www.bio.davidson.edu/courses/synthetic/papers/Keasling_malaria.pdf Ro D, et al. (2006). Production of the antimalarial drug precursor artemisinic acid in engineered yeast. Nature 440:940-43.]

Medical Applications of Synthetic Biology - Samantha Simpson

2007-12-06T03:06:29Z

Sasimpson: /* Paper */

== Project Proposal ==

My project will be on medical applications of synthetic biology. I will reference Anderson's paper on utilizing quorum-sensing and hypoxia-responsive genes coupled with invasin from ''Yersinia tuberculosis'' to invade cancer-causing cells. Coupled with Critchley's work, which describes a bacterial system that invades eukaryotic cells and delivers proteins coded for in the bacteria's genome, one could possibly create 'search and destroy' ''E.coli'' that can locate, invade, and kill tumor cells. Garmory's paper also highlights the possibility of using bacteria as a drug-delivering system, specifically vaccine vectors. Kobayashi describes cells that produce a protective biofilm layer after exposure to DNA-damaging agents. Ro engineers yeast to create an anti-malarial drug that can be created at a lower cost than the previous standard production process. These papers, and others that have a focus on direct medical applications such as novel cancer therapies, vaccination technology, biological protection, and the creation of new medicines, will be referenced to create a cohesive overview of applications of synthetic biology to the medical field.

== Paper ==

Synthetic biology is a rapidly emerging field that strives to re-design existing biological systems and components or fabricate novel biological systems and components. The end result of experimentation in synthetic biology is to help scientists understand a naturally occurring genetic pathway more fully or to create a new, useful pathway. Recent research efforts have focused on engineering bacteria to create a new form of biofuel or to make promoters of varying efficiencies for more exact gene expression. Other research efforts focus on engineering yeast to make antimalarial drugs or engineering ''E. coli'' to recognize and invade tumor-like cells, both of which are medical applications of synthetic biology. Advancements such as novel cancer therapies, vaccination technology, biological protection, and the creation of new medicines highlight synthetic biology’s potential to create breakthroughs in both the prevention and treatment side of medical science. This paper will examine various medical applications of synthetic biology and further development that needs to be done before current research can be used in hospitals and doctors’ offices.

'''Prevention'''

Engineering live bacteria to act as a vaccine vector is an area of interest in synthetic biology. Bacteria was first thought to be a good vector because it would not degrade at mucosal surface and it would survive the low pH in the gastrointestinal tract like most other orally administered antigens (Garmory, 2003). A particular strain of ''Salmonella'' with one or more deletions in the [http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&uid=15012217&cmd=showdetailview&indexed=google shikimate pathway], which creates a precursor for phenylalanine and tyrosine, is a promising vector because it gets into the body easily but does not harmfully effect the tissue and is gone within a week or two of introduction. Scientists engineered this strain to carry the [http://en.wikipedia.org/wiki/Yersinia_pestis ''Yersinia pestis''] V antigen, and studied its ability to protect mice from ''Y. pestis''. The V antigen is generally injected via an intramuscular route, however, in this study, 20 mice were given the V antigen via the intragastric route so scientists could test the effectiveness of an oral administration. Unfortunately, the inoculation was not extremely successful and only 6 of 20 mice infected with ''Y. pestis'' showed a strong response (Garmory, 2003). Garmory and colleagues cited several reasons for this: the copy number of the plasmid containing the V antigen might not have been high enough; the attenuated ''Salmonella'' may not have been able to reach cells that would be targeted by ''Y. pestis''; and the inability of the cell to secrete the antigen. The authors remain hopeful that attenuated bacteria vectors may someday produce an orally administered long-lasting response for protection against bubonic and pneumonic plague (Garmory, 2003). Many changes need to be made to live vector vaccine design before it is an effective method of treating or preventing disease, such as solving the issue of specifity of a vaccine administered orally, and creating a bacterial chassey that can secrete an antigen at an appropriate time.

Another method of disease prevention with a synthetic biology approach is related to skin cancer. ''E. coli'' was engineered to sense single-stranded DNA after DNA damage was coupled with a mechanism to produce a biofilm around the cell. The biofilm output module was tested with DNA damage done by mitomycin C, a DNA crosslinking agent, or UV irradiation, a known cause of skin cancer. Single-stranded DNA activates RecA, which in turn represses the CI repressor protein, thus allowing the transcription of the PL promoter (Kobayashi, 2004). The PL promoter is in charge of the ''traA'' gene, which controls biofilm formation (Fig. 1). The biofilm protection, when part of a toggle switch mechanism, lasted indefinitely when pulsed with UV light at 8 J/m2 for only 2 seconds (Kobayashi, 2004) (Fig. 2). The biofilm-production mechanism has not been engineered in eukaryotic cells, but a prokaryote-based system that could aid with human skin disease is imaginable. For example, a sunscreen that changes colors when the DNA damage may be too intense for a sunbather as a warning, or a sunscreen that becomes more protective as the possibility of genetic damage increased. Further research should focus on how to best implement the biofilm producing output module to protect human DNA.

<center>

{|
|-
|[[Image:Toggleswitch.JPG]]
|[[Image:UVinducedbiofilm.JPG]]
|-
|'''Fig. 1.''' The RecA / traA toggleswitch mechanism. (Kobayashi, 2004 - Permission Pending)
|'''Fig. 2.''' Crystal violet absorbance measures biofilm formation. (Kobayashi, 2004 - Permission Pending)
|}
</center>

'''Treatment'''

Synthetic biologists consider finding a cure for cancer a reachable goal. Previous to the development of synthetic biology as a field, it was known that three types of bacteria, ''Bifodobacterium'', ''Clostridium'', and ''Salmonella'' all preferentially infect the dense cells of tumors (Pawelek, 2003). All three types of bacteria are associated with reducing tumor size in patients who are infected with them, and it was noted that all three bacteria act anaerobically. Capitalizing on the idea that tumors create an unusually dense, anaerobic mass of cells, Christopher Voigt’s lab engineered bacteria to identify and invade cancer cells. They utilized the ''lux'' quorum sensing from ''Vibrio fischeri'' to identify cells growing at high densities (Fig. 3) and the ''fdhF'' promoter to identify cells growing in anaerobic conditions (Fig. 4) (Anderson, 2006). They coupled this identification mechanism with the invasin output module from ''Yersinia pseudotuberculosis'' so the ''E. coli'' could invade specific cells.

<center>

{|
|-
|[[Image:InvasinDensity.JPG]]
|[[Image:InvasinAnaerobic2.JPG]]
|-
|'''Fig. 3.''' The red line represents when the invasin output module was paired with the inducible promoter ''lux'' which senses high cell density, the black line is constitutively on invasin. (Anderson, 2006 - Permission Pending)
|'''Fig. 4.''' The invasin output module was paired with the inducible promoter ''fdhF'' which senses anaerobic conditions. (Anderson, 2006 - Permission Pending)
|}

</center>

Next, scientists hope to insert a gene that would destroy the invaded cells, making the complete cycle a search, invade, and destroy loop that would only be sensitive to cancer cells. One promising destroy mechanism has been implemented in humans via intratumoral injection, based on ''Salmonella'' that contains the ''E. coli'' cytosine deaminase gene that converts 5-fluorocytosine to 5-fluorouracil, a chemotherapy drug (Nemunaitis, 2003). Unfortunately, while the tumors did produce [http://en.wikipedia.org/wiki/Fluorouracil 5-fluorouracil] (5-FU), they did not produce it in high enough yields to cause regression of the cancer. Another experiment flooded mouse cells with 6-methylpurine-2’deoxyribose (6-MPDR), which was converted to the toxin [http://jpet.aspetjournals.org/cgi/content/abstract/304/3/1280 6-methyl purine] (MeP) by the enzyme purine nucleoside phosphorylase (PNP), which is naturally found in ''E. coli'' (Critchley, 2004) (Fig. 5).

<center>

[[Image:Tumor.JPG]]

'''Fig. 5.''' Tumor size after injections. Cancer cells were injected into mice, and 5 days later injections with PBS, 6-MPDR, and invasin-enhanced ''E-coli'' began. Tumor cells recieving injections of invasin-enhanced ''E.coli'' and 6-MPDR grew the slowest, but did not get smaller. (Critchley, 2004 - Permission Pending)

</center>

The problem with this is that all cells in the body might be exposed to the toxin MeP. In addition to the search and destroy mechanism, scientists hope to increase selectivity by combining the hypoxia-sensing and density-sensing units so that ''E. coli'' will only enter eukaryotic cells that exhibit both characteristics. This would make it more likely that invasin-enhanced ''E. coli'' would only invade cancer cells rather than muscle tissue that had low oxygen due to exercise (Anderson, 2006). To create a fully functional, synthetic biology-based approach to cancer therapy, scientists need to develop bacteria that discriminately invades cancer cells and destroys them. Most of the pieces are there – scientists have engineered ''E. coli'' that selectively invades hypoxic cells or dense cells and ''Salmonella'' that can destroy tumors in vivo – they just need to be put together effectively.

Finally, synthetic biology has made its mark on the field of medicine by creating drugs that are less expensive to produce than those made by pharmaceutical companies. Jay Keasling’s project involving the antimalarial drug artemisinin is the best example. Yeast was engineered to increase normal farnesyl pyrophosphate (FPP) production, convert FPP to amorphadiene, and oxidizing amorphadiene to artemisinic acid, which can then easily be separated from the yeast cells and converted to artemisinin in the laboratory (Fig. 6). This innovation allows the production of artemisinin at lower prices than those currently on the market, and without regard to environmental constraints (Ro, 2006). On a completely different tangent, Collins’s lab engineered T7 bacteriophages to deliver biofilm-degrading enzymes such as dispersin B after infecting and replicating in an ''E. coli'' biofilm. Dispersin B works to degrade an adhesin that is critical for biofilm formation. Phages that released dispersin B were found to be 99.997% effective at removing biofilm, which is 4.5 orders of magnitude higher than non-enzymatic phages (Lu, 2007) (Fig. 7). The specificity of phages for a certain type of bacteria make them a viable vector to be used in human treatments, which may range from removing dental plaques to cleaning inserted medical devices. Using synthetic biology to engineer an antimalarial drug in yeast or a biofilm-destroying enzyme in bacteriophages greatly increases the possible contributions to the field of medicine.

<center>

{|
|-
|[[Image:Artemesinin.JPG]]
|[[Image:BiofilmDegradation.JPG]]
|-
|'''Fig. 6.''' All the steps in the box indicate steps that were optimized in yeast for a maximum artemisinic acid output. (Ro, 2006 - Permission Pending)
|'''Fig. 7.''' The amount of biofilm was decreased when the dispersin B enzymes were incorporated into T7 bacteriophages. (Lu, 2007 - Permission Pending)
|}
</center>

'''Conclusion'''

Medical applications of synthetic biology are wide-ranging and eminently applicable to daily life. Synthetic biology based cancer treatments that utilize ''E.coli'' engineered to sense and attack tissues with high cell density and low oxygen availability are promising and need to be tested in vivo. Designing vectors out of attenuated bacteria may be a safe, effective method for disease treatment. Using biofilms to sense sun damage potential may decrease the likelihood of skin cancer. Finally, using engineered cells to produce medicines in a cost-effective, environmentally-friendly way may revolutionize the pharmaceutical industry. Investing more research efforts in the field of synthetic biology may turn out to be an investment in medical technology.

== Works Cited ==

-[http://www.bio.davidson.edu/courses/synthetic/papers/2006_anderson.pdf Anderson JC, Clarke EJ, Arkin, AP, and Voigt CA (2006). Environmentally controlled invasion of cancer cells by engineered bacteria. Journal of Molecular Biology 355:619-27.]

-[http://www.nature.com/gt/journal/v11/n15/abs/3302281a.html Critchley RJ, Jezzard S, Radford KJ, Goussard S, Lemoine NR, et al. (2004). Potential therapeutic applications of recombinant, invasive ''E. coli''. Gene Therapy 11:1224-33.]

-[http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&uid=15203915&cmd=showdetailview&indexed=google Garmory HS, Leary SEC, Griffin, KF, Williamson D, Brown, KA, and Titball RW (2003). The use of live attenuated bacteria as a delivery system for heterologous antigens. Journal of Drug Targeting 11:471-79.]

-[http://www.pnas.org/cgi/reprint/101/22/8414 Kobayashi H, et al. (2004). Programmable cells: Interfacing natural and engineered gene networks. PNAS 101: 8414-19.]

-[http://www.bio.davidson.edu/courses/synthetic/papers/Phage_BioFilms.pdf Lu, TK, and Collins JJ (2007). Dispersing biofilms with engineered enzymatic bacteriophage. PNAS 104: 11197-11202.]

-[http://www.nature.com/cgt/journal/v10/n10/abs/7700634a.html Nemunaitis J, Cunningham C, Senzer N, Kuhn J, Cramm J, Litz C, et al. (2003). Pilot trial of genetically modified, attenuated ''Salmonella'' expressing cytosine deaminase gene in refractory cancer patients. Cancer Gene Therapy 10:737-744.]

-[http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6W85-49FGKHC-N&_user=10&_coverDate=09%2F30%2F2003&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=d4529b45974bfe7d9ddab1fc865f088b Pawelek JM, Low KB, and Bermudes D (2003). Bacteria as tumour-targeting vectors. Lancet Oncology 4:548-56.]

-[http://www.bio.davidson.edu/courses/synthetic/papers/Keasling_malaria.pdf Ro D, et al. (2006). Production of the antimalarial drug precursor artemisinic acid in engineered yeast. Nature 440:940-43.]

Medical Applications of Synthetic Biology - Samantha Simpson

2007-12-06T03:05:37Z

Sasimpson: /* Paper */

== Project Proposal ==

My project will be on medical applications of synthetic biology. I will reference Anderson's paper on utilizing quorum-sensing and hypoxia-responsive genes coupled with invasin from ''Yersinia tuberculosis'' to invade cancer-causing cells. Coupled with Critchley's work, which describes a bacterial system that invades eukaryotic cells and delivers proteins coded for in the bacteria's genome, one could possibly create 'search and destroy' ''E.coli'' that can locate, invade, and kill tumor cells. Garmory's paper also highlights the possibility of using bacteria as a drug-delivering system, specifically vaccine vectors. Kobayashi describes cells that produce a protective biofilm layer after exposure to DNA-damaging agents. Ro engineers yeast to create an anti-malarial drug that can be created at a lower cost than the previous standard production process. These papers, and others that have a focus on direct medical applications such as novel cancer therapies, vaccination technology, biological protection, and the creation of new medicines, will be referenced to create a cohesive overview of applications of synthetic biology to the medical field.

== Paper ==

Synthetic biology is a rapidly emerging field that strives to re-design existing biological systems and components or fabricate novel biological systems and components. The end result of experimentation in synthetic biology is to help scientists understand a naturally occurring genetic pathway more fully or to create a new, useful pathway. Recent research efforts have focused on engineering bacteria to create a new form of biofuel or to make promoters of varying efficiencies for more exact gene expression. Other research efforts focus on engineering yeast to make antimalarial drugs or engineering ''E. coli'' to recognize and invade tumor-like cells, both of which are medical applications of synthetic biology. Advancements such as novel cancer therapies, vaccination technology, biological protection, and the creation of new medicines highlight synthetic biology’s potential to create breakthroughs in both the prevention and treatment side of medical science. This paper will examine various medical applications of synthetic biology and further development that needs to be done before current research can be used in hospitals and doctors’ offices.

'''Prevention'''

Engineering live bacteria to act as a vaccine vector is an area of interest in synthetic biology. Bacteria was first thought to be a good vector because it would not degrade at mucosal surface and it would survive the low pH in the gastrointestinal tract like most other orally administered antigens (Garmory, 2003). A particular strain of ''Salmonella'' with one or more deletions in the [http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&uid=15012217&cmd=showdetailview&indexed=google shikimate pathway], which creates a precursor for phenylalanine and tyrosine, is a promising vector because it gets into the body easily but does not harmfully effect the tissue and is gone within a week or two of introduction. Scientists engineered this strain to carry the [http://en.wikipedia.org/wiki/Yersinia_pestis ''Yersinia pestis''] V antigen, and studied its ability to protect mice from ''Y. pestis''. The V antigen is generally injected via an intramuscular route, however, in this study, 20 mice were given the V antigen via the intragastric route so scientists could test the effectiveness of an oral administration. Unfortunately, the inoculation was not extremely successful and only 6 of 20 mice infected with ''Y. pestis'' showed a strong response (Garmory, 2003). Garmory and colleagues cited several reasons for this: the copy number of the plasmid containing the V antigen might not have been high enough; the attenuated ''Salmonella'' may not have been able to reach cells that would be targeted by ''Y. pestis''; and the inability of the cell to secrete the antigen. The authors remain hopeful that attenuated bacteria vectors may someday produce an orally administered long-lasting response for protection against bubonic and pneumonic plague (Garmory, 2003). Many changes need to be made to live vector vaccine design before it is an effective method of treating or preventing disease, such as solving the issue of specifity of a vaccine administered orally, and creating a bacterial chassey that can secrete an antigen at an appropriate time.

Another method of disease prevention with a synthetic biology approach is related to skin cancer. ''E. coli'' engineered to sense single-stranded DNA after DNA damage was coupled with a mechanism to produce a biofilm around the cell. The biofilm output module was tested with DNA damage done by mitomycin C, a DNA crosslinking agent, or UV irradiation, a known cause of skin cancer. Single-stranded DNA activates RecA, which in turn represses the CI repressor protein, thus allowing the transcription of the PL promoter (Kobayashi, 2004). The PL promoter is in charge of the ''traA'' gene, which controls biofilm formation (Fig. 1). The biofilm protection, when part of a toggle switch mechanism, lasted indefinitely when pulsed with UV light at 8 J/m2 for only 2 seconds (Kobayashi, 2004) (Fig. 2). The biofilm-production mechanism has not been engineered in eukaryotic cells, but a prokaryote-based system that could aid with human skin disease is imaginable. For example, a sunscreen that changes colors when the DNA damage may be too intense for a sunbather as a warning, or a sunscreen that becomes more protective as the possibility of genetic damage increased. Further research should focus on how to best implement the biofilm producing output module to protect human DNA.

<center>

{|
|-
|[[Image:Toggleswitch.JPG]]
|[[Image:UVinducedbiofilm.JPG]]
|-
|'''Fig. 1.''' The RecA / traA toggleswitch mechanism. (Kobayashi, 2004 - Permission Pending)
|'''Fig. 2.''' Crystal violet absorbance measures biofilm formation. (Kobayashi, 2004 - Permission Pending)
|}
</center>

'''Treatment'''

Synthetic biologists consider finding a cure for cancer a reachable goal. Previous to the development of synthetic biology as a field, it was known that three types of bacteria, ''Bifodobacterium'', ''Clostridium'', and ''Salmonella'' all preferentially infect the dense cells of tumors (Pawelek, 2003). All three types of bacteria are associated with reducing tumor size in patients who are infected with them, and it was noted that all three bacteria act anaerobically. Capitalizing on the idea that tumors create an unusually dense, anaerobic mass of cells, Christopher Voigt’s lab engineered bacteria to identify and invade cancer cells. They utilized the ''lux'' quorum sensing from ''Vibrio fischeri'' to identify cells growing at high densities (Fig. 3) and the ''fdhF'' promoter to identify cells growing in anaerobic conditions (Fig. 4) (Anderson, 2006). They coupled this identification mechanism with the invasin output module from ''Yersinia pseudotuberculosis'' so the ''E. coli'' could invade specific cells.

<center>

{|
|-
|[[Image:InvasinDensity.JPG]]
|[[Image:InvasinAnaerobic2.JPG]]
|-
|'''Fig. 3.''' The red line represents when the invasin output module was paired with the inducible promoter ''lux'' which senses high cell density, the black line is constitutively on invasin. (Anderson, 2006 - Permission Pending)
|'''Fig. 4.''' The invasin output module was paired with the inducible promoter ''fdhF'' which senses anaerobic conditions. (Anderson, 2006 - Permission Pending)
|}

</center>

Next, scientists hope to insert a gene that would destroy the invaded cells, making the complete cycle a search, invade, and destroy loop that would only be sensitive to cancer cells. One promising destroy mechanism has been implemented in humans via intratumoral injection, based on ''Salmonella'' that contains the ''E. coli'' cytosine deaminase gene that converts 5-fluorocytosine to 5-fluorouracil, a chemotherapy drug (Nemunaitis, 2003). Unfortunately, while the tumors did produce [http://en.wikipedia.org/wiki/Fluorouracil 5-fluorouracil] (5-FU), they did not produce it in high enough yields to cause regression of the cancer. Another experiment flooded mouse cells with 6-methylpurine-2’deoxyribose (6-MPDR), which was converted to the toxin [http://jpet.aspetjournals.org/cgi/content/abstract/304/3/1280 6-methyl purine] (MeP) by the enzyme purine nucleoside phosphorylase (PNP), which is naturally found in ''E. coli'' (Critchley, 2004) (Fig. 5).

<center>

[[Image:Tumor.JPG]]

'''Fig. 5.''' Tumor size after injections. Cancer cells were injected into mice, and 5 days later injections with PBS, 6-MPDR, and invasin-enhanced ''E-coli'' began. Tumor cells recieving injections of invasin-enhanced ''E.coli'' and 6-MPDR grew the slowest, but did not get smaller. (Critchley, 2004 - Permission Pending)

</center>

The problem with this is that all cells in the body might be exposed to the toxin MeP. In addition to the search and destroy mechanism, scientists hope to increase selectivity by combining the hypoxia-sensing and density-sensing units so that ''E. coli'' will only enter eukaryotic cells that exhibit both characteristics. This would make it more likely that invasin-enhanced ''E. coli'' would only invade cancer cells rather than muscle tissue that had low oxygen due to exercise (Anderson, 2006). To create a fully functional, synthetic biology-based approach to cancer therapy, scientists need to develop bacteria that discriminately invades cancer cells and destroys them. Most of the pieces are there – scientists have engineered ''E. coli'' that selectively invades hypoxic cells or dense cells and ''Salmonella'' that can destroy tumors in vivo – they just need to be put together effectively.

Finally, synthetic biology has made its mark on the field of medicine by creating drugs that are less expensive to produce than those made by pharmaceutical companies. Jay Keasling’s project involving the antimalarial drug artemisinin is the best example. Yeast was engineered to increase normal farnesyl pyrophosphate (FPP) production, convert FPP to amorphadiene, and oxidizing amorphadiene to artemisinic acid, which can then easily be separated from the yeast cells and converted to artemisinin in the laboratory (Fig. 6). This innovation allows the production of artemisinin at lower prices than those currently on the market, and without regard to environmental constraints (Ro, 2006). On a completely different tangent, Collins’s lab engineered T7 bacteriophages to deliver biofilm-degrading enzymes such as dispersin B after infecting and replicating in an ''E. coli'' biofilm. Dispersin B works to degrade an adhesin that is critical for biofilm formation. Phages that released dispersin B were found to be 99.997% effective at removing biofilm, which is 4.5 orders of magnitude higher than non-enzymatic phages (Lu, 2007) (Fig. 7). The specificity of phages for a certain type of bacteria make them a viable vector to be used in human treatments, which may range from removing dental plaques to cleaning inserted medical devices. Using synthetic biology to engineer an antimalarial drug in yeast or a biofilm-destroying enzyme in bacteriophages greatly increases the possible contributions to the field of medicine.

<center>

{|
|-
|[[Image:Artemesinin.JPG]]
|[[Image:BiofilmDegradation.JPG]]
|-
|'''Fig. 6.''' All the steps in the box indicate steps that were optimized in yeast for a maximum artemisinic acid output. (Ro, 2006 - Permission Pending)
|'''Fig. 7.''' The amount of biofilm was decreased when the dispersin B enzymes were incorporated into T7 bacteriophages. (Lu, 2007 - Permission Pending)
|}
</center>

'''Conclusion'''

Medical applications of synthetic biology are wide-ranging and eminently applicable to daily life. Synthetic biology based cancer treatments that utilize ''E.coli'' engineered to sense and attack tissues with high cell density and low oxygen availability are promising and need to be tested in vivo. Designing vectors out of attenuated bacteria may be a safe, effective method for disease treatment. Using biofilms to sense sun damage potential may decrease the likelihood of skin cancer. Finally, using engineered cells to produce medicines in a cost-effective, environmentally-friendly way may revolutionize the pharmaceutical industry. Investing more research efforts in the field of synthetic biology may turn out to be an investment in medical technology.

== Works Cited ==

-[http://www.bio.davidson.edu/courses/synthetic/papers/2006_anderson.pdf Anderson JC, Clarke EJ, Arkin, AP, and Voigt CA (2006). Environmentally controlled invasion of cancer cells by engineered bacteria. Journal of Molecular Biology 355:619-27.]

-[http://www.nature.com/gt/journal/v11/n15/abs/3302281a.html Critchley RJ, Jezzard S, Radford KJ, Goussard S, Lemoine NR, et al. (2004). Potential therapeutic applications of recombinant, invasive ''E. coli''. Gene Therapy 11:1224-33.]

-[http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&uid=15203915&cmd=showdetailview&indexed=google Garmory HS, Leary SEC, Griffin, KF, Williamson D, Brown, KA, and Titball RW (2003). The use of live attenuated bacteria as a delivery system for heterologous antigens. Journal of Drug Targeting 11:471-79.]

-[http://www.pnas.org/cgi/reprint/101/22/8414 Kobayashi H, et al. (2004). Programmable cells: Interfacing natural and engineered gene networks. PNAS 101: 8414-19.]

-[http://www.bio.davidson.edu/courses/synthetic/papers/Phage_BioFilms.pdf Lu, TK, and Collins JJ (2007). Dispersing biofilms with engineered enzymatic bacteriophage. PNAS 104: 11197-11202.]

-[http://www.nature.com/cgt/journal/v10/n10/abs/7700634a.html Nemunaitis J, Cunningham C, Senzer N, Kuhn J, Cramm J, Litz C, et al. (2003). Pilot trial of genetically modified, attenuated ''Salmonella'' expressing cytosine deaminase gene in refractory cancer patients. Cancer Gene Therapy 10:737-744.]

-[http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6W85-49FGKHC-N&_user=10&_coverDate=09%2F30%2F2003&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=d4529b45974bfe7d9ddab1fc865f088b Pawelek JM, Low KB, and Bermudes D (2003). Bacteria as tumour-targeting vectors. Lancet Oncology 4:548-56.]

-[http://www.bio.davidson.edu/courses/synthetic/papers/Keasling_malaria.pdf Ro D, et al. (2006). Production of the antimalarial drug precursor artemisinic acid in engineered yeast. Nature 440:940-43.]

Medical Applications of Synthetic Biology - Samantha Simpson

2007-12-06T02:59:03Z

Sasimpson: /* Paper */

== Project Proposal ==

My project will be on medical applications of synthetic biology. I will reference Anderson's paper on utilizing quorum-sensing and hypoxia-responsive genes coupled with invasin from ''Yersinia tuberculosis'' to invade cancer-causing cells. Coupled with Critchley's work, which describes a bacterial system that invades eukaryotic cells and delivers proteins coded for in the bacteria's genome, one could possibly create 'search and destroy' ''E.coli'' that can locate, invade, and kill tumor cells. Garmory's paper also highlights the possibility of using bacteria as a drug-delivering system, specifically vaccine vectors. Kobayashi describes cells that produce a protective biofilm layer after exposure to DNA-damaging agents. Ro engineers yeast to create an anti-malarial drug that can be created at a lower cost than the previous standard production process. These papers, and others that have a focus on direct medical applications such as novel cancer therapies, vaccination technology, biological protection, and the creation of new medicines, will be referenced to create a cohesive overview of applications of synthetic biology to the medical field.

== Paper ==

Synthetic biology is a rapidly emerging field that strives to re-design existing biological systems and components or fabricate novel biological systems and components. The end result of experimentation in synthetic biology is to help scientists understand a naturally occurring genetic pathway more fully or to create a new, useful pathway. Recent research efforts have focused on engineering bacteria to create a new form of biofuel or to make promoters of varying efficiencies for more exact gene expression. Other research efforts focus on engineering yeast to make antimalarial drugs or engineering ''E. coli'' to recognize and invade tumor-like cells, both of which are medical applications of synthetic biology. Advancements such as novel cancer therapies, vaccination technology, biological protection, and the creation of new medicines highlight synthetic biology’s potential to create breakthroughs in both the prevention and treatment side of medical science. This paper will examine various medical applications of synthetic biology and further development that needs to be done before current research can be used in hospitals and doctors’ offices.

'''Prevention'''

Engineering live bacteria to act as a vaccine vector is an area of interest in synthetic biology. Bacteria was first thought to be a good vector because it would not degrade at mucosal surface and it would survive the low pH in the gastrointestinal tract like most other orally administered antigens (Garmory, 2003). A particular strain of ''Salmonella'' with one or more deletions in the [http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&uid=15012217&cmd=showdetailview&indexed=google shikimate pathway], which creates a precursor for phenylalanine and tyrosine, is a promising vector because it gets into the body easily but does not harmfully effect the tissue and is gone within a week or two of introduction. Scientists engineered this strain to carry the [http://en.wikipedia.org/wiki/Yersinia_pestis ''Yersinia pestis''] V antigen, and studied its ability to protect mice from ''Y. pestis''. The V antigen is generally injected via an intramuscular route, however, in this study, 20 mice were given the V antigen via the intragastric route so scientists could test the effectiveness of an oral administration. Unfortunately, the inoculation was not extremely successful and only 6 of 20 mice infected with ''Y. pestis'' showed a strong response (Garmory, 2003). Garmory and colleagues cited several reasons for this: the copy number of the plasmid containing the V antigen might not have been high enough; the attenuated ''Salmonella'' may not have been able to reach cells that would be targeted by ''Y. pestis''; and the inability of the cell to secrete the antigen. Many changes need to be made to live vector vaccine design before it is an effective method of treating or preventing disease. Using bacteria as a vaccine vector has similar issues of specificity and implementation in a human system.

Another method of disease prevention with a synthetic biology approach is related to skin cancer. ''E. coli'' engineered to sense single-stranded DNA after DNA damage was coupled with a mechanism to produce a biofilm around the cell. The biofilm output module was tested with DNA damage done by mitomycin C, a DNA crosslinking agent, or UV irradiation, a known cause of skin cancer. Single-stranded DNA activates RecA, which in turn represses the CI repressor protein, thus allowing the transcription of the PL promoter (Kobayashi, 2004). The PL promoter is in charge of the ''traA'' gene, which controls biofilm formation (Fig. 1). The biofilm protection, when part of a toggle switch mechanism, lasted indefinitely when pulsed with UV light at 8 J/m2 for only 2 seconds (Kobayashi, 2004) (Fig. 2). The biofilm-production mechanism has not been engineered in eukaryotic cells, but a prokaryote-based system that could aid with human skin disease is imaginable. For example, a sunscreen that changes colors when the DNA damage may be too intense for a sunbather as a warning, or a sunscreen that becomes more protective as the possibility of genetic damage increased. Further research should focus on how to best implement the biofilm producing output module to protect human DNA.

<center>

{|
|-
|[[Image:Toggleswitch.JPG]]
|[[Image:UVinducedbiofilm.JPG]]
|-
|'''Fig. 1.''' The RecA / traA toggleswitch mechanism. (Kobayashi, 2004 - Permission Pending)
|'''Fig. 2.''' Crystal violet absorbance measures biofilm formation. (Kobayashi, 2004 - Permission Pending)
|}
</center>

'''Treatment'''

Synthetic biologists consider finding a cure for cancer a reachable goal. Previous to the development of synthetic biology as a field, it was known that three types of bacteria, ''Bifodobacterium'', ''Clostridium'', and ''Salmonella'' all preferentially infect the dense cells of tumors (Pawelek, 2003). All three types of bacteria are associated with reducing tumor size in patients who are infected with them, and it was noted that all three bacteria act anaerobically. Capitalizing on the idea that tumors create an unusually dense, anaerobic mass of cells, Christopher Voigt’s lab engineered bacteria to identify and invade cancer cells. They utilized the ''lux'' quorum sensing from ''Vibrio fischeri'' to identify cells growing at high densities (Fig. 3) and the ''fdhF'' promoter to identify cells growing in anaerobic conditions (Fig. 4) (Anderson, 2006). They coupled this identification mechanism with the invasin output module from ''Yersinia pseudotuberculosis'' so the ''E. coli'' could invade specific cells.

<center>

{|
|-
|[[Image:InvasinDensity.JPG]]
|[[Image:InvasinAnaerobic2.JPG]]
|-
|'''Fig. 3.''' The red line represents when the invasin output module was paired with the inducible promoter ''lux'' which senses high cell density, the black line is constitutively on invasin. (Anderson, 2006 - Permission Pending)
|'''Fig. 4.''' The invasin output module was paired with the inducible promoter ''fdhF'' which senses anaerobic conditions. (Anderson, 2006 - Permission Pending)
|}

</center>

Next, scientists hope to insert a gene that would destroy the invaded cells, making the complete cycle a search, invade, and destroy loop that would only be sensitive to cancer cells. One promising destroy mechanism has been implemented in humans via intratumoral injection, based on ''Salmonella'' that contains the ''E. coli'' cytosine deaminase gene that converts 5-fluorocytosine to 5-fluorouracil, a chemotherapy drug (Nemunaitis, 2003). Unfortunately, while the tumors did produce [http://en.wikipedia.org/wiki/Fluorouracil 5-fluorouracil] (5-FU), they did not produce it in high enough yields to cause regression of the cancer. Another experiment flooded mouse cells with 6-methylpurine-2’deoxyribose (6-MPDR), which was converted to the toxin [http://jpet.aspetjournals.org/cgi/content/abstract/304/3/1280 6-methyl purine] (MeP) by the enzyme purine nucleoside phosphorylase (PNP), which is naturally found in ''E. coli'' (Critchley, 2004) (Fig. 5).

<center>

[[Image:Tumor.JPG]]

'''Fig. 5.''' Tumor size after injections. Cancer cells were injected into mice, and 5 days later injections with PBS, 6-MPDR, and invasin-enhanced ''E-coli'' began. Tumor cells recieving injections of invasin-enhanced ''E.coli'' and 6-MPDR grew the slowest, but did not get smaller. (Critchley, 2004 - Permission Pending)

</center>

The problem with this is that all cells in the body might be exposed to the toxin MeP. In addition to the search and destroy mechanism, scientists hope to increase selectivity by combining the hypoxia-sensing and density-sensing units so that ''E. coli'' will only enter eukaryotic cells that exhibit both characteristics. This would make it more likely that invasin-enhanced ''E. coli'' would only invade cancer cells rather than muscle tissue that had low oxygen due to exercise (Anderson, 2006). To create a fully functional, synthetic biology-based approach to cancer therapy, scientists need to develop bacteria that discriminately invades cancer cells and destroys them. Most of the pieces are there – scientists have engineered ''E. coli'' that selectively invades hypoxic cells or dense cells and ''Salmonella'' that can destroy tumors in vivo – they just need to be put together effectively.

Finally, synthetic biology has made its mark on the field of medicine by creating drugs that are less expensive to produce than those made by pharmaceutical companies. Jay Keasling’s project involving the antimalarial drug artemisinin is the best example. Yeast was engineered to increase normal farnesyl pyrophosphate (FPP) production, convert FPP to amorphadiene, and oxidizing amorphadiene to artemisinic acid, which can then easily be separated from the yeast cells and converted to artemisinin in the laboratory (Fig. 6). This innovation allows the production of artemisinin at lower prices than those currently on the market, and without regard to environmental constraints (Ro, 2006). On a completely different tangent, Collins’s lab engineered T7 bacteriophages to deliver biofilm-degrading enzymes such as dispersin B after infecting and replicating in an ''E. coli'' biofilm. Dispersin B works to degrade an adhesin that is critical for biofilm formation. Phages that released dispersin B were found to be 99.997% effective at removing biofilm, which is 4.5 orders of magnitude higher than non-enzymatic phages (Lu, 2007) (Fig. 7). The specificity of phages for a certain type of bacteria make them a viable vector to be used in human treatments, which may range from removing dental plaques to cleaning inserted medical devices. Using synthetic biology to engineer an antimalarial drug in yeast or a biofilm-destroying enzyme in bacteriophages greatly increases the possible contributions to the field of medicine.

<center>

{|
|-
|[[Image:Artemesinin.JPG]]
|[[Image:BiofilmDegradation.JPG]]
|-
|'''Fig. 6.''' All the steps in the box indicate steps that were optimized in yeast for a maximum artemisinic acid output. (Ro, 2006 - Permission Pending)
|'''Fig. 7.''' The amount of biofilm was decreased when the dispersin B enzymes were incorporated into T7 bacteriophages. (Lu, 2007 - Permission Pending)
|}
</center>

'''Conclusion'''

Medical applications of synthetic biology are wide-ranging and eminently applicable to daily life. Synthetic biology based cancer treatments that utilize ''E.coli'' engineered to sense and attack tissues with high cell density and low oxygen availability are promising and need to be tested in vivo. Designing vectors out of attenuated bacteria may be a safe, effective method for disease treatment. Using biofilms to sense sun damage potential may decrease the likelihood of skin cancer. Finally, using engineered cells to produce medicines in a cost-effective, environmentally-friendly way may revolutionize the pharmaceutical industry. Investing more research efforts in the field of synthetic biology may turn out to be an investment in medical technology.

== Works Cited ==

-[http://www.bio.davidson.edu/courses/synthetic/papers/2006_anderson.pdf Anderson JC, Clarke EJ, Arkin, AP, and Voigt CA (2006). Environmentally controlled invasion of cancer cells by engineered bacteria. Journal of Molecular Biology 355:619-27.]

-[http://www.nature.com/gt/journal/v11/n15/abs/3302281a.html Critchley RJ, Jezzard S, Radford KJ, Goussard S, Lemoine NR, et al. (2004). Potential therapeutic applications of recombinant, invasive ''E. coli''. Gene Therapy 11:1224-33.]

-[http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&uid=15203915&cmd=showdetailview&indexed=google Garmory HS, Leary SEC, Griffin, KF, Williamson D, Brown, KA, and Titball RW (2003). The use of live attenuated bacteria as a delivery system for heterologous antigens. Journal of Drug Targeting 11:471-79.]

-[http://www.pnas.org/cgi/reprint/101/22/8414 Kobayashi H, et al. (2004). Programmable cells: Interfacing natural and engineered gene networks. PNAS 101: 8414-19.]

-[http://www.bio.davidson.edu/courses/synthetic/papers/Phage_BioFilms.pdf Lu, TK, and Collins JJ (2007). Dispersing biofilms with engineered enzymatic bacteriophage. PNAS 104: 11197-11202.]

-[http://www.nature.com/cgt/journal/v10/n10/abs/7700634a.html Nemunaitis J, Cunningham C, Senzer N, Kuhn J, Cramm J, Litz C, et al. (2003). Pilot trial of genetically modified, attenuated ''Salmonella'' expressing cytosine deaminase gene in refractory cancer patients. Cancer Gene Therapy 10:737-744.]

-[http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6W85-49FGKHC-N&_user=10&_coverDate=09%2F30%2F2003&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=d4529b45974bfe7d9ddab1fc865f088b Pawelek JM, Low KB, and Bermudes D (2003). Bacteria as tumour-targeting vectors. Lancet Oncology 4:548-56.]

-[http://www.bio.davidson.edu/courses/synthetic/papers/Keasling_malaria.pdf Ro D, et al. (2006). Production of the antimalarial drug precursor artemisinic acid in engineered yeast. Nature 440:940-43.]

Medical Applications of Synthetic Biology - Samantha Simpson

2007-12-06T02:47:58Z

Sasimpson: /* Paper */

== Project Proposal ==

My project will be on medical applications of synthetic biology. I will reference Anderson's paper on utilizing quorum-sensing and hypoxia-responsive genes coupled with invasin from ''Yersinia tuberculosis'' to invade cancer-causing cells. Coupled with Critchley's work, which describes a bacterial system that invades eukaryotic cells and delivers proteins coded for in the bacteria's genome, one could possibly create 'search and destroy' ''E.coli'' that can locate, invade, and kill tumor cells. Garmory's paper also highlights the possibility of using bacteria as a drug-delivering system, specifically vaccine vectors. Kobayashi describes cells that produce a protective biofilm layer after exposure to DNA-damaging agents. Ro engineers yeast to create an anti-malarial drug that can be created at a lower cost than the previous standard production process. These papers, and others that have a focus on direct medical applications such as novel cancer therapies, vaccination technology, biological protection, and the creation of new medicines, will be referenced to create a cohesive overview of applications of synthetic biology to the medical field.

== Paper ==

Synthetic biology is a rapidly emerging field that strives to re-design existing biological systems and components or fabricate novel biological systems and components. The end result of experimentation in synthetic biology is to help scientists understand a naturally occurring genetic pathway more fully or to create a new, useful pathway. Recent research efforts have focused on engineering bacteria to create a new form of biofuel or to make promoters of varying efficiencies for more exact gene expression. Other research efforts focus on engineering yeast to make antimalarial drugs or engineering ''E. coli'' to recognize and invade tumor-like cells, both of which are medical applications of synthetic biology. Advancements such as novel cancer therapies, vaccination technology, biological protection, and the creation of new medicines highlight synthetic biology’s potential to create breakthroughs in both the prevention and treatment side of medical science. This paper will examine various medical applications of synthetic biology and further development that needs to be done before current research can be used in hospitals and doctors’ offices.

'''Prevention'''

Engineering live bacteria to act as a vaccine vector is an area of interest in synthetic biology. Bacteria was first thought to be a good vector because it would not degrade at mucosal surface and it would survive the low pH in the gastrointestinal tract like most other orally administered antigens (Garmory, 2003). A particular strain of ''Salmonella'' with one or more deletions in the [http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&uid=15012217&cmd=showdetailview&indexed=google shikimate pathway], which creates a precursor for phenylalanine and tyrosine, is a promising vector because it gets into the body easily but does not harmfully effect the tissue and is gone within a week or two of introduction. Scientists engineered this strain to carry the [[http://en.wikipedia.org/wiki/Yersinia_pestis ''Yersinia pestis'']] V antigen, and studied its ability to protect mice from ''Y. pestis''. Unfortunately, the inoculation was not extremely successful and only 6 of 20 mice showed a strong response (Garmory, 2003). Garmory and colleagues cited several reasons for this: the copy number of the plasmid containing the V antigen might not have been high enough; the attenuated ''Salmonella'' may not have been able to reach cells that would be targeted by ''Y. pestis''; and the inability of the cell to secrete the antigen. Many changes need to be made to live vector vaccine design before it is an effective method of treating or preventing disease. Using bacteria as a vaccine vector has similar issues of specificity and implementation in a human system.

Another method of disease prevention with a synthetic biology approach is related to skin cancer. ''E. coli'' engineered to sense single-stranded DNA after DNA damage was coupled with a mechanism to produce a biofilm around the cell. The biofilm output module was tested with DNA damage done by mitomycin C, a DNA crosslinking agent, or UV irradiation, a known cause of skin cancer. Single-stranded DNA activates RecA, which in turn represses the CI repressor protein, thus allowing the transcription of the PL promoter (Kobayashi, 2004). The PL promoter is in charge of the ''traA'' gene, which controls biofilm formation (Fig. 1). The biofilm protection, when part of a toggle switch mechanism, lasted indefinitely when pulsed with UV light at 8 J/m2 for only 2 seconds (Kobayashi, 2004) (Fig. 2). The biofilm-production mechanism has not been engineered in eukaryotic cells, but a prokaryote-based system that could aid with human skin disease is imaginable. For example, a sunscreen that changes colors when the DNA damage may be too intense for a sunbather as a warning, or a sunscreen that becomes more protective as the possibility of genetic damage increased. Further research should focus on how to best implement the biofilm producing output module to protect human DNA.

<center>

{|
|-
|[[Image:Toggleswitch.JPG]]
|[[Image:UVinducedbiofilm.JPG]]
|-
|'''Fig. 1.''' The RecA / traA toggleswitch mechanism. (Kobayashi, 2004 - Permission Pending)
|'''Fig. 2.''' Crystal violet absorbance measures biofilm formation. (Kobayashi, 2004 - Permission Pending)
|}
</center>

'''Treatment'''

Synthetic biologists consider finding a cure for cancer a reachable goal. Previous to the development of synthetic biology as a field, it was known that three types of bacteria, ''Bifodobacterium'', ''Clostridium'', and ''Salmonella'' all preferentially infect the dense cells of tumors (Pawelek, 2003). All three types of bacteria are associated with reducing tumor size in patients who are infected with them, and it was noted that all three bacteria act anaerobically. Capitalizing on the idea that tumors create an unusually dense, anaerobic mass of cells, Christopher Voigt’s lab engineered bacteria to identify and invade cancer cells. They utilized the ''lux'' quorum sensing from ''Vibrio fischeri'' to identify cells growing at high densities (Fig. 3) and the ''fdhF'' promoter to identify cells growing in anaerobic conditions (Fig. 4) (Anderson, 2006). They coupled this identification mechanism with the invasin output module from ''Yersinia pseudotuberculosis'' so the ''E. coli'' could invade specific cells.

<center>

{|
|-
|[[Image:InvasinDensity.JPG]]
|[[Image:InvasinAnaerobic2.JPG]]
|-
|'''Fig. 3.''' The red line represents when the invasin output module was paired with the inducible promoter ''lux'' which senses high cell density, the black line is constitutively on invasin. (Anderson, 2006 - Permission Pending)
|'''Fig. 4.''' The invasin output module was paired with the inducible promoter ''fdhF'' which senses anaerobic conditions. (Anderson, 2006 - Permission Pending)
|}

</center>

Next, scientists hope to insert a gene that would destroy the invaded cells, making the complete cycle a search, invade, and destroy loop that would only be sensitive to cancer cells. One promising destroy mechanism has been implemented in humans via intratumoral injection, based on ''Salmonella'' that contains the ''E. coli'' cytosine deaminase gene that converts 5-fluorocytosine to 5-fluorouracil, a chemotherapy drug (Nemunaitis, 2003). Unfortunately, while the tumors did produce [http://en.wikipedia.org/wiki/Fluorouracil 5-fluorouracil] (5-FU), they did not produce it in high enough yields to cause regression of the cancer. Another experiment flooded mouse cells with 6-methylpurine-2’deoxyribose (6-MPDR), which was converted to the toxin [http://jpet.aspetjournals.org/cgi/content/abstract/304/3/1280 6-methyl purine] (MeP) by the enzyme purine nucleoside phosphorylase (PNP), which is naturally found in ''E. coli'' (Critchley, 2004) (Fig. 5).

<center>

[[Image:Tumor.JPG]]

'''Fig. 5.''' Tumor size after injections. Cancer cells were injected into mice, and 5 days later injections with PBS, 6-MPDR, and invasin-enhanced ''E-coli'' began. Tumor cells recieving injections of invasin-enhanced ''E.coli'' and 6-MPDR grew the slowest, but did not get smaller. (Critchley, 2004 - Permission Pending)

</center>

The problem with this is that all cells in the body might be exposed to the toxin MeP. In addition to the search and destroy mechanism, scientists hope to increase selectivity by combining the hypoxia-sensing and density-sensing units so that ''E. coli'' will only enter eukaryotic cells that exhibit both characteristics. This would make it more likely that invasin-enhanced ''E. coli'' would only invade cancer cells rather than muscle tissue that had low oxygen due to exercise (Anderson, 2006). To create a fully functional, synthetic biology-based approach to cancer therapy, scientists need to develop bacteria that discriminately invades cancer cells and destroys them. Most of the pieces are there – scientists have engineered ''E. coli'' that selectively invades hypoxic cells or dense cells and ''Salmonella'' that can destroy tumors in vivo – they just need to be put together effectively.

Finally, synthetic biology has made its mark on the field of medicine by creating drugs that are less expensive to produce than those made by pharmaceutical companies. Jay Keasling’s project involving the antimalarial drug artemisinin is the best example. Yeast was engineered to increase normal farnesyl pyrophosphate (FPP) production, convert FPP to amorphadiene, and oxidizing amorphadiene to artemisinic acid, which can then easily be separated from the yeast cells and converted to artemisinin in the laboratory (Fig. 6). This innovation allows the production of artemisinin at lower prices than those currently on the market, and without regard to environmental constraints (Ro, 2006). On a completely different tangent, Collins’s lab engineered T7 bacteriophages to deliver biofilm-degrading enzymes such as dispersin B after infecting and replicating in an ''E. coli'' biofilm. Dispersin B works to degrade an adhesin that is critical for biofilm formation. Phages that released dispersin B were found to be 99.997% effective at removing biofilm, which is 4.5 orders of magnitude higher than non-enzymatic phages (Lu, 2007) (Fig. 7). The specificity of phages for a certain type of bacteria make them a viable vector to be used in human treatments, which may range from removing dental plaques to cleaning inserted medical devices. Using synthetic biology to engineer an antimalarial drug in yeast or a biofilm-destroying enzyme in bacteriophages greatly increases the possible contributions to the field of medicine.

<center>

{|
|-
|[[Image:Artemesinin.JPG]]
|[[Image:BiofilmDegradation.JPG]]
|-
|'''Fig. 6.''' All the steps in the box indicate steps that were optimized in yeast for a maximum artemisinic acid output. (Ro, 2006 - Permission Pending)
|'''Fig. 7.''' The amount of biofilm was decreased when the dispersin B enzymes were incorporated into T7 bacteriophages. (Lu, 2007 - Permission Pending)
|}
</center>

'''Conclusion'''

Medical applications of synthetic biology are wide-ranging and eminently applicable to daily life. Synthetic biology based cancer treatments that utilize ''E.coli'' engineered to sense and attack tissues with high cell density and low oxygen availability are promising and need to be tested in vivo. Designing vectors out of attenuated bacteria may be a safe, effective method for disease treatment. Using biofilms to sense sun damage potential may decrease the likelihood of skin cancer. Finally, using engineered cells to produce medicines in a cost-effective, environmentally-friendly way may revolutionize the pharmaceutical industry. Investing more research efforts in the field of synthetic biology may turn out to be an investment in medical technology.

== Works Cited ==

-[http://www.bio.davidson.edu/courses/synthetic/papers/2006_anderson.pdf Anderson JC, Clarke EJ, Arkin, AP, and Voigt CA (2006). Environmentally controlled invasion of cancer cells by engineered bacteria. Journal of Molecular Biology 355:619-27.]

-[http://www.nature.com/gt/journal/v11/n15/abs/3302281a.html Critchley RJ, Jezzard S, Radford KJ, Goussard S, Lemoine NR, et al. (2004). Potential therapeutic applications of recombinant, invasive ''E. coli''. Gene Therapy 11:1224-33.]

-[http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&uid=15203915&cmd=showdetailview&indexed=google Garmory HS, Leary SEC, Griffin, KF, Williamson D, Brown, KA, and Titball RW (2003). The use of live attenuated bacteria as a delivery system for heterologous antigens. Journal of Drug Targeting 11:471-79.]

-[http://www.pnas.org/cgi/reprint/101/22/8414 Kobayashi H, et al. (2004). Programmable cells: Interfacing natural and engineered gene networks. PNAS 101: 8414-19.]

-[http://www.bio.davidson.edu/courses/synthetic/papers/Phage_BioFilms.pdf Lu, TK, and Collins JJ (2007). Dispersing biofilms with engineered enzymatic bacteriophage. PNAS 104: 11197-11202.]

-[http://www.nature.com/cgt/journal/v10/n10/abs/7700634a.html Nemunaitis J, Cunningham C, Senzer N, Kuhn J, Cramm J, Litz C, et al. (2003). Pilot trial of genetically modified, attenuated ''Salmonella'' expressing cytosine deaminase gene in refractory cancer patients. Cancer Gene Therapy 10:737-744.]

-[http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6W85-49FGKHC-N&_user=10&_coverDate=09%2F30%2F2003&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=d4529b45974bfe7d9ddab1fc865f088b Pawelek JM, Low KB, and Bermudes D (2003). Bacteria as tumour-targeting vectors. Lancet Oncology 4:548-56.]

-[http://www.bio.davidson.edu/courses/synthetic/papers/Keasling_malaria.pdf Ro D, et al. (2006). Production of the antimalarial drug precursor artemisinic acid in engineered yeast. Nature 440:940-43.]

Medical Applications of Synthetic Biology - Samantha Simpson

2007-12-06T02:44:59Z

Sasimpson: /* Paper */

== Project Proposal ==

My project will be on medical applications of synthetic biology. I will reference Anderson's paper on utilizing quorum-sensing and hypoxia-responsive genes coupled with invasin from ''Yersinia tuberculosis'' to invade cancer-causing cells. Coupled with Critchley's work, which describes a bacterial system that invades eukaryotic cells and delivers proteins coded for in the bacteria's genome, one could possibly create 'search and destroy' ''E.coli'' that can locate, invade, and kill tumor cells. Garmory's paper also highlights the possibility of using bacteria as a drug-delivering system, specifically vaccine vectors. Kobayashi describes cells that produce a protective biofilm layer after exposure to DNA-damaging agents. Ro engineers yeast to create an anti-malarial drug that can be created at a lower cost than the previous standard production process. These papers, and others that have a focus on direct medical applications such as novel cancer therapies, vaccination technology, biological protection, and the creation of new medicines, will be referenced to create a cohesive overview of applications of synthetic biology to the medical field.

== Paper ==

Synthetic biology is a rapidly emerging field that strives to re-design existing biological systems and components or fabricate novel biological systems and components. The end result of experimentation in synthetic biology is to help scientists understand a naturally occurring genetic pathway more fully or to create a new, useful pathway. Recent research efforts have focused on engineering bacteria to create a new form of biofuel or to make promoters of varying efficiencies for more exact gene expression. Other research efforts focus on engineering yeast to make antimalarial drugs or engineering ''E. coli'' to recognize and invade tumor-like cells, both of which are medical applications of synthetic biology. Advancements such as novel cancer therapies, vaccination technology, biological protection, and the creation of new medicines highlight synthetic biology’s potential to create breakthroughs in both the prevention and treatment side of medical science. This paper will examine various medical applications of synthetic biology and further development that needs to be done before current research can be used in hospitals and doctors’ offices.

'''Prevention'''

Bacteria was first thought to be a good vector because it would not degrade at mucosal surface and it would survive the low pH in the gastrointestinal tract like most other orally administered antigens (Garmory, 2003). A particular strain of ''Salmonella'' with one or more deletions in the [http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&uid=15012217&cmd=showdetailview&indexed=google shikimate pathway], which creates a precursor for phenylalanine and tyrosine, is a promising vector because it gets into the body easily but does not harmfully effect the tissue and is gone within a week or two of introduction. Scientists engineered this strain to carry the ''Yersinia pestis'' V antigen, and studied its ability to protect mice from ''Y. pestis''. Unfortunately, the inoculation was not extremely successful and only 6 of 20 mice showed a strong response (Garmory, 2003). Garmory and colleagues cited several reasons for this: the copy number of the plasmid containing the V antigen might not have been high enough; the attenuated ''Salmonella'' may not have been able to reach cells that would be targeted by ''Y. pestis''; and the inability of the cell to secrete the antigen. Many changes need to be made to live vector vaccine design before it is an effective method of treating or preventing disease. Using bacteria as a vaccine vector has similar issues of specificity and implementation in a human system.

Another method of disease prevention with a synthetic biology approach is related to skin cancer. ''E. coli'' engineered to sense single-stranded DNA after DNA damage was coupled with a mechanism to produce a biofilm around the cell. The biofilm output module was tested with DNA damage done by mitomycin C, a DNA crosslinking agent, or UV irradiation, a known cause of skin cancer. Single-stranded DNA activates RecA, which in turn represses the CI repressor protein, thus allowing the transcription of the PL promoter (Kobayashi, 2004). The PL promoter is in charge of the ''traA'' gene, which controls biofilm formation (Fig. 1). The biofilm protection, when part of a toggle switch mechanism, lasted indefinitely when pulsed with UV light at 8 J/m2 for only 2 seconds (Kobayashi, 2004) (Fig. 2). The biofilm-production mechanism has not been engineered in eukaryotic cells, but a prokaryote-based system that could aid with human skin disease is imaginable. For example, a sunscreen that changes colors when the DNA damage may be too intense for a sunbather as a warning, or a sunscreen that becomes more protective as the possibility of genetic damage increased. Further research should focus on how to best implement the biofilm producing output module to protect human DNA.

<center>

{|
|-
|[[Image:Toggleswitch.JPG]]
|[[Image:UVinducedbiofilm.JPG]]
|-
|'''Fig. 1.''' The RecA / traA toggleswitch mechanism. (Kobayashi, 2004 - Permission Pending)
|'''Fig. 2.''' Crystal violet absorbance measures biofilm formation. (Kobayashi, 2004 - Permission Pending)
|}
</center>

'''Treatment'''

Synthetic biologists consider finding a cure for cancer a reachable goal. Previous to the development of synthetic biology as a field, it was known that three types of bacteria, ''Bifodobacterium'', ''Clostridium'', and ''Salmonella'' all preferentially infect the dense cells of tumors (Pawelek, 2003). All three types of bacteria are associated with reducing tumor size in patients who are infected with them, and it was noted that all three bacteria act anaerobically. Capitalizing on the idea that tumors create an unusually dense, anaerobic mass of cells, Christopher Voigt’s lab engineered bacteria to identify and invade cancer cells. They utilized the ''lux'' quorum sensing from ''Vibrio fischeri'' to identify cells growing at high densities (Fig. 3) and the ''fdhF'' promoter to identify cells growing in anaerobic conditions (Fig. 4) (Anderson, 2006). They coupled this identification mechanism with the invasin output module from ''Yersinia pseudotuberculosis'' so the ''E. coli'' could invade specific cells.

<center>

{|
|-
|[[Image:InvasinDensity.JPG]]
|[[Image:InvasinAnaerobic2.JPG]]
|-
|'''Fig. 3.''' The red line represents when the invasin output module was paired with the inducible promoter ''lux'' which senses high cell density, the black line is constitutively on invasin. (Anderson, 2006 - Permission Pending)
|'''Fig. 4.''' The invasin output module was paired with the inducible promoter ''fdhF'' which senses anaerobic conditions. (Anderson, 2006 - Permission Pending)
|}

</center>

Next, scientists hope to insert a gene that would destroy the invaded cells, making the complete cycle a search, invade, and destroy loop that would only be sensitive to cancer cells. One promising destroy mechanism has been implemented in humans via intratumoral injection, based on ''Salmonella'' that contains the ''E. coli'' cytosine deaminase gene that converts 5-fluorocytosine to 5-fluorouracil, a chemotherapy drug (Nemunaitis, 2003). Unfortunately, while the tumors did produce [http://en.wikipedia.org/wiki/Fluorouracil 5-fluorouracil] (5-FU), they did not produce it in high enough yields to cause regression of the cancer. Another experiment flooded mouse cells with 6-methylpurine-2’deoxyribose (6-MPDR), which was converted to the toxin [http://jpet.aspetjournals.org/cgi/content/abstract/304/3/1280 6-methyl purine] (MeP) by the enzyme purine nucleoside phosphorylase (PNP), which is naturally found in ''E. coli'' (Critchley, 2004) (Fig. 5).

<center>

[[Image:Tumor.JPG]]

'''Fig. 5.''' Tumor size after injections. Cancer cells were injected into mice, and 5 days later injections with PBS, 6-MPDR, and invasin-enhanced ''E-coli'' began. Tumor cells recieving injections of invasin-enhanced ''E.coli'' and 6-MPDR grew the slowest, but did not get smaller. (Critchley, 2004 - Permission Pending)

</center>

The problem with this is that all cells in the body might be exposed to the toxin MeP. In addition to the search and destroy mechanism, scientists hope to increase selectivity by combining the hypoxia-sensing and density-sensing units so that ''E. coli'' will only enter eukaryotic cells that exhibit both characteristics. This would make it more likely that invasin-enhanced ''E. coli'' would only invade cancer cells rather than muscle tissue that had low oxygen due to exercise (Anderson, 2006). To create a fully functional, synthetic biology-based approach to cancer therapy, scientists need to develop bacteria that discriminately invades cancer cells and destroys them. Most of the pieces are there – scientists have engineered ''E. coli'' that selectively invades hypoxic cells or dense cells and ''Salmonella'' that can destroy tumors in vivo – they just need to be put together effectively.

Finally, synthetic biology has made its mark on the field of medicine by creating drugs that are less expensive to produce than those made by pharmaceutical companies. Jay Keasling’s project involving the antimalarial drug artemisinin is the best example. Yeast was engineered to increase normal farnesyl pyrophosphate (FPP) production, convert FPP to amorphadiene, and oxidizing amorphadiene to artemisinic acid, which can then easily be separated from the yeast cells and converted to artemisinin in the laboratory (Fig. 6). This innovation allows the production of artemisinin at lower prices than those currently on the market, and without regard to environmental constraints (Ro, 2006). On a completely different tangent, Collins’s lab engineered T7 bacteriophages to deliver biofilm-degrading enzymes such as dispersin B after infecting and replicating in an ''E. coli'' biofilm. Dispersin B works to degrade an adhesin that is critical for biofilm formation. Phages that released dispersin B were found to be 99.997% effective at removing biofilm, which is 4.5 orders of magnitude higher than non-enzymatic phages (Lu, 2007) (Fig. 7). The specificity of phages for a certain type of bacteria make them a viable vector to be used in human treatments, which may range from removing dental plaques to cleaning inserted medical devices. Using synthetic biology to engineer an antimalarial drug in yeast or a biofilm-destroying enzyme in bacteriophages greatly increases the possible contributions to the field of medicine.

<center>

{|
|-
|[[Image:Artemesinin.JPG]]
|[[Image:BiofilmDegradation.JPG]]
|-
|'''Fig. 6.''' All the steps in the box indicate steps that were optimized in yeast for a maximum artemisinic acid output. (Ro, 2006 - Permission Pending)
|'''Fig. 7.''' The amount of biofilm was decreased when the dispersin B enzymes were incorporated into T7 bacteriophages. (Lu, 2007 - Permission Pending)
|}
</center>

'''Conclusion'''

Medical applications of synthetic biology are wide-ranging and eminently applicable to daily life. Synthetic biology based cancer treatments that utilize ''E.coli'' engineered to sense and attack tissues with high cell density and low oxygen availability are promising and need to be tested in vivo. Designing vectors out of attenuated bacteria may be a safe, effective method for disease treatment. Using biofilms to sense sun damage potential may decrease the likelihood of skin cancer. Finally, using engineered cells to produce medicines in a cost-effective, environmentally-friendly way may revolutionize the pharmaceutical industry. Investing more research efforts in the field of synthetic biology may turn out to be an investment in medical technology.

== Works Cited ==

-[http://www.bio.davidson.edu/courses/synthetic/papers/2006_anderson.pdf Anderson JC, Clarke EJ, Arkin, AP, and Voigt CA (2006). Environmentally controlled invasion of cancer cells by engineered bacteria. Journal of Molecular Biology 355:619-27.]

-[http://www.nature.com/gt/journal/v11/n15/abs/3302281a.html Critchley RJ, Jezzard S, Radford KJ, Goussard S, Lemoine NR, et al. (2004). Potential therapeutic applications of recombinant, invasive ''E. coli''. Gene Therapy 11:1224-33.]

-[http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&uid=15203915&cmd=showdetailview&indexed=google Garmory HS, Leary SEC, Griffin, KF, Williamson D, Brown, KA, and Titball RW (2003). The use of live attenuated bacteria as a delivery system for heterologous antigens. Journal of Drug Targeting 11:471-79.]

-[http://www.pnas.org/cgi/reprint/101/22/8414 Kobayashi H, et al. (2004). Programmable cells: Interfacing natural and engineered gene networks. PNAS 101: 8414-19.]

-[http://www.bio.davidson.edu/courses/synthetic/papers/Phage_BioFilms.pdf Lu, TK, and Collins JJ (2007). Dispersing biofilms with engineered enzymatic bacteriophage. PNAS 104: 11197-11202.]

-[http://www.nature.com/cgt/journal/v10/n10/abs/7700634a.html Nemunaitis J, Cunningham C, Senzer N, Kuhn J, Cramm J, Litz C, et al. (2003). Pilot trial of genetically modified, attenuated ''Salmonella'' expressing cytosine deaminase gene in refractory cancer patients. Cancer Gene Therapy 10:737-744.]

-[http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6W85-49FGKHC-N&_user=10&_coverDate=09%2F30%2F2003&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=d4529b45974bfe7d9ddab1fc865f088b Pawelek JM, Low KB, and Bermudes D (2003). Bacteria as tumour-targeting vectors. Lancet Oncology 4:548-56.]

-[http://www.bio.davidson.edu/courses/synthetic/papers/Keasling_malaria.pdf Ro D, et al. (2006). Production of the antimalarial drug precursor artemisinic acid in engineered yeast. Nature 440:940-43.]

Medical Applications of Synthetic Biology - Samantha Simpson

2007-12-06T01:08:00Z

Sasimpson: /* Paper */

== Project Proposal ==

My project will be on medical applications of synthetic biology. I will reference Anderson's paper on utilizing quorum-sensing and hypoxia-responsive genes coupled with invasin from ''Yersinia tuberculosis'' to invade cancer-causing cells. Coupled with Critchley's work, which describes a bacterial system that invades eukaryotic cells and delivers proteins coded for in the bacteria's genome, one could possibly create 'search and destroy' ''E.coli'' that can locate, invade, and kill tumor cells. Garmory's paper also highlights the possibility of using bacteria as a drug-delivering system, specifically vaccine vectors. Kobayashi describes cells that produce a protective biofilm layer after exposure to DNA-damaging agents. Ro engineers yeast to create an anti-malarial drug that can be created at a lower cost than the previous standard production process. These papers, and others that have a focus on direct medical applications such as novel cancer therapies, vaccination technology, biological protection, and the creation of new medicines, will be referenced to create a cohesive overview of applications of synthetic biology to the medical field.

== Paper ==

Synthetic biology is a rapidly emerging field that strives to re-design existing biological systems and components or fabricate novel biological systems and components. The end result of experimentation in synthetic biology is to help scientists understand a naturally occurring genetic pathway more fully or to create a new, useful pathway. Recent research efforts have focused on engineering bacteria to create a new form of biofuel or to make promoters of varying efficiencies for more exact gene expression. Other research efforts focus on engineering yeast to make antimalarial drugs or engineering ''E. coli'' to recognize and invade tumor-like cells, both of which are medical applications of synthetic biology. Advancements such as novel cancer therapies, vaccination technology, biological protection, and the creation of new medicines highlight synthetic biology’s potential to create breakthroughs in both the prevention and treatment side of medical science. This paper will examine various medical applications of synthetic biology and further development that needs to be done before current research can be used in hospitals and doctors’ offices.

'''Prevention'''

Using bacteria as a vaccine vector has similar issues of specificity and implementation in a human system. Bacteria was first thought to be a good vector because it would not degrade at mucosal surface and it would survive the low pH in the gastrointestinal tract (Garmory, 2003). A particular strain of ''Salmonella'' with one or more deletions in the [http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&uid=15012217&cmd=showdetailview&indexed=google shikimate pathway], which creates a precursor for phenylalanine and tyrosine, is a promising vector because it gets into the body easily but does not harmfully effect the tissue and is gone within a week or two of introduction. Scientists engineered this strain to carry the ''Yersinia pestis'' V antigen, and studied its ability to protect mice from ''Y. pestis''. Unfortunately, the inoculation was not extremely successful and only 6 of 20 mice showed a strong response (Garmory, 2003). Garmory and colleagues cited several reasons for this: the copy number of the plasmid containing the V antigen might not have been high enough; the attenuated ''Salmonella'' may not have been able to reach cells that would be targeted by ''Y. pestis''; and the inability of the cell to secrete the antigen. Many changes need to be made to live vector vaccine design before it is an effective method of treating or preventing disease.

Another method of disease prevention with a synthetic biology approach is related to skin cancer. ''E. coli'' engineered to sense single-stranded DNA after DNA damage was coupled with a mechanism to produce a biofilm around the cell. The biofilm output module was tested with DNA damage done by mitomycin C, a DNA crosslinking agent, or UV irradiation, a known cause of skin cancer. Single-stranded DNA activates RecA, which in turn represses the CI repressor protein, thus allowing the transcription of the PL promoter (Kobayashi, 2004). The PL promoter is in charge of the ''traA'' gene, which controls biofilm formation (Fig. 1). The biofilm protection, when part of a toggle switch mechanism, lasted indefinitely when pulsed with UV light at 8 J/m2 for only 2 seconds (Kobayashi, 2004) (Fig. 2). The biofilm-production mechanism has not been engineered in eukaryotic cells, but a prokaryote-based system that could aid with human skin disease is imaginable. For example, a sunscreen that changes colors when the DNA damage may be too intense for a sunbather as a warning, or a sunscreen that becomes more protective as the possibility of genetic damage increased. Further research should focus on how to best implement the biofilm producing output module to protect human DNA.

<center>

{|
|-
|[[Image:Toggleswitch.JPG]]
|[[Image:UVinducedbiofilm.JPG]]
|-
|'''Fig. 1.''' The RecA / traA toggleswitch mechanism. (Kobayashi, 2004 - Permission Pending)
|'''Fig. 2.''' Crystal violet absorbance measures biofilm formation. (Kobayashi, 2004 - Permission Pending)
|}
</center>

'''Treatment'''

Synthetic biologists consider finding a cure for cancer a reachable goal. Previous to the development of synthetic biology as a field, it was known that three types of bacteria, ''Bifodobacterium'', ''Clostridium'', and ''Salmonella'' all preferentially infect the dense cells of tumors (Pawelek, 2003). All three types of bacteria are associated with reducing tumor size in patients who are infected with them, and it was noted that all three bacteria act anaerobically. Capitalizing on the idea that tumors create an unusually dense, anaerobic mass of cells, Christopher Voigt’s lab engineered bacteria to identify and invade cancer cells. They utilized the ''lux'' quorum sensing from ''Vibrio fischeri'' to identify cells growing at high densities (Fig. 3) and the ''fdhF'' promoter to identify cells growing in anaerobic conditions (Fig. 4) (Anderson, 2006). They coupled this identification mechanism with the invasin output module from ''Yersinia pseudotuberculosis'' so the ''E. coli'' could invade specific cells.

<center>

{|
|-
|[[Image:InvasinDensity.JPG]]
|[[Image:InvasinAnaerobic2.JPG]]
|-
|'''Fig. 3.''' The red line represents when the invasin output module was paired with the inducible promoter ''lux'' which senses high cell density, the black line is constitutively on invasin. (Anderson, 2006 - Permission Pending)
|'''Fig. 4.''' The invasin output module was paired with the inducible promoter ''fdhF'' which senses anaerobic conditions. (Anderson, 2006 - Permission Pending)
|}

</center>

Next, scientists hope to insert a gene that would destroy the invaded cells, making the complete cycle a search, invade, and destroy loop that would only be sensitive to cancer cells. One promising destroy mechanism has been implemented in humans via intratumoral injection, based on ''Salmonella'' that contains the ''E. coli'' cytosine deaminase gene that converts 5-fluorocytosine to 5-fluorouracil, a chemotherapy drug (Nemunaitis, 2003). Unfortunately, while the tumors did produce [http://en.wikipedia.org/wiki/Fluorouracil 5-fluorouracil] (5-FU), they did not produce it in high enough yields to cause regression of the cancer. Another experiment flooded mouse cells with 6-methylpurine-2’deoxyribose (6-MPDR), which was converted to the toxin [http://jpet.aspetjournals.org/cgi/content/abstract/304/3/1280 6-methyl purine] (MeP) by the enzyme purine nucleoside phosphorylase (PNP), which is naturally found in ''E. coli'' (Critchley, 2004) (Fig. 5).

<center>

[[Image:Tumor.JPG]]

'''Fig. 5.''' Tumor size after injections. Cancer cells were injected into mice, and 5 days later injections with PBS, 6-MPDR, and invasin-enhanced ''E-coli'' began. Tumor cells recieving injections of invasin-enhanced ''E.coli'' and 6-MPDR grew the slowest, but did not get smaller. (Critchley, 2004 - Permission Pending)

</center>

The problem with this is that all cells in the body might be exposed to the toxin MeP. In addition to the search and destroy mechanism, scientists hope to increase selectivity by combining the hypoxia-sensing and density-sensing units so that ''E. coli'' will only enter eukaryotic cells that exhibit both characteristics. This would make it more likely that invasin-enhanced ''E. coli'' would only invade cancer cells rather than muscle tissue that had low oxygen due to exercise (Anderson, 2006). To create a fully functional, synthetic biology-based approach to cancer therapy, scientists need to develop bacteria that discriminately invades cancer cells and destroys them. Most of the pieces are there – scientists have engineered ''E. coli'' that selectively invades hypoxic cells or dense cells and ''Salmonella'' that can destroy tumors in vivo – they just need to be put together effectively.

Finally, synthetic biology has made its mark on the field of medicine by creating drugs that are less expensive to produce than those made by pharmaceutical companies. Jay Keasling’s project involving the antimalarial drug artemisinin is the best example. Yeast was engineered to increase normal farnesyl pyrophosphate (FPP) production, convert FPP to amorphadiene, and oxidizing amorphadiene to artemisinic acid, which can then easily be separated from the yeast cells and converted to artemisinin in the laboratory (Fig. 6). This innovation allows the production of artemisinin at lower prices than those currently on the market, and without regard to environmental constraints (Ro, 2006). On a completely different tangent, Collins’s lab engineered T7 bacteriophages to deliver biofilm-degrading enzymes such as dispersin B after infecting and replicating in an ''E. coli'' biofilm. Dispersin B works to degrade an adhesin that is critical for biofilm formation. Phages that released dispersin B were found to be 99.997% effective at removing biofilm, which is 4.5 orders of magnitude higher than non-enzymatic phages (Lu, 2007) (Fig. 7). The specificity of phages for a certain type of bacteria make them a viable vector to be used in human treatments, which may range from removing dental plaques to cleaning inserted medical devices. Using synthetic biology to engineer an antimalarial drug in yeast or a biofilm-destroying enzyme in bacteriophages greatly increases the possible contributions to the field of medicine.

<center>

{|
|-
|[[Image:Artemesinin.JPG]]
|[[Image:BiofilmDegradation.JPG]]
|-
|'''Fig. 6.''' All the steps in the box indicate steps that were optimized in yeast for a maximum artemisinic acid output. (Ro, 2006 - Permission Pending)
|'''Fig. 7.''' The amount of biofilm was decreased when the dispersin B enzymes were incorporated into T7 bacteriophages. (Lu, 2007 - Permission Pending)
|}
</center>

'''Conclusion'''

Medical applications of synthetic biology are wide-ranging and eminently applicable to daily life. Synthetic biology based cancer treatments that utilize ''E.coli'' engineered to sense and attack tissues with high cell density and low oxygen availability are promising and need to be tested in vivo. Designing vectors out of attenuated bacteria may be a safe, effective method for disease treatment. Using biofilms to sense sun damage potential may decrease the likelihood of skin cancer. Finally, using engineered cells to produce medicines in a cost-effective, environmentally-friendly way may revolutionize the pharmaceutical industry. Investing more research efforts in the field of synthetic biology may turn out to be an investment in medical technology.

== Works Cited ==

-[http://www.bio.davidson.edu/courses/synthetic/papers/2006_anderson.pdf Anderson JC, Clarke EJ, Arkin, AP, and Voigt CA (2006). Environmentally controlled invasion of cancer cells by engineered bacteria. Journal of Molecular Biology 355:619-27.]

-[http://www.nature.com/gt/journal/v11/n15/abs/3302281a.html Critchley RJ, Jezzard S, Radford KJ, Goussard S, Lemoine NR, et al. (2004). Potential therapeutic applications of recombinant, invasive ''E. coli''. Gene Therapy 11:1224-33.]

-[http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&uid=15203915&cmd=showdetailview&indexed=google Garmory HS, Leary SEC, Griffin, KF, Williamson D, Brown, KA, and Titball RW (2003). The use of live attenuated bacteria as a delivery system for heterologous antigens. Journal of Drug Targeting 11:471-79.]

-[http://www.pnas.org/cgi/reprint/101/22/8414 Kobayashi H, et al. (2004). Programmable cells: Interfacing natural and engineered gene networks. PNAS 101: 8414-19.]

-[http://www.bio.davidson.edu/courses/synthetic/papers/Phage_BioFilms.pdf Lu, TK, and Collins JJ (2007). Dispersing biofilms with engineered enzymatic bacteriophage. PNAS 104: 11197-11202.]

-[http://www.nature.com/cgt/journal/v10/n10/abs/7700634a.html Nemunaitis J, Cunningham C, Senzer N, Kuhn J, Cramm J, Litz C, et al. (2003). Pilot trial of genetically modified, attenuated ''Salmonella'' expressing cytosine deaminase gene in refractory cancer patients. Cancer Gene Therapy 10:737-744.]

-[http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6W85-49FGKHC-N&_user=10&_coverDate=09%2F30%2F2003&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=d4529b45974bfe7d9ddab1fc865f088b Pawelek JM, Low KB, and Bermudes D (2003). Bacteria as tumour-targeting vectors. Lancet Oncology 4:548-56.]

-[http://www.bio.davidson.edu/courses/synthetic/papers/Keasling_malaria.pdf Ro D, et al. (2006). Production of the antimalarial drug precursor artemisinic acid in engineered yeast. Nature 440:940-43.]

Medical Applications of Synthetic Biology - Samantha Simpson

2007-11-24T15:38:11Z

Sasimpson:

== Project Proposal ==

My project will be on medical applications of synthetic biology. I will reference Anderson's paper on utilizing quorum-sensing and hypoxia-responsive genes coupled with invasin from ''Yersinia tuberculosis'' to invade cancer-causing cells. Coupled with Critchley's work, which describes a bacterial system that invades eukaryotic cells and delivers proteins coded for in the bacteria's genome, one could possibly create 'search and destroy' ''E.coli'' that can locate, invade, and kill tumor cells. Garmory's paper also highlights the possibility of using bacteria as a drug-delivering system, specifically vaccine vectors. Kobayashi describes cells that produce a protective biofilm layer after exposure to DNA-damaging agents. Ro engineers yeast to create an anti-malarial drug that can be created at a lower cost than the previous standard production process. These papers, and others that have a focus on direct medical applications such as novel cancer therapies, vaccination technology, biological protection, and the creation of new medicines, will be referenced to create a cohesive overview of applications of synthetic biology to the medical field.

== Paper ==

Synthetic biology is a rapidly emerging field that strives to re-design existing biological systems and components or fabricate novel biological systems and components. The end result of experimentation in synthetic biology is to help scientists understand a naturally occurring genetic pathway more fully or to create a new, useful pathway. Recent research efforts have focused on engineering bacteria to create a new form of biofuel or to make promoters of varying efficiencies for more exact gene expression. Other research efforts focus on engineering yeast to make antimalarial drugs or engineering ''E. coli'' to recognize and invade tumor-like cells, both of which are medical applications of synthetic biology. Advancements such as novel cancer therapies, vaccination technology, biological protection, and the creation of new medicines highlight synthetic biology’s potential to create breakthroughs in both the prevention and treatment side of medical science. This paper will examine various medical applications of synthetic biology and further development that needs to be done before current research can be used in hospitals and doctors’ offices.

'''Prevention'''

Using bacteria as a vaccine vector has similar issues of specificity and implementation in a human system. Bacteria was first thought to be a good vector because they would not degrade at mucosal surfaces, they would absorb better and survive the low pH in the gastrointestinal tract (Garmory, 2003). A particular strain of ''Salmonella'' with one or more deletions in the [http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&uid=15012217&cmd=showdetailview&indexed=google shikimate pathway], which creates a precursor for phenylalanine and tyrosine, is a promising vector because it gets into the body easily but does not harmfully effect the tissue and is gone within a week or two of introduction. Scientists engineered this strain to carry the ''Yersinia pestis'' V antigen, and studied its ability to protect mice from ''Y. pestis''. Unfortunately, the inoculation was not extremely successful and only 6 of 20 mice showed a strong response (Garmory, 2003). Garmory and colleagues cited several reasons for this: the copy number of the plasmid containing the V antigen might not have been high enough; the attenuated ''Salmonella'' may not have been able to reach cells that would be targeted by ''Y. pestis''; and the inability of the cell to secrete the antigen. Many changes need to be made to live vector vaccine design before it is an effective method of treating or preventing disease.

Another method of disease prevention with a synthetic biology approach is related to skin cancer. ''E. coli'' engineered to sense single-stranded DNA after DNA damage was coupled with a mechanism to produce a biofilm around the cell. The biofilm output module was tested with DNA damage done by mitomycin C, a DNA crosslinking agent, or UV irradiation, a known cause of skin cancer. Single-stranded DNA activates RecA, which in turn represses the CI repressor protein, thus allowing the transcription of the PL promoter (Kobayashi, 2004). The PL promoter is in charge of the ''traA'' gene, which controls biofilm formation (Fig. 1). The biofilm protection, when part of a toggle switch mechanism, lasted indefinitely when pulsed with UV light at 8 J/m2 for only 2 seconds (Kobayashi, 2004) (Fig. 2). The biofilm-production mechanism has not been engineered in eukaryotic cells, but a prokaryote-based system that could aid with human skin disease is imaginable. For example, a sunscreen that changes colors when the DNA damage may be too intense for a sunbather as a warning, or a sunscreen that becomes more protective as the possibility of genetic damage increased. Further research should focus on how to best implement the biofilm producing output module to protect human DNA.

<center>

{|
|-
|[[Image:Toggleswitch.JPG]]
|[[Image:UVinducedbiofilm.JPG]]
|-
|'''Fig. 1.''' The RecA / traA toggleswitch mechanism. (Kobayashi, 2004 - Permission Pending)
|'''Fig. 2.''' Crystal violet absorbance measures biofilm formation. (Kobayashi, 2004 - Permission Pending)
|}
</center>

'''Treatment'''

Synthetic biologists consider finding a cure for cancer a reachable goal. Previous to the development of synthetic biology as a field, it was known that three types of bacteria, ''Bifodobacterium'', ''Clostridium'', and ''Salmonella'' all preferentially infect the dense cells of tumors (Pawelek, 2003). All three types of bacteria are associated with reducing tumor size in patients who are infected with them, and it was noted that all three bacteria act anaerobically. Capitalizing on the idea that tumors create an unusually dense, anaerobic mass of cells, Christopher Voigt’s lab engineered bacteria to identify and invade cancer cells. They utilized the ''lux'' quorum sensing from ''Vibrio fischeri'' to identify cells growing at high densities (Fig. 3) and the ''fdhF'' promoter to identify cells growing in anaerobic conditions (Fig. 4) (Anderson, 2006). They coupled this identification mechanism with the invasin output module from ''Yersinia pseudotuberculosis'' so the ''E. coli'' could invade specific cells.

<center>

{|
|-
|[[Image:InvasinDensity.JPG]]
|[[Image:InvasinAnaerobic2.JPG]]
|-
|'''Fig. 3.''' The red line represents when the invasin output module was paired with the inducible promoter ''lux'' which senses high cell density, the black line is constitutively on invasin. (Anderson, 2006 - Permission Pending)
|'''Fig. 4.''' The invasin output module was paired with the inducible promoter ''fdhF'' which senses anaerobic conditions. (Anderson, 2006 - Permission Pending)
|}

</center>

Next, scientists hope to insert a gene that would destroy the invaded cells, making the complete cycle a search, invade, and destroy loop that would only be sensitive to cancer cells. One promising destroy mechanism has been implemented in humans via intratumoral injection, based on ''Salmonella'' that contains the ''E. coli'' cytosine deaminase gene that converts 5-fluorocytosine to 5-fluorouracil, a chemotherapy drug (Nemunaitis, 2003). Unfortunately, while the tumors did produce [http://en.wikipedia.org/wiki/Fluorouracil 5-fluorouracil] (5-FU), they did not produce it in high enough yields to cause regression of the cancer. Another experiment flooded mouse cells with 6-methylpurine-2’deoxyribose (6-MPDR), which was converted to the toxin [http://jpet.aspetjournals.org/cgi/content/abstract/304/3/1280 6-methyl purine] (MeP) by the enzyme purine nucleoside phosphorylase (PNP), which is naturally found in ''E. coli'' (Critchley, 2004) (Fig. 5).

<center>

[[Image:Tumor.JPG]]

'''Fig. 5.''' Tumor size after injections. Cancer cells were injected into mice, and 5 days later injections with PBS, 6-MPDR, and invasin-enhanced ''E-coli'' began. Tumor cells recieving injections of invasin-enhanced ''E.coli'' and 6-MPDR grew the slowest, but did not get smaller. (Critchley, 2004 - Permission Pending)

</center>

The problem with this is that all cells in the body might be exposed to the toxin MeP. In addition to the search and destroy mechanism, scientists hope to increase selectivity by combining the hypoxia-sensing and density-sensing units so that ''E. coli'' will only enter eukaryotic cells that exhibit both characteristics. This would make it more likely that invasin-enhanced ''E. coli'' would only invade cancer cells rather than muscle tissue that had low oxygen due to exercise (Anderson, 2006). To create a fully functional, synthetic biology-based approach to cancer therapy, scientists need to develop bacteria that discriminately invades cancer cells and destroys them. Most of the pieces are there – scientists have engineered ''E. coli'' that selectively invades hypoxic cells or dense cells and ''Salmonella'' that can destroy tumors in vivo – they just need to be put together effectively.

Finally, synthetic biology has made its mark on the field of medicine by creating drugs that are less expensive to produce than those made by pharmaceutical companies. Jay Keasling’s project involving the antimalarial drug artemisinin is the best example. Yeast was engineered to increase normal farnesyl pyrophosphate (FPP) production, convert FPP to amorphadiene, and oxidizing amorphadiene to artemisinic acid, which can then easily be separated from the yeast cells and converted to artemisinin in the laboratory (Fig. 6). This innovation allows the production of artemisinin at lower prices than those currently on the market, and without regard to environmental constraints (Ro, 2006). On a completely different tangent, Collins’s lab engineered T7 bacteriophages to deliver biofilm-degrading enzymes such as dispersin B after infecting and replicating in an ''E. coli'' biofilm. Dispersin B works to degrade an adhesin that is critical for biofilm formation. Phages that released dispersin B were found to be 99.997% effective at removing biofilm, which is 4.5 orders of magnitude higher than non-enzymatic phages (Lu, 2007) (Fig. 7). The specificity of phages for a certain type of bacteria make them a viable vector to be used in human treatments, which may range from removing dental plaques to cleaning inserted medical devices. Using synthetic biology to engineer an antimalarial drug in yeast or a biofilm-destroying enzyme in bacteriophages greatly increases the possible contributions to the field of medicine.

<center>

{|
|-
|[[Image:Artemesinin.JPG]]
|[[Image:BiofilmDegradation.JPG]]
|-
|'''Fig. 6.''' All the steps in the box indicate steps that were optimized in yeast for a maximum artemisinic acid output. (Ro, 2006 - Permission Pending)
|'''Fig. 7.''' The amount of biofilm was decreased when the dispersin B enzymes were incorporated into T7 bacteriophages. (Lu, 2007 - Permission Pending)
|}
</center>

'''Conclusion'''

Medical applications of synthetic biology are wide-ranging and eminently applicable to daily life. Synthetic biology based cancer treatments that utilize ''E.coli'' engineered to sense and attack tissues with high cell density and low oxygen availability are promising and need to be tested in vivo. Designing vectors out of attenuated bacteria may be a safe, effective method for disease treatment. Using biofilms to sense sun damage potential may decrease the likelihood of skin cancer. Finally, using engineered cells to produce medicines in a cost-effective, environmentally-friendly way may revolutionize the pharmaceutical industry. Investing more research efforts in the field of synthetic biology may turn out to be an investment in medical technology.

== Works Cited ==

-[http://www.bio.davidson.edu/courses/synthetic/papers/2006_anderson.pdf Anderson JC, Clarke EJ, Arkin, AP, and Voigt CA (2006). Environmentally controlled invasion of cancer cells by engineered bacteria. Journal of Molecular Biology 355:619-27.]

-[http://www.nature.com/gt/journal/v11/n15/abs/3302281a.html Critchley RJ, Jezzard S, Radford KJ, Goussard S, Lemoine NR, et al. (2004). Potential therapeutic applications of recombinant, invasive ''E. coli''. Gene Therapy 11:1224-33.]

-[http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&uid=15203915&cmd=showdetailview&indexed=google Garmory HS, Leary SEC, Griffin, KF, Williamson D, Brown, KA, and Titball RW (2003). The use of live attenuated bacteria as a delivery system for heterologous antigens. Journal of Drug Targeting 11:471-79.]

-[http://www.pnas.org/cgi/reprint/101/22/8414 Kobayashi H, et al. (2004). Programmable cells: Interfacing natural and engineered gene networks. PNAS 101: 8414-19.]

-[http://www.bio.davidson.edu/courses/synthetic/papers/Phage_BioFilms.pdf Lu, TK, and Collins JJ (2007). Dispersing biofilms with engineered enzymatic bacteriophage. PNAS 104: 11197-11202.]

-[http://www.nature.com/cgt/journal/v10/n10/abs/7700634a.html Nemunaitis J, Cunningham C, Senzer N, Kuhn J, Cramm J, Litz C, et al. (2003). Pilot trial of genetically modified, attenuated ''Salmonella'' expressing cytosine deaminase gene in refractory cancer patients. Cancer Gene Therapy 10:737-744.]

-[http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6W85-49FGKHC-N&_user=10&_coverDate=09%2F30%2F2003&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=d4529b45974bfe7d9ddab1fc865f088b Pawelek JM, Low KB, and Bermudes D (2003). Bacteria as tumour-targeting vectors. Lancet Oncology 4:548-56.]

-[http://www.bio.davidson.edu/courses/synthetic/papers/Keasling_malaria.pdf Ro D, et al. (2006). Production of the antimalarial drug precursor artemisinic acid in engineered yeast. Nature 440:940-43.]

Medical Applications of Synthetic Biology - Samantha Simpson

2007-11-24T15:30:35Z

Sasimpson:

== Project Proposal ==

My project will be on medical applications of synthetic biology. I will reference Anderson's paper on utilizing quorum-sensing and hypoxia-responsive genes coupled with invasin from ''Yersinia tuberculosis'' to invade cancer-causing cells. Coupled with Critchley's work, which describes a bacterial system that invades eukaryotic cells and delivers proteins coded for in the bacteria's genome, one could possibly create 'search and destroy' ''E.coli'' that can locate, invade, and kill tumor cells. Garmory's paper also highlights the possibility of using bacteria as a drug-delivering system, specifically vaccine vectors. Kobayashi describes cells that produce a protective biofilm layer after exposure to DNA-damaging agents. Ro engineers yeast to create an anti-malarial drug that can be created at a lower cost than the previous standard production process. These papers, and others that have a focus on direct medical applications such as novel cancer therapies, vaccination technology, biological protection, and the creation of new medicines, will be referenced to create a cohesive overview of applications of synthetic biology to the medical field.

== Paper ==

Synthetic biology is a rapidly emerging field that strives to re-design existing biological systems and components or fabricate novel biological systems and components. The end result of experimentation in synthetic biology is to help scientists understand a naturally occurring genetic pathway more fully or to create a new, useful pathway. Recent research efforts have focused on engineering bacteria to create a new form of biofuel or to make promoters of varying efficiencies for more exact gene expression. Other research efforts focus on engineering yeast to make antimalarial drugs or engineering ''E. coli'' to recognize and invade tumor-like cells, both of which are medical applications of synthetic biology. Advancements such as novel cancer therapies, vaccination technology, biological protection, and the creation of new medicines highlight synthetic biology’s potential to create breakthroughs in both the prevention and treatment side of medical science. This paper will examine various medical applications of synthetic biology and further development that needs to be done before current research can be used in hospitals and doctors’ offices.

'''Prevention'''

Using bacteria as a vaccine vector has similar issues of specificity and implementation in a human system. Bacteria was first thought to be a good vector because they would not degrade at mucosal surfaces, they would absorb better and survive the low pH in the gastrointestinal tract (Garmory, 2003). A particular strain of ''Salmonella'' with one or more deletions in the [http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&uid=15012217&cmd=showdetailview&indexed=google shikimate pathway], which creates a precursor for phenylalanine and tyrosine, is a promising vector because it gets into the body easily but does not harmfully effect the tissue and is gone within a week or two of introduction. Scientists engineered this strain to carry the ''Yersinia pestis'' V antigen, and studied its ability to protect mice from ''Y. pestis''. Unfortunately, the inoculation was not extremely successful and only 6 of 20 mice showed a strong response (Garmory, 2003). Garmory and colleagues cited several reasons for this: the copy number of the plasmid containing the V antigen might not have been high enough; the attenuated ''Salmonella'' may not have been able to reach cells that would be targeted by ''Y. pestis''; and the inability of the cell to secrete the antigen. Many changes need to be made to live vector vaccine design before it is an effective method of treating or preventing disease.

Another method of disease prevention with a synthetic biology approach is related to skin cancer. ''E. coli'' engineered to sense single-stranded DNA after DNA damage was coupled with a mechanism to produce a biofilm around the cell. The biofilm output module was tested with DNA damage done by mitomycin C, a DNA crosslinking agent, or UV irradiation, a known cause of skin cancer. Single-stranded DNA activates RecA, which in turn represses the CI repressor protein, thus allowing the transcription of the PL promoter (Kobayashi, 2004). The PL promoter is in charge of the ''traA'' gene, which controls biofilm formation (Fig. 1). The biofilm protection, when part of a toggle switch mechanism, lasted indefinitely when pulsed with UV light at 8 J/m2 for only 2 seconds (Kobayashi, 2004) (Fig. 2). The biofilm-production mechanism has not been engineered in eukaryotic cells, but a prokaryote-based system that could aid with human skin disease is imaginable. For example, a sunscreen that changes colors when the DNA damage may be too intense for a sunbather as a warning, or a sunscreen that becomes more protective as the possibility of genetic damage increased. Further research should focus on how to best implement the biofilm producing output module to protect human DNA.

<center>

{|
|-
|[[Image:Toggleswitch.JPG]]
|[[Image:UVinducedbiofilm.JPG]]
|-
|'''Fig. 1.''' The RecA / traA toggleswitch mechanism. (Kobayashi, 2004 - Permission Pending)
|'''Fig. 2.''' Crystal violet absorbance measures biofilm formation. (Kobayashi, 2004 - Permission Pending)
|}
</center>

'''Treatment'''

Synthetic biologists consider finding a cure for cancer a reachable goal. Previous to the development of synthetic biology as a field, it was known that three types of bacteria, ''Bifodobacterium'', ''Clostridium'', and ''Salmonella'' all preferentially infect the dense cells of tumors (Pawelek, 2003). All three types of bacteria are associated with reducing tumor size in patients who are infected with them, and it was noted that all three bacteria act anaerobically. Capitalizing on the idea that tumors create an unusually dense, anaerobic mass of cells, Christopher Voigt’s lab engineered bacteria to identify and invade cancer cells. They utilized the ''lux'' quorum sensing from ''Vibrio fischeri'' to identify cells growing at high densities (Fig. 3) and the ''fdhF'' promoter to identify cells growing in anaerobic conditions (Fig. 4) (Anderson, 2006). They coupled this identification mechanism with the invasin output module from ''Yersinia pseudotuberculosis'' so the ''E. coli'' could invade specific cells.

<center>

{|
|-
|[[Image:InvasinDensity.JPG]]
|[[Image:InvasinAnaerobic2.JPG]]
|-
|'''Fig. 3.''' The red line represents when the invasin output module was paired with the inducible promoter ''lux'' which senses high cell density, the black line is constitutively on invasin. (Anderson, 2006 - Permission Pending)
|'''Fig. 4.''' The invasin output module was paired with the inducible promoter ''fdhF'' which senses anaerobic conditions. (Anderson, 2006 - Permission Pending)
|}

</center>

Next, scientists hope to insert a gene that would destroy the invaded cells, making the complete cycle a search, invade, and destroy loop that would only be sensitive to cancer cells. One promising destroy mechanism has been implemented in humans via intratumoral injection, based on ''Salmonella'' that contains the ''E. coli'' cytosine deaminase gene that converts 5-fluorocytosine to 5-fluorouracil, a chemotherapy drug (Nemunaitis, 2003). Unfortunately, while the tumors did produce 5-fluorouracil (5-FU), they did not produce it in high enough yields to cause regression of the cancer. Another experiment flooded mouse cells with 6-methylpurine-2’deoxyribose (6-MPDR), which was converted to the toxin 6-methyl purine (MeP) by the enzyme purine nucleoside phosphorylase (PNP), which is naturally found in ''E. coli'' (Critchley, 2004) (Fig. 5).

<center>

[[Image:Tumor.JPG]]

'''Fig. 5.''' Tumor size after injections. Cancer cells were injected into mice, and 5 days later injections with PBS, 6-MPDR, and invasin-enhanced ''E-coli'' began. Tumor cells recieving injections of invasin-enhanced ''E.coli'' and 6-MPDR grew the slowest, but did not get smaller. (Critchley, 2004 - Permission Pending)

</center>

The problem with this is that all cells in the body might be exposed to the toxin MeP. In addition to the search and destroy mechanism, scientists hope to increase selectivity by combining the hypoxia-sensing and density-sensing units so that ''E. coli'' will only enter eukaryotic cells that exhibit both characteristics. This would make it more likely that invasin-enhanced ''E. coli'' would only invade cancer cells rather than muscle tissue that had low oxygen due to exercise (Anderson, 2006). To create a fully functional, synthetic biology-based approach to cancer therapy, scientists need to develop bacteria that discriminately invades cancer cells and destroys them. Most of the pieces are there – scientists have engineered ''E. coli'' that selectively invades hypoxic cells or dense cells and ''Salmonella'' that can destroy tumors in vivo – they just need to be put together effectively.

Finally, synthetic biology has made its mark on the field of medicine by creating drugs that are less expensive to produce than those made by pharmaceutical companies. Jay Keasling’s project involving the antimalarial drug artemisinin is the best example. Yeast was engineered to increase normal farnesyl pyrophosphate (FPP) production, convert FPP to amorphadiene, and oxidizing amorphadiene to artemisinic acid, which can then easily be separated from the yeast cells and converted to artemisinin in the laboratory (Fig. 6). This innovation allows the production of artemisinin at lower prices than those currently on the market, and without regard to environmental constraints (Ro, 2006). On a completely different tangent, Collins’s lab engineered T7 bacteriophages to deliver biofilm-degrading enzymes such as dispersin B after infecting and replicating in an ''E. coli'' biofilm. Dispersin B works to degrade an adhesin that is critical for biofilm formation. Phages that released dispersin B were found to be 99.997% effective at removing biofilm, which is 4.5 orders of magnitude higher than non-enzymatic phages (Lu, 2007) (Fig. 7). The specificity of phages for a certain type of bacteria make them a viable vector to be used in human treatments, which may range from removing dental plaques to cleaning inserted medical devices. Using synthetic biology to engineer an antimalarial drug in yeast or a biofilm-destroying enzyme in bacteriophages greatly increases the possible contributions to the field of medicine.

<center>

{|
|-
|[[Image:Artemesinin.JPG]]
|[[Image:BiofilmDegradation.JPG]]
|-
|'''Fig. 6.''' All the steps in the box indicate steps that were optimized in yeast for a maximum artemisinic acid output. (Ro, 2006 - Permission Pending)
|'''Fig. 7.''' The amount of biofilm was decreased when the dispersin B enzymes were incorporated into T7 bacteriophages. (Lu, 2007 - Permission Pending)
|}
</center>

'''Conclusion'''

Medical applications of synthetic biology are wide-ranging and eminently applicable to daily life. Synthetic biology based cancer treatments that utilize ''E.coli'' engineered to sense and attack tissues with high cell density and low oxygen availability are promising and need to be tested in vivo. Designing vectors out of attenuated bacteria may be a safe, effective method for disease treatment. Using biofilms to sense sun damage potential may decrease the likelihood of skin cancer. Finally, using engineered cells to produce medicines in a cost-effective, environmentally-friendly way may revolutionize the pharmaceutical industry. Investing more research efforts in the field of synthetic biology may turn out to be an investment in medical technology.

== Works Cited ==

-[http://www.bio.davidson.edu/courses/synthetic/papers/2006_anderson.pdf Anderson JC, Clarke EJ, Arkin, AP, and Voigt CA (2006). Environmentally controlled invasion of cancer cells by engineered bacteria. Journal of Molecular Biology 355:619-27.]

-[http://www.nature.com/gt/journal/v11/n15/abs/3302281a.html Critchley RJ, Jezzard S, Radford KJ, Goussard S, Lemoine NR, et al. (2004). Potential therapeutic applications of recombinant, invasive ''E. coli''. Gene Therapy 11:1224-33.]

-[http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&uid=15203915&cmd=showdetailview&indexed=google Garmory HS, Leary SEC, Griffin, KF, Williamson D, Brown, KA, and Titball RW (2003). The use of live attenuated bacteria as a delivery system for heterologous antigens. Journal of Drug Targeting 11:471-79.]

-[http://www.pnas.org/cgi/reprint/101/22/8414 Kobayashi H, et al. (2004). Programmable cells: Interfacing natural and engineered gene networks. PNAS 101: 8414-19.]

-[http://www.bio.davidson.edu/courses/synthetic/papers/Phage_BioFilms.pdf Lu, TK, and Collins JJ (2007). Dispersing biofilms with engineered enzymatic bacteriophage. PNAS 104: 11197-11202.]

-[http://www.nature.com/cgt/journal/v10/n10/abs/7700634a.html Nemunaitis J, Cunningham C, Senzer N, Kuhn J, Cramm J, Litz C, et al. (2003). Pilot trial of genetically modified, attenuated ''Salmonella'' expressing cytosine deaminase gene in refractory cancer patients. Cancer Gene Therapy 10:737-744.]

-[http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6W85-49FGKHC-N&_user=10&_coverDate=09%2F30%2F2003&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=d4529b45974bfe7d9ddab1fc865f088b Pawelek JM, Low KB, and Bermudes D (2003). Bacteria as tumour-targeting vectors. Lancet Oncology 4:548-56.]

-[http://www.bio.davidson.edu/courses/synthetic/papers/Keasling_malaria.pdf Ro D, et al. (2006). Production of the antimalarial drug precursor artemisinic acid in engineered yeast. Nature 440:940-43.]

Medical Applications of Synthetic Biology - Samantha Simpson

2007-11-24T15:26:44Z

Sasimpson:

== Project Proposal ==

My project will be on medical applications of synthetic biology. I will reference Anderson's paper on utilizing quorum-sensing and hypoxia-responsive genes coupled with invasin from ''Yersinia tuberculosis'' to invade cancer-causing cells. Coupled with Critchley's work, which describes a bacterial system that invades eukaryotic cells and delivers proteins coded for in the bacteria's genome, one could possibly create 'search and destroy' ''E.coli'' that can locate, invade, and kill tumor cells. Garmory's paper also highlights the possibility of using bacteria as a drug-delivering system, specifically vaccine vectors. Kobayashi describes cells that produce a protective biofilm layer after exposure to DNA-damaging agents. Ro engineers yeast to create an anti-malarial drug that can be created at a lower cost than the previous standard production process. These papers, and others that have a focus on direct medical applications such as novel cancer therapies, vaccination technology, biological protection, and the creation of new medicines, will be referenced to create a cohesive overview of applications of synthetic biology to the medical field.

== Paper ==

Synthetic biology is a rapidly emerging field that strives to re-design existing biological systems and components or fabricate novel biological systems and components. The end result of experimentation in synthetic biology is to help scientists understand a naturally occurring genetic pathway more fully or to create a new, useful pathway. Recent research efforts have focused on engineering bacteria to create a new form of biofuel or to make promoters of varying efficiencies for more exact gene expression. Other research efforts focus on engineering yeast to make antimalarial drugs or engineering ''E. coli'' to recognize and invade tumor-like cells, both of which are medical applications of synthetic biology. Advancements such as novel cancer therapies, vaccination technology, biological protection, and the creation of new medicines highlight synthetic biology’s potential to create breakthroughs in both the prevention and treatment side of medical science. This paper will examine various medical applications of synthetic biology and further development that needs to be done before current research can be used in hospitals and doctors’ offices.

'''Prevention'''

Using bacteria as a vaccine vector has similar issues of specificity and implementation in a human system. Bacteria was first thought to be a good vector because they would not degrade at mucosal surfaces, they would absorb better and survive the low pH in the gastrointestinal tract (Garmory, 2003). A particular strain of ''Salmonella'' with one or more deletions in the shikimate pathway, which creates a precursor for phenylalanine and tyrosine, is a promising vector because it gets into the body easily but does not harmfully effect the tissue and is gone within a week or two of introduction. Scientists engineered this strain to carry the ''Yersinia pestis'' V antigen, and studied its ability to protect mice from ''Y. pestis''. Unfortunately, the inoculation was not extremely successful and only 6 of 20 mice showed a strong response (Garmory, 2003). Garmory and colleagues cited several reasons for this: the copy number of the plasmid containing the V antigen might not have been high enough; the attenuated ''Salmonella'' may not have been able to reach cells that would be targeted by ''Y. pestis''; and the inability of the cell to secrete the antigen. Many changes need to be made to live vector vaccine design before it is an effective method of treating or preventing disease.

Another method of disease prevention with a synthetic biology approach is related to skin cancer. ''E. coli'' engineered to sense single-stranded DNA after DNA damage was coupled with a mechanism to produce a biofilm around the cell. The biofilm output module was tested with DNA damage done by mitomycin C, a DNA crosslinking agent, or UV irradiation, a known cause of skin cancer. Single-stranded DNA activates RecA, which in turn represses the CI repressor protein, thus allowing the transcription of the PL promoter (Kobayashi, 2004). The PL promoter is in charge of the ''traA'' gene, which controls biofilm formation (Fig. 1). The biofilm protection, when part of a toggle switch mechanism, lasted indefinitely when pulsed with UV light at 8 J/m2 for only 2 seconds (Kobayashi, 2004) (Fig. 2). The biofilm-production mechanism has not been engineered in eukaryotic cells, but a prokaryote-based system that could aid with human skin disease is imaginable. For example, a sunscreen that changes colors when the DNA damage may be too intense for a sunbather as a warning, or a sunscreen that becomes more protective as the possibility of genetic damage increased. Further research should focus on how to best implement the biofilm producing output module to protect human DNA.

<center>

{|
|-
|[[Image:Toggleswitch.JPG]]
|[[Image:UVinducedbiofilm.JPG]]
|-
|'''Fig. 1.''' The RecA / traA toggleswitch mechanism. (Kobayashi, 2004 - Permission Pending)
|'''Fig. 2.''' Crystal violet absorbance measures biofilm formation. (Kobayashi, 2004 - Permission Pending)
|}
</center>

'''Treatment'''

Synthetic biologists consider finding a cure for cancer a reachable goal. Previous to the development of synthetic biology as a field, it was known that three types of bacteria, ''Bifodobacterium'', ''Clostridium'', and ''Salmonella'' all preferentially infect the dense cells of tumors (Pawelek, 2003). All three types of bacteria are associated with reducing tumor size in patients who are infected with them, and it was noted that all three bacteria act anaerobically. Capitalizing on the idea that tumors create an unusually dense, anaerobic mass of cells, Christopher Voigt’s lab engineered bacteria to identify and invade cancer cells. They utilized the ''lux'' quorum sensing from ''Vibrio fischeri'' to identify cells growing at high densities (Fig. 3) and the ''fdhF'' promoter to identify cells growing in anaerobic conditions (Fig. 4) (Anderson, 2006). They coupled this identification mechanism with the invasin output module from ''Yersinia pseudotuberculosis'' so the ''E. coli'' could invade specific cells.

<center>

{|
|-
|[[Image:InvasinDensity.JPG]]
|[[Image:InvasinAnaerobic2.JPG]]
|-
|'''Fig. 3.''' The red line represents when the invasin output module was paired with the inducible promoter ''lux'' which senses high cell density, the black line is constitutively on invasin. (Anderson, 2006 - Permission Pending)
|'''Fig. 4.''' The invasin output module was paired with the inducible promoter ''fdhF'' which senses anaerobic conditions. (Anderson, 2006 - Permission Pending)
|}

</center>

Next, scientists hope to insert a gene that would destroy the invaded cells, making the complete cycle a search, invade, and destroy loop that would only be sensitive to cancer cells. One promising destroy mechanism has been implemented in humans via intratumoral injection, based on ''Salmonella'' that contains the ''E. coli'' cytosine deaminase gene that converts 5-fluorocytosine to 5-fluorouracil, a chemotherapy drug (Nemunaitis, 2003). Unfortunately, while the tumors did produce 5-fluorouracil (5-FU), they did not produce it in high enough yields to cause regression of the cancer. Another experiment flooded mouse cells with 6-methylpurine-2’deoxyribose (6-MPDR), which was converted to the toxin 6-methyl purine (MeP) by the enzyme purine nucleoside phosphorylase (PNP), which is naturally found in ''E. coli'' (Critchley, 2004) (Fig. 5).

<center>

[[Image:Tumor.JPG]]

'''Fig. 5.''' Tumor size after injections. Cancer cells were injected into mice, and 5 days later injections with PBS, 6-MPDR, and invasin-enhanced ''E-coli'' began. Tumor cells recieving injections of invasin-enhanced ''E.coli'' and 6-MPDR grew the slowest, but did not get smaller. (Critchley, 2004 - Permission Pending)

</center>

The problem with this is that all cells in the body might be exposed to the toxin MeP. In addition to the search and destroy mechanism, scientists hope to increase selectivity by combining the hypoxia-sensing and density-sensing units so that ''E. coli'' will only enter eukaryotic cells that exhibit both characteristics. This would make it more likely that invasin-enhanced ''E. coli'' would only invade cancer cells rather than muscle tissue that had low oxygen due to exercise (Anderson, 2006). To create a fully functional, synthetic biology-based approach to cancer therapy, scientists need to develop bacteria that discriminately invades cancer cells and destroys them. Most of the pieces are there – scientists have engineered ''E. coli'' that selectively invades hypoxic cells or dense cells and ''Salmonella'' that can destroy tumors in vivo – they just need to be put together effectively.

Finally, synthetic biology has made its mark on the field of medicine by creating drugs that are less expensive to produce than those made by pharmaceutical companies. Jay Keasling’s project involving the antimalarial drug artemisinin is the best example. Yeast was engineered to increase normal farnesyl pyrophosphate (FPP) production, convert FPP to amorphadiene, and oxidizing amorphadiene to artemisinic acid, which can then easily be separated from the yeast cells and converted to artemisinin in the laboratory (Fig. 6). This innovation allows the production of artemisinin at lower prices than those currently on the market, and without regard to environmental constraints (Ro, 2006). On a completely different tangent, Collins’s lab engineered T7 bacteriophages to deliver biofilm-degrading enzymes such as dispersin B after infecting and replicating in an ''E. coli'' biofilm. Dispersin B works to degrade an adhesin that is critical for biofilm formation. Phages that released dispersin B were found to be 99.997% effective at removing biofilm, which is 4.5 orders of magnitude higher than non-enzymatic phages (Lu, 2007) (Fig. 7). The specificity of phages for a certain type of bacteria make them a viable vector to be used in human treatments, which may range from removing dental plaques to cleaning inserted medical devices. Using synthetic biology to engineer an antimalarial drug in yeast or a biofilm-destroying enzyme in bacteriophages greatly increases the possible contributions to the field of medicine.

<center>

{|
|-
|[[Image:Artemesinin.JPG]]
|[[Image:BiofilmDegradation.JPG]]
|-
|'''Fig. 6.''' All the steps in the box indicate steps that were optimized in yeast for a maximum artemisinic acid output. (Ro, 2006 - Permission Pending)
|'''Fig. 7.''' The amount of biofilm was decreased when the dispersin B enzymes were incorporated into T7 bacteriophages. (Lu, 2007 - Permission Pending)
|}
</center>

'''Conclusion'''

Medical applications of synthetic biology are wide-ranging and eminently applicable to daily life. Synthetic biology based cancer treatments that utilize ''E.coli'' engineered to sense and attack tissues with high cell density and low oxygen availability are promising and need to be tested in vivo. Designing vectors out of attenuated bacteria may be a safe, effective method for disease treatment. Using biofilms to sense sun damage potential may decrease the likelihood of skin cancer. Finally, using engineered cells to produce medicines in a cost-effective, environmentally-friendly way may revolutionize the pharmaceutical industry. Investing more research efforts in the field of synthetic biology may turn out to be an investment in medical technology.

== Works Cited ==

-[http://www.bio.davidson.edu/courses/synthetic/papers/2006_anderson.pdf Anderson JC, Clarke EJ, Arkin, AP, and Voigt CA (2006). Environmentally controlled invasion of cancer cells by engineered bacteria. Journal of Molecular Biology 355:619-27.]

-[http://www.nature.com/gt/journal/v11/n15/abs/3302281a.html Critchley RJ, Jezzard S, Radford KJ, Goussard S, Lemoine NR, et al. (2004). Potential therapeutic applications of recombinant, invasive ''E. coli''. Gene Therapy 11:1224-33.]

-[http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&uid=15203915&cmd=showdetailview&indexed=google Garmory HS, Leary SEC, Griffin, KF, Williamson D, Brown, KA, and Titball RW (2003). The use of live attenuated bacteria as a delivery system for heterologous antigens. Journal of Drug Targeting 11:471-79.]

-[http://www.pnas.org/cgi/reprint/101/22/8414 Kobayashi H, et al. (2004). Programmable cells: Interfacing natural and engineered gene networks. PNAS 101: 8414-19.]

-[http://www.bio.davidson.edu/courses/synthetic/papers/Phage_BioFilms.pdf Lu, TK, and Collins JJ (2007). Dispersing biofilms with engineered enzymatic bacteriophage. PNAS 104: 11197-11202.]

-[http://www.nature.com/cgt/journal/v10/n10/abs/7700634a.html Nemunaitis J, Cunningham C, Senzer N, Kuhn J, Cramm J, Litz C, et al. (2003). Pilot trial of genetically modified, attenuated ''Salmonella'' expressing cytosine deaminase gene in refractory cancer patients. Cancer Gene Therapy 10:737-744.]

-[http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6W85-49FGKHC-N&_user=10&_coverDate=09%2F30%2F2003&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=d4529b45974bfe7d9ddab1fc865f088b Pawelek JM, Low KB, and Bermudes D (2003). Bacteria as tumour-targeting vectors. Lancet Oncology 4:548-56.]

-[http://www.bio.davidson.edu/courses/synthetic/papers/Keasling_malaria.pdf Ro D, et al. (2006). Production of the antimalarial drug precursor artemisinic acid in engineered yeast. Nature 440:940-43.]

Medical Applications of Synthetic Biology - Samantha Simpson

2007-11-24T15:21:05Z

Sasimpson:

'''Project Proposal'''

My project will be on medical applications of synthetic biology. I will reference Anderson's paper on utilizing quorum-sensing and hypoxia-responsive genes coupled with invasin from ''Yersinia tuberculosis'' to invade cancer-causing cells. Coupled with Critchley's work, which describes a bacterial system that invades eukaryotic cells and delivers proteins coded for in the bacteria's genome, one could possibly create 'search and destroy' ''E.coli'' that can locate, invade, and kill tumor cells. Garmory's paper also highlights the possibility of using bacteria as a drug-delivering system, specifically vaccine vectors. Kobayashi describes cells that produce a protective biofilm layer after exposure to DNA-damaging agents. Ro engineers yeast to create an anti-malarial drug that can be created at a lower cost than the previous standard production process. These papers, and others that have a focus on direct medical applications such as novel cancer therapies, vaccination technology, biological protection, and the creation of new medicines, will be referenced to create a cohesive overview of applications of synthetic biology to the medical field.

'''Paper'''

Synthetic biology is a rapidly emerging field that strives to re-design existing biological systems and components or fabricate novel biological systems and components. The end result of experimentation in synthetic biology is to help scientists understand a naturally occurring genetic pathway more fully or to create a new, useful pathway. Recent research efforts have focused on engineering bacteria to create a new form of biofuel or to make promoters of varying efficiencies for more exact gene expression. Other research efforts focus on engineering yeast to make antimalarial drugs or engineering ''E. coli'' to recognize and invade tumor-like cells, both of which are medical applications of synthetic biology. Advancements such as novel cancer therapies, vaccination technology, biological protection, and the creation of new medicines highlight synthetic biology’s potential to create breakthroughs in medical science. This paper will examine various medical applications of synthetic biology and further development that needs to be done before current research can be used in hospitals and doctors’ offices.

Synthetic biologists consider finding a cure for cancer a reachable goal. Previous to the development of synthetic biology as a field, it was known that three types of bacteria, ''Bifodobacterium'', ''Clostridium'', and ''Salmonella'' all preferentially infect the dense cells of tumors (Pawelek, 2003). All three types of bacteria are associated with reducing tumor size in patients who are infected with them, and it was noted that all three bacteria act anaerobically. Capitalizing on the idea that tumors create an unusually dense, anaerobic mass of cells, Christopher Voigt’s lab engineered bacteria to identify and invade cancer cells. They utilized the ''lux'' quorum sensing from ''Vibrio fischeri'' to identify cells growing at high densities (Fig. 1) and the ''fdhF'' promoter to identify cells growing in anaerobic conditions (Fig. 2) (Anderson, 2006). They coupled this identification mechanism with the invasin output module from ''Yersinia pseudotuberculosis'' so the ''E. coli'' could invade specific cells.

<center>

{|
|-
|[[Image:InvasinDensity.JPG]]
|[[Image:InvasinAnaerobic2.JPG]]
|-
|'''Fig. 1.''' The red line represents when the invasin output module was paired with the inducible promoter ''lux'' which senses high cell density, the black line is constitutively on invasin. (Anderson, 2006 - Permission Pending)
|'''Fig. 2.''' The invasin output module was paired with the inducible promoter ''fdhF'' which senses anaerobic conditions. (Anderson, 2006 - Permission Pending)
|}

</center>

Next, scientists hope to insert a gene that would destroy the invaded cells, making the complete cycle a search, invade, and destroy loop that would only be sensitive to cancer cells. One promising destroy mechanism has been implemented in humans via intratumoral injection, based on ''Salmonella'' that contains the ''E. coli'' cytosine deaminase gene that converts 5-fluorocytosine to 5-fluorouracil, a chemotherapy drug (Nemunaitis, 2003). Unfortunately, while the tumors did produce 5-fluorouracil (5-FU), they did not produce it in high enough yields to cause regression of the cancer. Another experiment flooded mouse cells with 6-methylpurine-2’deoxyribose (6-MPDR), which was converted to the toxin 6-methyl purine (MeP) by the enzyme purine nucleoside phosphorylase (PNP), which is naturally found in ''E. coli'' (Critchley, 2004) (Fig. 3).

<center>

[[Image:Tumor.JPG]]

'''Fig. 3.''' Tumor size after injections. Cancer cells were injected into mice, and 5 days later injections with PBS, 6-MPDR, and invasin-enhanced ''E-coli'' began. Tumor cells recieving injections of invasin-enhanced ''E.coli'' and 6-MPDR grew the slowest, but did not get smaller. (Critchley, 2004 - Permission Pending)

</center>

The problem with this is that all cells in the body might be exposed to the toxin MeP. In addition to the search and destroy mechanism, scientists hope to increase selectivity by combining the hypoxia-sensing and density-sensing units so that ''E. coli'' will only enter eukaryotic cells that exhibit both characteristics. This would make it more likely that invasin-enhanced ''E. coli'' would only invade cancer cells rather than muscle tissue that had low oxygen due to exercise (Anderson, 2006). To create a fully functional, synthetic biology-based approach to cancer therapy, scientists need to develop bacteria that discriminately invades cancer cells and destroys them. Most of the pieces are there – scientists have engineered ''E. coli'' that selectively invades hypoxic cells or dense cells and ''Salmonella'' that can destroy tumors in vivo – they just need to be put together effectively.

Using bacteria as a vaccine vector has similar issues of specificity and implementation in a human system. Bacteria was first thought to be a good vector because they would not degrade at mucosal surfaces, they would absorb better and survive the low pH in the gastrointestinal tract (Garmory, 2003). A particular strain of ''Salmonella'' with one or more deletions in the shikimate pathway, which creates a precursor for phenylalanine and tyrosine, is a promising vector because it gets into the body easily but does not harmfully effect the tissue and is gone within a week or two of introduction. Scientists engineered this strain to carry the ''Yersinia pestis'' V antigen, and studied its ability to protect mice from ''Y. pestis''. Unfortunately, the inoculation was not extremely successful and only 6 of 20 mice showed a strong response (Garmory, 2003). Garmory and colleagues cited several reasons for this: the copy number of the plasmid containing the V antigen might not have been high enough; the attenuated ''Salmonella'' may not have been able to reach cells that would be targeted by ''Y. pestis''; and the inability of the cell to secrete the antigen. Many changes need to be made to live vector vaccine design before it is an effective method of treating or preventing disease.

Another method of disease prevention with a synthetic biology approach is related to skin cancer. ''E. coli'' engineered to sense single-stranded DNA after DNA damage was coupled with a mechanism to produce a biofilm around the cell. The biofilm output module was tested with DNA damage done by mitomycin C, a DNA crosslinking agent, or UV irradiation, a known cause of skin cancer. Single-stranded DNA activates RecA, which in turn represses the CI repressor protein, thus allowing the transcription of the PL promoter (Kobayashi, 2004). The PL promoter is in charge of the ''traA'' gene, which controls biofilm formation (Fig. 4). The biofilm protection, when part of a toggle switch mechanism, lasted indefinitely when pulsed with UV light at 8 J/m2 for only 2 seconds (Kobayashi, 2004) (Fig. 5). The biofilm-production mechanism has not been engineered in eukaryotic cells, but a prokaryote-based system that could aid with human skin disease is imaginable. For example, a sunscreen that changes colors when the DNA damage may be too intense for a sunbather as a warning, or a sunscreen that becomes more protective as the possibility of genetic damage increased. Further research should focus on how to best implement the biofilm producing output module to protect human DNA.

<center>

{|
|-
|[[Image:Toggleswitch.JPG]]
|[[Image:UVinducedbiofilm.JPG]]
|-
|'''Fig. 4.''' The RecA / traA toggleswitch mechanism. (Kobayashi, 2004 - Permission Pending)
|'''Fig. 5.''' Crystal violet absorbance measures biofilm formation. (Kobayashi, 2004 - Permission Pending)
|}
</center>

Finally, synthetic biology has made its mark on the field of medicine by creating drugs that are less expensive to produce than those made by pharmaceutical companies. Jay Keasling’s project involving the antimalarial drug artemisinin is the best example. Yeast was engineered to increase normal farnesyl pyrophosphate (FPP) production, convert FPP to amorphadiene, and oxidizing amorphadiene to artemisinic acid, which can then easily be separated from the yeast cells and converted to artemisinin in the laboratory (Fig. 6). This innovation allows the production of artemisinin at lower prices than those currently on the market, and without regard to environmental constraints (Ro, 2006). On a completely different tangent, Collins’s lab engineered T7 bacteriophages to deliver biofilm-degrading enzymes such as dispersin B after infecting and replicating in an ''E. coli'' biofilm. Dispersin B works to degrade an adhesin that is critical for biofilm formation. Phages that released dispersin B were found to be 99.997% effective at removing biofilm, which is 4.5 orders of magnitude higher than non-enzymatic phages (Lu, 2007) (Fig. 7). The specificity of phages for a certain type of bacteria make them a viable vector to be used in human treatments, which may range from removing dental plaques to cleaning inserted medical devices. Using synthetic biology to engineer an antimalarial drug in yeast or a biofilm-destroying enzyme in bacteriophages greatly increases the possible contributions to the field of medicine.

<center>

{|
|-
|[[Image:Artemesinin.JPG]]
|[[Image:BiofilmDegradation.JPG]]
|-
|'''Fig. 6.''' All the steps in the box indicate steps that were optimized in yeast for a maximum artemisinic acid output. (Ro, 2006 - Permission Pending)
|'''Fig. 7.''' The amount of biofilm was decreased when the dispersin B enzymes were incorporated into T7 bacteriophages. (Lu, 2007 - Permission Pending)
|}
</center>

Medical applications of synthetic biology are wide-ranging and eminently applicable to daily life. Synthetic biology based cancer treatments that utilize ''E.coli'' engineered to sense and attack tissues with high cell density and low oxygen availability are promising and need to be tested in vivo. Designing vectors out of attenuated bacteria may be a safe, effective method for disease treatment. Using biofilms to sense sun damage potential may decrease the likelihood of skin cancer. Finally, using engineered cells to produce medicines in a cost-effective, environmentally-friendly way may revolutionize the pharmaceutical industry. Investing more research efforts in the field of synthetic biology may turn out to be an investment in medical technology.

'''Works Cited'''

-[http://www.bio.davidson.edu/courses/synthetic/papers/2006_anderson.pdf Anderson JC, Clarke EJ, Arkin, AP, and Voigt CA (2006). Environmentally controlled invasion of cancer cells by engineered bacteria. Journal of Molecular Biology 355:619-27.]

-[http://www.nature.com/gt/journal/v11/n15/abs/3302281a.html Critchley RJ, Jezzard S, Radford KJ, Goussard S, Lemoine NR, et al. (2004). Potential therapeutic applications of recombinant, invasive ''E. coli''. Gene Therapy 11:1224-33.]

-[http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&uid=15203915&cmd=showdetailview&indexed=google Garmory HS, Leary SEC, Griffin, KF, Williamson D, Brown, KA, and Titball RW (2003). The use of live attenuated bacteria as a delivery system for heterologous antigens. Journal of Drug Targeting 11:471-79.]

-[http://www.pnas.org/cgi/reprint/101/22/8414 Kobayashi H, et al. (2004). Programmable cells: Interfacing natural and engineered gene networks. PNAS 101: 8414-19.]

-[http://www.bio.davidson.edu/courses/synthetic/papers/Phage_BioFilms.pdf Lu, TK, and Collins JJ (2007). Dispersing biofilms with engineered enzymatic bacteriophage. PNAS 104: 11197-11202.]

-[http://www.nature.com/cgt/journal/v10/n10/abs/7700634a.html Nemunaitis J, Cunningham C, Senzer N, Kuhn J, Cramm J, Litz C, et al. (2003). Pilot trial of genetically modified, attenuated ''Salmonella'' expressing cytosine deaminase gene in refractory cancer patients. Cancer Gene Therapy 10:737-744.]

-[http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6W85-49FGKHC-N&_user=10&_coverDate=09%2F30%2F2003&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=d4529b45974bfe7d9ddab1fc865f088b Pawelek JM, Low KB, and Bermudes D (2003). Bacteria as tumour-targeting vectors. Lancet Oncology 4:548-56.]

-[http://www.bio.davidson.edu/courses/synthetic/papers/Keasling_malaria.pdf Ro D, et al. (2006). Production of the antimalarial drug precursor artemisinic acid in engineered yeast. Nature 440:940-43.]

Medical Applications of Synthetic Biology - Samantha Simpson

2007-11-24T15:20:04Z

Sasimpson:

'''Project Proposal'''

My project will be on medical applications of synthetic biology. I will reference Anderson's paper on utilizing quorum-sensing and hypoxia-responsive genes coupled with invasin from ''Yersinia tuberculosis'' to invade cancer-causing cells. Coupled with Critchley's work, which describes a bacterial system that invades eukaryotic cells and delivers proteins coded for in the bacteria's genome, one could possibly create 'search and destroy' ''E.coli'' that can locate, invade, and kill tumor cells. Garmory's paper also highlights the possibility of using bacteria as a drug-delivering system, specifically vaccine vectors. Kobayashi describes cells that produce a protective biofilm layer after exposure to DNA-damaging agents. Ro engineers yeast to create an anti-malarial drug that can be created at a lower cost than the previous standard production process. These papers, and others that have a focus on direct medical applications such as novel cancer therapies, vaccination technology, biological protection, and the creation of new medicines, will be referenced to create a cohesive overview of applications of synthetic biology to the medical field.

'''Paper'''

Synthetic biology is a rapidly emerging field that strives to re-design existing biological systems and components or fabricate novel biological systems and components. The end result of experimentation in synthetic biology is to help scientists understand a naturally occurring genetic pathway more fully or to create a new, useful pathway. Recent research efforts have focused on engineering bacteria to create a new form of biofuel or to make promoters of varying efficiencies for more exact gene expression. Other research efforts focus on engineering yeast to make antimalarial drugs or engineering ''E. coli'' to recognize and invade tumor-like cells, both of which are medical applications of synthetic biology. Advancements such as novel cancer therapies, vaccination technology, biological protection, and the creation of new medicines highlight synthetic biology’s potential to create breakthroughs in medical science. This paper will examine various medical applications of synthetic biology and further development that needs to be done before current research can be used in hospitals and doctors’ offices.

Synthetic biologists consider finding a cure for cancer a reachable goal. Previous to the development of synthetic biology as a field, it was known that three types of bacteria, ''Bifodobacterium'', ''Clostridium'', and ''Salmonella'' all preferentially infect the dense cells of tumors (Pawelek, 2003). All three types of bacteria are associated with reducing tumor size in patients who are infected with them, and it was noted that all three bacteria act anaerobically. Capitalizing on the idea that tumors create an unusually dense, anaerobic mass of cells, Christopher Voigt’s lab engineered bacteria to identify and invade cancer cells. They utilized the ''lux'' quorum sensing from ''Vibrio fischeri'' to identify cells growing at high densities (Fig. 1) and the ''fdhF'' promoter to identify cells growing in anaerobic conditions (Fig. 2) (Anderson, 2006). They coupled this identification mechanism with the invasin output module from ''Yersinia pseudotuberculosis'' so the ''E. coli'' could invade specific cells.

<center>

{|
|-
|[[Image:InvasinDensity.JPG]]
|[[Image:InvasinAnaerobic2.JPG]]
|-
|'''Fig. 1.''' The red line represents when the invasin output module was paired with the inducible promoter ''lux'' which senses high cell density, the black line is constitutively on invasin. (Anderson, 2006 - Permission Pending)
|'''Fig. 2.''' The invasin output module was paired with the inducible promoter ''fdhF'' which senses anaerobic conditions. (Anderson, 2006 - Permission Pending)
|}

</center>

Next, scientists hope to insert a gene that would destroy the invaded cells, making the complete cycle a search, invade, and destroy loop that would only be sensitive to cancer cells. One promising destroy mechanism has been implemented in humans via intratumoral injection, based on ''Salmonella'' that contains the ''E. coli'' cytosine deaminase gene that converts 5-fluorocytosine to 5-fluorouracil, a chemotherapy drug (Nemunaitis, 2003). Unfortunately, while the tumors did produce 5-fluorouracil (5-FU), they did not produce it in high enough yields to cause regression of the cancer. Another experiment flooded mouse cells with 6-methylpurine-2’deoxyribose (6-MPDR), which was converted to the toxin 6-methyl purine (MeP) by the enzyme purine nucleoside phosphorylase (PNP), which is naturally found in ''E. coli'' (Critchley, 2004) (Fig. 3).

<center>

[[Image:Tumor.JPG]]

'''Fig. 3.''' Tumor size after injections. Cancer cells were injected into mice, and 5 days later injections with PBS, 6-MPDR, and invasin-enhanced ''E-coli'' began. Tumor cells recieving injections of invasin-enhanced ''E.coli'' and 6-MPDR grew the slowest, but did not get smaller. (Critchley, 2004 - Permission Pending)

</center>

The problem with this is that all cells in the body might be exposed to the toxin MeP. In addition to the search and destroy mechanism, scientists hope to increase selectivity by combining the hypoxia-sensing and density-sensing units so that ''E. coli'' will only enter eukaryotic cells that exhibit both characteristics. This would make it more likely that invasin-enhanced ''E. coli'' would only invade cancer cells rather than muscle tissue that had low oxygen due to exercise (Anderson, 2006). To create a fully functional, synthetic biology-based approach to cancer therapy, scientists need to develop bacteria that discriminately invades cancer cells and destroys them. Most of the pieces are there – scientists have engineered ''E. coli'' that selectively invades hypoxic cells or dense cells and ''Salmonella'' that can destroy tumors in vivo – they just need to be put together effectively.

Using bacteria as a vaccine vector has similar issues of specificity and implementation in a human system. Bacteria was first thought to be a good vector because they would not degrade at mucosal surfaces, they would absorb better and survive the low pH in the gastrointestinal tract (Garmory, 2003). A particular strain of ''Salmonella'' with one or more deletions in the shikimate pathway, which creates a precursor for phenylalanine and tyrosine, is a promising vector because it gets into the body easily but does not harmfully effect the tissue and is gone within a week or two of introduction. Scientists engineered this strain to carry the ''Yersinia pestis'' V antigen, and studied its ability to protect mice from ''Y. pestis''. Unfortunately, the inoculation was not extremely successful and only 6 of 20 mice showed a strong response (Garmory, 2003). Garmory and colleagues cited several reasons for this: the copy number of the plasmid containing the V antigen might not have been high enough; the attenuated ''Salmonella'' may not have been able to reach cells that would be targeted by ''Y. pestis''; and the inability of the cell to secrete the antigen. Many changes need to be made to live vector vaccine design before it is an effective method of treating or preventing disease.

Another method of disease prevention with a synthetic biology approach is related to skin cancer. ''E. coli'' engineered to sense single-stranded DNA after DNA damage was coupled with a mechanism to produce a biofilm around the cell. The biofilm output module was tested with DNA damage done by mitomycin C, a DNA crosslinking agent, or UV irradiation, a known cause of skin cancer. Single-stranded DNA activates RecA, which in turn represses the CI repressor protein, thus allowing the transcription of the PL promoter (Kobayashi, 2004). The PL promoter is in charge of the ''traA'' gene, which controls biofilm formation (Fig. 4). The biofilm protection, when part of a toggle switch mechanism, lasted indefinitely when pulsed with UV light at 8 J/m2 for only 2 seconds (Kobayashi, 2004) (Fig. 5). The biofilm-production mechanism has not been engineered in eukaryotic cells, but a prokaryote-based system that could aid with human skin disease is imaginable. For example, a sunscreen that changes colors when the DNA damage may be too intense for a sunbather as a warning, or a sunscreen that becomes more protective as the possibility of genetic damage increased. Further research should focus on how to best implement the biofilm producing output module to protect human DNA.
<center>

{|
|-
|[[Image:Toggleswitch.JPG]]
|[[Image:UVinducedbiofilm.JPG]]
|-
|'''Fig. 4.''' The RecA / traA toggleswitch mechanism. (Kobayashi, 2004 - Permission Pending)
|'''Fig. 5.''' Crystal violet absorbance measures biofilm formation. (Kobayashi, 2004 - Permission Pending)
|}
</center>
Finally, synthetic biology has made its mark on the field of medicine by creating drugs that are less expensive to produce than those made by pharmaceutical companies. Jay Keasling’s project involving the antimalarial drug artemisinin is the best example. Yeast was engineered to increase normal farnesyl pyrophosphate (FPP) production, convert FPP to amorphadiene, and oxidizing amorphadiene to artemisinic acid, which can then easily be separated from the yeast cells and converted to artemisinin in the laboratory (Fig. 6). This innovation allows the production of artemisinin at lower prices than those currently on the market, and without regard to environmental constraints (Ro, 2006). On a completely different tangent, Collins’s lab engineered T7 bacteriophages to deliver biofilm-degrading enzymes such as dispersin B after infecting and replicating in an ''E. coli'' biofilm. Dispersin B works to degrade an adhesin that is critical for biofilm formation. Phages that released dispersin B were found to be 99.997% effective at removing biofilm, which is 4.5 orders of magnitude higher than non-enzymatic phages (Lu, 2007) (Fig. 7). The specificity of phages for a certain type of bacteria make them a viable vector to be used in human treatments, which may range from removing dental plaques to cleaning inserted medical devices. Using synthetic biology to engineer an antimalarial drug in yeast or a biofilm-destroying enzyme in bacteriophages greatly increases the possible contributions to the field of medicine.

<center>

{|
|-
|[[Image:Artemesinin.JPG]]
|[[Image:BiofilmDegradation.JPG]]
|-
|'''Fig. 6.''' All the steps in the box indicate steps that were optimized in yeast for a maximum artemisinic acid output. (Ro, 2006 - Permission Pending)
|'''Fig. 7.''' The amount of biofilm was decreased when the dispersin B enzymes were incorporated into T7 bacteriophages. (Lu, 2007 - Permission Pending)
|}
</center>

Medical applications of synthetic biology are wide-ranging and eminently applicable to daily life. Synthetic biology based cancer treatments that utilize ''E.coli'' engineered to sense and attack tissues with high cell density and low oxygen availability are promising and need to be tested in vivo. Designing vectors out of attenuated bacteria may be a safe, effective method for disease treatment. Using biofilms to sense sun damage potential may decrease the likelihood of skin cancer. Finally, using engineered cells to produce medicines in a cost-effective, environmentally-friendly way may revolutionize the pharmaceutical industry. Investing more research efforts in the field of synthetic biology may turn out to be an investment in medical technology.

'''Works Cited'''

-[http://www.bio.davidson.edu/courses/synthetic/papers/2006_anderson.pdf Anderson JC, Clarke EJ, Arkin, AP, and Voigt CA (2006). Environmentally controlled invasion of cancer cells by engineered bacteria. Journal of Molecular Biology 355:619-27.]

-[http://www.nature.com/gt/journal/v11/n15/abs/3302281a.html Critchley RJ, Jezzard S, Radford KJ, Goussard S, Lemoine NR, et al. (2004). Potential therapeutic applications of recombinant, invasive ''E. coli''. Gene Therapy 11:1224-33.]

-[http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&uid=15203915&cmd=showdetailview&indexed=google Garmory HS, Leary SEC, Griffin, KF, Williamson D, Brown, KA, and Titball RW (2003). The use of live attenuated bacteria as a delivery system for heterologous antigens. Journal of Drug Targeting 11:471-79.]

-[http://www.pnas.org/cgi/reprint/101/22/8414 Kobayashi H, et al. (2004). Programmable cells: Interfacing natural and engineered gene networks. PNAS 101: 8414-19.]

-[http://www.bio.davidson.edu/courses/synthetic/papers/Phage_BioFilms.pdf Lu, TK, and Collins JJ (2007). Dispersing biofilms with engineered enzymatic bacteriophage. PNAS 104: 11197-11202.]

-[http://www.nature.com/cgt/journal/v10/n10/abs/7700634a.html Nemunaitis J, Cunningham C, Senzer N, Kuhn J, Cramm J, Litz C, et al. (2003). Pilot trial of genetically modified, attenuated ''Salmonella'' expressing cytosine deaminase gene in refractory cancer patients. Cancer Gene Therapy 10:737-744.]

-[http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6W85-49FGKHC-N&_user=10&_coverDate=09%2F30%2F2003&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=d4529b45974bfe7d9ddab1fc865f088b Pawelek JM, Low KB, and Bermudes D (2003). Bacteria as tumour-targeting vectors. Lancet Oncology 4:548-56.]

-[http://www.bio.davidson.edu/courses/synthetic/papers/Keasling_malaria.pdf Ro D, et al. (2006). Production of the antimalarial drug precursor artemisinic acid in engineered yeast. Nature 440:940-43.]

Medical Applications of Synthetic Biology - Samantha Simpson

2007-11-24T15:19:16Z

Sasimpson:

'''Project Proposal'''

My project will be on medical applications of synthetic biology. I will reference Anderson's paper on utilizing quorum-sensing and hypoxia-responsive genes coupled with invasin from ''Yersinia tuberculosis'' to invade cancer-causing cells. Coupled with Critchley's work, which describes a bacterial system that invades eukaryotic cells and delivers proteins coded for in the bacteria's genome, one could possibly create 'search and destroy' ''E.coli'' that can locate, invade, and kill tumor cells. Garmory's paper also highlights the possibility of using bacteria as a drug-delivering system, specifically vaccine vectors. Kobayashi describes cells that produce a protective biofilm layer after exposure to DNA-damaging agents. Ro engineers yeast to create an anti-malarial drug that can be created at a lower cost than the previous standard production process. These papers, and others that have a focus on direct medical applications such as novel cancer therapies, vaccination technology, biological protection, and the creation of new medicines, will be referenced to create a cohesive overview of applications of synthetic biology to the medical field.

'''Paper'''

Synthetic biology is a rapidly emerging field that strives to re-design existing biological systems and components or fabricate novel biological systems and components. The end result of experimentation in synthetic biology is to help scientists understand a naturally occurring genetic pathway more fully or to create a new, useful pathway. Recent research efforts have focused on engineering bacteria to create a new form of biofuel or to make promoters of varying efficiencies for more exact gene expression. Other research efforts focus on engineering yeast to make antimalarial drugs or engineering ''E. coli'' to recognize and invade tumor-like cells, both of which are medical applications of synthetic biology. Advancements such as novel cancer therapies, vaccination technology, biological protection, and the creation of new medicines highlight synthetic biology’s potential to create breakthroughs in medical science. This paper will examine various medical applications of synthetic biology and further development that needs to be done before current research can be used in hospitals and doctors’ offices.

Synthetic biologists consider finding a cure for cancer a reachable goal. Previous to the development of synthetic biology as a field, it was known that three types of bacteria, ''Bifodobacterium'', ''Clostridium'', and ''Salmonella'' all preferentially infect the dense cells of tumors (Pawelek, 2003). All three types of bacteria are associated with reducing tumor size in patients who are infected with them, and it was noted that all three bacteria act anaerobically. Capitalizing on the idea that tumors create an unusually dense, anaerobic mass of cells, Christopher Voigt’s lab engineered bacteria to identify and invade cancer cells. They utilized the ''lux'' quorum sensing from ''Vibrio fischeri'' to identify cells growing at high densities (Fig. 1) and the ''fdhF'' promoter to identify cells growing in anaerobic conditions (Fig. 2) (Anderson, 2006). They coupled this identification mechanism with the invasin output module from ''Yersinia pseudotuberculosis'' so the ''E. coli'' could invade specific cells.

<center>

{|
|-
|[[Image:InvasinDensity.JPG]]
|[[Image:InvasinAnaerobic2.JPG]]
|-
|'''Fig. 1.''' The red line represents when the invasin output module was paired with the inducible promoter ''lux'' which senses high cell density, the black line is constitutively on invasin. (Anderson, 2006 - Permission Pending)
|'''Fig. 2.''' The invasin output module was paired with the inducible promoter ''fdhF'' which senses anaerobic conditions. (Anderson, 2006 - Permission Pending)
|}

</center>

Next, scientists hope to insert a gene that would destroy the invaded cells, making the complete cycle a search, invade, and destroy loop that would only be sensitive to cancer cells. One promising destroy mechanism has been implemented in humans via intratumoral injection, based on ''Salmonella'' that contains the ''E. coli'' cytosine deaminase gene that converts 5-fluorocytosine to 5-fluorouracil, a chemotherapy drug (Nemunaitis, 2003). Unfortunately, while the tumors did produce 5-fluorouracil (5-FU), they did not produce it in high enough yields to cause regression of the cancer. Another experiment flooded mouse cells with 6-methylpurine-2’deoxyribose (6-MPDR), which was converted to the toxin 6-methyl purine (MeP) by the enzyme purine nucleoside phosphorylase (PNP), which is naturally found in ''E. coli'' (Critchley, 2004) (Fig. 3).

<center>

[[Image:Tumor.JPG]]
'''Fig. 3.''' Tumor size after injections. Cancer cells were injected into mice, and 5 days later injections with PBS, 6-MPDR, and invasin-enhanced ''E-coli'' began. Tumor cells recieving injections of invasin-enhanced ''E.coli'' and 6-MPDR grew the slowest, but did not get smaller. (Critchley, 2004 - Permission Pending)

</center>

The problem with this is that all cells in the body might be exposed to the toxin MeP. In addition to the search and destroy mechanism, scientists hope to increase selectivity by combining the hypoxia-sensing and density-sensing units so that ''E. coli'' will only enter eukaryotic cells that exhibit both characteristics. This would make it more likely that invasin-enhanced ''E. coli'' would only invade cancer cells rather than muscle tissue that had low oxygen due to exercise (Anderson, 2006). To create a fully functional, synthetic biology-based approach to cancer therapy, scientists need to develop bacteria that discriminately invades cancer cells and destroys them. Most of the pieces are there – scientists have engineered ''E. coli'' that selectively invades hypoxic cells or dense cells and ''Salmonella'' that can destroy tumors in vivo – they just need to be put together effectively.

Using bacteria as a vaccine vector has similar issues of specificity and implementation in a human system. Bacteria was first thought to be a good vector because they would not degrade at mucosal surfaces, they would absorb better and survive the low pH in the gastrointestinal tract (Garmory, 2003). A particular strain of ''Salmonella'' with one or more deletions in the shikimate pathway, which creates a precursor for phenylalanine and tyrosine, is a promising vector because it gets into the body easily but does not harmfully effect the tissue and is gone within a week or two of introduction. Scientists engineered this strain to carry the ''Yersinia pestis'' V antigen, and studied its ability to protect mice from ''Y. pestis''. Unfortunately, the inoculation was not extremely successful and only 6 of 20 mice showed a strong response (Garmory, 2003). Garmory and colleagues cited several reasons for this: the copy number of the plasmid containing the V antigen might not have been high enough; the attenuated ''Salmonella'' may not have been able to reach cells that would be targeted by ''Y. pestis''; and the inability of the cell to secrete the antigen. Many changes need to be made to live vector vaccine design before it is an effective method of treating or preventing disease.

Another method of disease prevention with a synthetic biology approach is related to skin cancer. ''E. coli'' engineered to sense single-stranded DNA after DNA damage was coupled with a mechanism to produce a biofilm around the cell. The biofilm output module was tested with DNA damage done by mitomycin C, a DNA crosslinking agent, or UV irradiation, a known cause of skin cancer. Single-stranded DNA activates RecA, which in turn represses the CI repressor protein, thus allowing the transcription of the PL promoter (Kobayashi, 2004). The PL promoter is in charge of the ''traA'' gene, which controls biofilm formation (Fig. 4). The biofilm protection, when part of a toggle switch mechanism, lasted indefinitely when pulsed with UV light at 8 J/m2 for only 2 seconds (Kobayashi, 2004) (Fig. 5). The biofilm-production mechanism has not been engineered in eukaryotic cells, but a prokaryote-based system that could aid with human skin disease is imaginable. For example, a sunscreen that changes colors when the DNA damage may be too intense for a sunbather as a warning, or a sunscreen that becomes more protective as the possibility of genetic damage increased. Further research should focus on how to best implement the biofilm producing output module to protect human DNA.
<center>

{|
|-
|[[Image:Toggleswitch.JPG]]
|[[Image:UVinducedbiofilm.JPG]]
|-
|'''Fig. 4.''' The RecA / traA toggleswitch mechanism. (Kobayashi, 2004 - Permission Pending)
|'''Fig. 5.''' Crystal violet absorbance measures biofilm formation. (Kobayashi, 2004 - Permission Pending)
|}
</center>
Finally, synthetic biology has made its mark on the field of medicine by creating drugs that are less expensive to produce than those made by pharmaceutical companies. Jay Keasling’s project involving the antimalarial drug artemisinin is the best example. Yeast was engineered to increase normal farnesyl pyrophosphate (FPP) production, convert FPP to amorphadiene, and oxidizing amorphadiene to artemisinic acid, which can then easily be separated from the yeast cells and converted to artemisinin in the laboratory (Fig. 6). This innovation allows the production of artemisinin at lower prices than those currently on the market, and without regard to environmental constraints (Ro, 2006). On a completely different tangent, Collins’s lab engineered T7 bacteriophages to deliver biofilm-degrading enzymes such as dispersin B after infecting and replicating in an ''E. coli'' biofilm. Dispersin B works to degrade an adhesin that is critical for biofilm formation. Phages that released dispersin B were found to be 99.997% effective at removing biofilm, which is 4.5 orders of magnitude higher than non-enzymatic phages (Lu, 2007) (Fig. 7). The specificity of phages for a certain type of bacteria make them a viable vector to be used in human treatments, which may range from removing dental plaques to cleaning inserted medical devices. Using synthetic biology to engineer an antimalarial drug in yeast or a biofilm-destroying enzyme in bacteriophages greatly increases the possible contributions to the field of medicine.

<center>

{|
|-
|[[Image:Artemesinin.JPG]]
|[[Image:BiofilmDegradation.JPG]]
|-
|'''Fig. 6.''' All the steps in the box indicate steps that were optimized in yeast for a maximum artemisinic acid output. (Ro, 2006 - Permission Pending)
|'''Fig. 7.''' The amount of biofilm was decreased when the dispersin B enzymes were incorporated into T7 bacteriophages. (Lu, 2007 - Permission Pending)
|}
</center>

Medical applications of synthetic biology are wide-ranging and eminently applicable to daily life. Synthetic biology based cancer treatments that utilize ''E.coli'' engineered to sense and attack tissues with high cell density and low oxygen availability are promising and need to be tested in vivo. Designing vectors out of attenuated bacteria may be a safe, effective method for disease treatment. Using biofilms to sense sun damage potential may decrease the likelihood of skin cancer. Finally, using engineered cells to produce medicines in a cost-effective, environmentally-friendly way may revolutionize the pharmaceutical industry. Investing more research efforts in the field of synthetic biology may turn out to be an investment in medical technology.

'''Works Cited'''

-[http://www.bio.davidson.edu/courses/synthetic/papers/2006_anderson.pdf Anderson JC, Clarke EJ, Arkin, AP, and Voigt CA (2006). Environmentally controlled invasion of cancer cells by engineered bacteria. Journal of Molecular Biology 355:619-27.]

-[http://www.nature.com/gt/journal/v11/n15/abs/3302281a.html Critchley RJ, Jezzard S, Radford KJ, Goussard S, Lemoine NR, et al. (2004). Potential therapeutic applications of recombinant, invasive ''E. coli''. Gene Therapy 11:1224-33.]

-[http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&uid=15203915&cmd=showdetailview&indexed=google Garmory HS, Leary SEC, Griffin, KF, Williamson D, Brown, KA, and Titball RW (2003). The use of live attenuated bacteria as a delivery system for heterologous antigens. Journal of Drug Targeting 11:471-79.]

-[http://www.pnas.org/cgi/reprint/101/22/8414 Kobayashi H, et al. (2004). Programmable cells: Interfacing natural and engineered gene networks. PNAS 101: 8414-19.]

-[http://www.bio.davidson.edu/courses/synthetic/papers/Phage_BioFilms.pdf Lu, TK, and Collins JJ (2007). Dispersing biofilms with engineered enzymatic bacteriophage. PNAS 104: 11197-11202.]

-[http://www.nature.com/cgt/journal/v10/n10/abs/7700634a.html Nemunaitis J, Cunningham C, Senzer N, Kuhn J, Cramm J, Litz C, et al. (2003). Pilot trial of genetically modified, attenuated ''Salmonella'' expressing cytosine deaminase gene in refractory cancer patients. Cancer Gene Therapy 10:737-744.]

-[http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6W85-49FGKHC-N&_user=10&_coverDate=09%2F30%2F2003&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=d4529b45974bfe7d9ddab1fc865f088b Pawelek JM, Low KB, and Bermudes D (2003). Bacteria as tumour-targeting vectors. Lancet Oncology 4:548-56.]

-[http://www.bio.davidson.edu/courses/synthetic/papers/Keasling_malaria.pdf Ro D, et al. (2006). Production of the antimalarial drug precursor artemisinic acid in engineered yeast. Nature 440:940-43.]

Medical Applications of Synthetic Biology - Samantha Simpson

2007-11-24T15:14:29Z

Sasimpson:

'''Project Proposal'''

My project will be on medical applications of synthetic biology. I will reference Anderson's paper on utilizing quorum-sensing and hypoxia-responsive genes coupled with invasin from ''Yersinia tuberculosis'' to invade cancer-causing cells. Coupled with Critchley's work, which describes a bacterial system that invades eukaryotic cells and delivers proteins coded for in the bacteria's genome, one could possibly create 'search and destroy' ''E.coli'' that can locate, invade, and kill tumor cells. Garmory's paper also highlights the possibility of using bacteria as a drug-delivering system, specifically vaccine vectors. Kobayashi describes cells that produce a protective biofilm layer after exposure to DNA-damaging agents. Ro engineers yeast to create an anti-malarial drug that can be created at a lower cost than the previous standard production process. These papers, and others that have a focus on direct medical applications such as novel cancer therapies, vaccination technology, biological protection, and the creation of new medicines, will be referenced to create a cohesive overview of applications of synthetic biology to the medical field.

'''Paper'''

Synthetic biology is a rapidly emerging field that strives to re-design existing biological systems and components or fabricate novel biological systems and components. The end result of experimentation in synthetic biology is to help scientists understand a naturally occurring genetic pathway more fully or to create a new, useful pathway. Recent research efforts have focused on engineering bacteria to create a new form of biofuel or to make promoters of varying efficiencies for more exact gene expression. Other research efforts focus on engineering yeast to make antimalarial drugs or engineering ''E. coli'' to recognize and invade tumor-like cells, both of which are medical applications of synthetic biology. Advancements such as novel cancer therapies, vaccination technology, biological protection, and the creation of new medicines highlight synthetic biology’s potential to create breakthroughs in medical science. This paper will examine various medical applications of synthetic biology and further development that needs to be done before current research can be used in hospitals and doctors’ offices.

Synthetic biologists consider finding a cure for cancer a reachable goal. Previous to the development of synthetic biology as a field, it was known that three types of bacteria, ''Bifodobacterium'', ''Clostridium'', and ''Salmonella'' all preferentially infect the dense cells of tumors (Pawelek, 2003). All three types of bacteria are associated with reducing tumor size in patients who are infected with them, and it was noted that all three bacteria act anaerobically. Capitalizing on the idea that tumors create an unusually dense, anaerobic mass of cells, Christopher Voigt’s lab engineered bacteria to identify and invade cancer cells. They utilized the ''lux'' quorum sensing from ''Vibrio fischeri'' to identify cells growing at high densities (Fig. 1) and the ''fdhF'' promoter to identify cells growing in anaerobic conditions (Fig. 2) (Anderson, 2006). They coupled this identification mechanism with the invasin output module from ''Yersinia pseudotuberculosis'' so the ''E. coli'' could invade specific cells. Next, scientists hope to insert a gene that would destroy the invaded cells, making the complete cycle a search, invade, and destroy loop that would only be sensitive to cancer cells. One promising destroy mechanism has been implemented in humans via intratumoral injection, based on ''Salmonella'' that contains the ''E. coli'' cytosine deaminase gene that converts 5-fluorocytosine to 5-fluorouracil, a chemotherapy drug (Nemunaitis, 2003). Unfortunately, while the tumors did produce 5-fluorouracil (5-FU), they did not produce it in high enough yields to cause regression of the cancer. Another experiment flooded mouse cells with 6-methylpurine-2’deoxyribose (6-MPDR), which was converted to the toxin 6-methyl purine (MeP) by the enzyme purine nucleoside phosphorylase (PNP), which is naturally found in ''E. coli'' (Critchley, 2004) (Fig. 3). The problem with this is that all cells in the body might be exposed to the toxin MeP. In addition to the search and destroy mechanism, scientists hope to increase selectivity by combining the hypoxia-sensing and density-sensing units so that ''E. coli'' will only enter eukaryotic cells that exhibit both characteristics. This would make it more likely that invasin-enhanced ''E. coli'' would only invade cancer cells rather than muscle tissue that had low oxygen due to exercise (Anderson, 2006). To create a fully functional, synthetic biology-based approach to cancer therapy, scientists need to develop bacteria that discriminately invades cancer cells and destroys them. Most of the pieces are there – scientists have engineered ''E. coli'' that selectively invades hypoxic cells or dense cells and ''Salmonella'' that can destroy tumors in vivo – they just need to be put together effectively.

<center>

{|
|-
|[[Image:InvasinDensity.JPG]]
|[[Image:InvasinAnaerobic2.JPG]]
|[[Image:Tumor.JPG]]
|-
|'''Fig. 1.''' The red line represents when the invasin output module was paired with the inducible promoter ''lux'' which senses high cell density, the black line is constitutively on invasin. (Anderson, 2006 - Permission Pending)
|'''Fig. 2.''' The invasin output module was paired with the inducible promoter ''fdhF'' which senses anaerobic conditions. (Anderson, 2006 - Permission Pending)
|'''Fig. 3.''' Tumor size after injections. Cancer cells were injected into mice, and 5 days later injections with PBS, 6-MPDR, and invasin-enhanced ''E-coli'' began. Tumor cells recieving injections of invasin-enhanced ''E.coli'' and 6-MPDR grew the slowest, but did not get smaller. (Critchley, 2004 - Permission Pending)
|}
</center>

Using bacteria as a vaccine vector has similar issues of specificity and implementation in a human system. Bacteria was first thought to be a good vector because they would not degrade at mucosal surfaces, they would absorb better and survive the low pH in the gastrointestinal tract (Garmory, 2003). A particular strain of ''Salmonella'' with one or more deletions in the shikimate pathway, which creates a precursor for phenylalanine and tyrosine, is a promising vector because it gets into the body easily but does not harmfully effect the tissue and is gone within a week or two of introduction. Scientists engineered this strain to carry the ''Yersinia pestis'' V antigen, and studied its ability to protect mice from ''Y. pestis''. Unfortunately, the inoculation was not extremely successful and only 6 of 20 mice showed a strong response (Garmory, 2003). Garmory and colleagues cited several reasons for this: the copy number of the plasmid containing the V antigen might not have been high enough; the attenuated ''Salmonella'' may not have been able to reach cells that would be targeted by ''Y. pestis''; and the inability of the cell to secrete the antigen. Many changes need to be made to live vector vaccine design before it is an effective method of treating or preventing disease.

Another method of disease prevention with a synthetic biology approach is related to skin cancer. ''E. coli'' engineered to sense single-stranded DNA after DNA damage was coupled with a mechanism to produce a biofilm around the cell. The biofilm output module was tested with DNA damage done by mitomycin C, a DNA crosslinking agent, or UV irradiation, a known cause of skin cancer. Single-stranded DNA activates RecA, which in turn represses the CI repressor protein, thus allowing the transcription of the PL promoter (Kobayashi, 2004). The PL promoter is in charge of the ''traA'' gene, which controls biofilm formation (Fig. 4). The biofilm protection, when part of a toggle switch mechanism, lasted indefinitely when pulsed with UV light at 8 J/m2 for only 2 seconds (Kobayashi, 2004) (Fig. 5). The biofilm-production mechanism has not been engineered in eukaryotic cells, but a prokaryote-based system that could aid with human skin disease is imaginable. For example, a sunscreen that changes colors when the DNA damage may be too intense for a sunbather as a warning, or a sunscreen that becomes more protective as the possibility of genetic damage increased. Further research should focus on how to best implement the biofilm producing output module to protect human DNA.
<center>

{|
|-
|[[Image:Toggleswitch.JPG]]
|[[Image:UVinducedbiofilm.JPG]]
|-
|'''Fig. 4.''' The RecA / traA toggleswitch mechanism. (Kobayashi, 2004 - Permission Pending)
|'''Fig. 5.''' Crystal violet absorbance measures biofilm formation. (Kobayashi, 2004 - Permission Pending)
|}
</center>
Finally, synthetic biology has made its mark on the field of medicine by creating drugs that are less expensive to produce than those made by pharmaceutical companies. Jay Keasling’s project involving the antimalarial drug artemisinin is the best example. Yeast was engineered to increase normal farnesyl pyrophosphate (FPP) production, convert FPP to amorphadiene, and oxidizing amorphadiene to artemisinic acid, which can then easily be separated from the yeast cells and converted to artemisinin in the laboratory (Fig. 6). This innovation allows the production of artemisinin at lower prices than those currently on the market, and without regard to environmental constraints (Ro, 2006). On a completely different tangent, Collins’s lab engineered T7 bacteriophages to deliver biofilm-degrading enzymes such as dispersin B after infecting and replicating in an ''E. coli'' biofilm. Dispersin B works to degrade an adhesin that is critical for biofilm formation. Phages that released dispersin B were found to be 99.997% effective at removing biofilm, which is 4.5 orders of magnitude higher than non-enzymatic phages (Lu, 2007) (Fig. 7). The specificity of phages for a certain type of bacteria make them a viable vector to be used in human treatments, which may range from removing dental plaques to cleaning inserted medical devices. Using synthetic biology to engineer an antimalarial drug in yeast or a biofilm-destroying enzyme in bacteriophages greatly increases the possible contributions to the field of medicine.

<center>

{|
|-
|[[Image:Artemesinin.JPG]]
|[[Image:BiofilmDegradation.JPG]]
|-
|'''Fig. 6.''' All the steps in the box indicate steps that were optimized in yeast for a maximum artemisinic acid output. (Ro, 2006 - Permission Pending)
|'''Fig. 7.''' The amount of biofilm was decreased when the dispersin B enzymes were incorporated into T7 bacteriophages. (Lu, 2007 - Permission Pending)
|}
</center>

Medical applications of synthetic biology are wide-ranging and eminently applicable to daily life. Synthetic biology based cancer treatments that utilize ''E.coli'' engineered to sense and attack tissues with high cell density and low oxygen availability are promising and need to be tested in vivo. Designing vectors out of attenuated bacteria may be a safe, effective method for disease treatment. Using biofilms to sense sun damage potential may decrease the likelihood of skin cancer. Finally, using engineered cells to produce medicines in a cost-effective, environmentally-friendly way may revolutionize the pharmaceutical industry. Investing more research efforts in the field of synthetic biology may turn out to be an investment in medical technology.

'''Works Cited'''

-[http://www.bio.davidson.edu/courses/synthetic/papers/2006_anderson.pdf Anderson JC, Clarke EJ, Arkin, AP, and Voigt CA (2006). Environmentally controlled invasion of cancer cells by engineered bacteria. Journal of Molecular Biology 355:619-27.]

-[http://www.nature.com/gt/journal/v11/n15/abs/3302281a.html Critchley RJ, Jezzard S, Radford KJ, Goussard S, Lemoine NR, et al. (2004). Potential therapeutic applications of recombinant, invasive ''E. coli''. Gene Therapy 11:1224-33.]

-[http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&uid=15203915&cmd=showdetailview&indexed=google Garmory HS, Leary SEC, Griffin, KF, Williamson D, Brown, KA, and Titball RW (2003). The use of live attenuated bacteria as a delivery system for heterologous antigens. Journal of Drug Targeting 11:471-79.]

-[http://www.pnas.org/cgi/reprint/101/22/8414 Kobayashi H, et al. (2004). Programmable cells: Interfacing natural and engineered gene networks. PNAS 101: 8414-19.]

-[http://www.bio.davidson.edu/courses/synthetic/papers/Phage_BioFilms.pdf Lu, TK, and Collins JJ (2007). Dispersing biofilms with engineered enzymatic bacteriophage. PNAS 104: 11197-11202.]

-[http://www.nature.com/cgt/journal/v10/n10/abs/7700634a.html Nemunaitis J, Cunningham C, Senzer N, Kuhn J, Cramm J, Litz C, et al. (2003). Pilot trial of genetically modified, attenuated ''Salmonella'' expressing cytosine deaminase gene in refractory cancer patients. Cancer Gene Therapy 10:737-744.]

-[http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6W85-49FGKHC-N&_user=10&_coverDate=09%2F30%2F2003&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=d4529b45974bfe7d9ddab1fc865f088b Pawelek JM, Low KB, and Bermudes D (2003). Bacteria as tumour-targeting vectors. Lancet Oncology 4:548-56.]

-[http://www.bio.davidson.edu/courses/synthetic/papers/Keasling_malaria.pdf Ro D, et al. (2006). Production of the antimalarial drug precursor artemisinic acid in engineered yeast. Nature 440:940-43.]

Medical Applications of Synthetic Biology - Samantha Simpson

2007-11-23T21:36:27Z

Sasimpson:

'''Project Proposal'''

My project will be on medical applications of synthetic biology. I will reference Anderson's paper on utilizing quorum-sensing and hypoxia-responsive genes coupled with invasin from ''Yersinia tuberculosis'' to invade cancer-causing cells. Coupled with Critchley's work, which describes a bacterial system that invades eukaryotic cells and delivers proteins coded for in the bacteria's genome, one could possibly create 'search and destroy' ''E.coli'' that can locate, invade, and kill tumor cells. Garmory's paper also highlights the possibility of using bacteria as a drug-delivering system, specifically vaccine vectors. Kobayashi describes cells that produce a protective biofilm layer after exposure to DNA-damaging agents. Ro engineers yeast to create an anti-malarial drug that can be created at a lower cost than the previous standard production process. These papers, and others that have a focus on direct medical applications such as novel cancer therapies, vaccination technology, biological protection, and the creation of new medicines, will be referenced to create a cohesive overview of applications of synthetic biology to the medical field.

'''Paper'''

Synthetic biology is a rapidly emerging field that strives to re-design existing biological systems and components or fabricate novel biological systems and components. The end result of experimentation in synthetic biology is to help scientists understand a naturally occurring genetic pathway more fully or to create a new, useful pathway. Recent research efforts have focused on engineering bacteria to create a new form of biofuel or to make promoters of varying efficiencies for more exact gene expression. Other research efforts focus on engineering yeast to make antimalarial drugs or engineering ''E. coli'' to recognize and invade tumor-like cells, both of which are medical applications of synthetic biology. Advancements such as novel cancer therapies, vaccination technology, biological protection, and the creation of new medicines highlight synthetic biology’s potential to create breakthroughs in medical science. This paper will examine various medical applications of synthetic biology and further development that needs to be done before current research can be used in hospitals and doctors’ offices.

Synthetic biologists consider finding a cure for cancer a reachable goal. Previous to the development of synthetic biology as a field, it was known that three types of bacteria, ''Bifodobacterium'', ''Clostridium'', and ''Salmonella'' all preferentially infect the dense cells of tumors (Pawelek 548). All three types of bacteria are associated with reducing tumor size in patients who are infected with them, and it was noted that all three bacteria act anaerobically. Capitalizing on the idea that tumors create an unusually dense, anaerobic mass of cells, Christopher Voigt’s lab engineered bacteria to identify and invade cancer cells. They utilized the ''lux'' quorum sensing from ''Vibrio fischeri'' to identify cells growing at high densities (Fig. 1) and the ''fdhF'' promoter to identify cells growing in anaerobic conditions (Fig. 2) (Anderson 619). They coupled this identification mechanism with the invasin output module from ''Yersinia pseudotuberculosis'' so the ''E. coli'' could invade specific cells. Next, scientists hope to insert a gene that would destroy the invaded cells, making the complete cycle a search, invade, and destroy loop that would only be sensitive to cancer cells. One promising destroy mechanism has been implemented in humans via intratumoral injection, based on ''Salmonella'' that contains the ''E. coli'' cytosine deaminase gene that converts 5-fluorocytosine to 5-fluorouracil, a chemotherapy drug (Nemunaitis 737). Unfortunately, while the tumors did produce 5-fluorouracil (5-FU), they did not produce it in high enough yields to cause regression of the cancer. Another experiment flooded mouse cells with 6-methylpurine-2’deoxyribose (6-MPDR), which was converted to the toxin 6-methyl purine (MeP) by the enzyme purine nucleoside phosphorylase (PNP), which is naturally found in ''E. coli'' (Critchley 1226) (Fig. 3). The problem with this is that all cells in the body might be exposed to the toxin MeP. In addition to the search and destroy mechanism, scientists hope to increase selectivity by combining the hypoxia-sensing and density-sensing units so that ''E. coli'' will only enter eukaryotic cells that exhibit both characteristics. This would make it more likely that invasin-enhanced ''E. coli'' would only invade cancer cells rather than muscle tissue that had low oxygen due to exercise (Anderson 624). To create a fully functional, synthetic biology-based approach to cancer therapy, scientists need to develop bacteria that discriminately invades cancer cells and destroys them. Most of the pieces are there – scientists have engineered ''E. coli'' that selectively invades hypoxic cells or dense cells and ''Salmonella'' that can destroy tumors in vivo – they just need to be put together effectively.

<center>

{|
|-
|[[Image:InvasinDensity.JPG]]
|[[Image:InvasinAnaerobic2.JPG]]
|[[Image:Tumor.JPG]]
|-
|'''Fig. 1.''' The red line represents when the invasin output module was paired with the inducible promoter ''lux'' which senses high cell density, the black line is constitutively on invasin. (Anderson - Permission Pending)
|'''Fig. 2.''' The invasin output module was paired with the inducible promoter ''fdhF'' which senses anaerobic conditions. (Anderson - Permission Pending)
|'''Fig. 3.''' Tumor size after injections. Cancer cells were injected into mice, and 5 days later injections with PBS, 6-MPDR, and invasin-enhanced ''E-coli'' began. Tumor cells recieving injections of invasin-enhanced ''E.coli'' and 6-MPDR grew the slowest, but did not get smaller. (Critchley - Permission Pending)
|}
</center>

Using bacteria as a vaccine vector has similar issues of specificity and implementation in a human system. Bacteria was first thought to be a good vector because they would not degrade at mucosal surfaces, they would absorb better and survive the low pH in the gastrointestinal tract (Garmory 471). A particular strain of ''Salmonella'' with one or more deletions in the shikimate pathway, which creates a precursor for phenylalanine and tyrosine, is a promising vector because it gets into the body easily but does not harmfully effect the tissue and is gone within a week or two of introduction. Scientists engineered this strain to carry the ''Yersinia pestis'' V antigen, and studied its ability to protect mice from ''Y. pestis''. Unfortunately, the inoculation was not extremely successful and only 6 of 20 mice showed a strong response (Garmory 473). Garmory and colleagues cited several reasons for this: the copy number of the plasmid containing the V antigen might not have been high enough; the attenuated ''Salmonella'' may not have been able to reach cells that would be targeted by ''Y. pestis''; and the inability of the cell to secrete the antigen. Many changes need to be made to live vector vaccine design before it is an effective method of treating or preventing disease.

Another method of disease prevention with a synthetic biology approach is related to skin cancer. ''E. coli'' engineered to sense single-stranded DNA after DNA damage was coupled with a mechanism to produce a biofilm around the cell. The biofilm output module was tested with DNA damage done by mitomycin C, a DNA crosslinking agent, or UV irradiation, a known cause of skin cancer. Single-stranded DNA activates RecA, which in turn represses the CI repressor protein, thus allowing the transcription of the PL promoter (Kobayashi 8416). The PL promoter is in charge of the ''traA'' gene, which controls biofilm formation (Fig. 4). The biofilm protection, when part of a toggle switch mechanism, lasted indefinitely when pulsed with UV light at 8 J/m2 for only 2 seconds (Kobayashi 8417) (Fig. 5). The biofilm-production mechanism has not been engineered in eukaryotic cells, but a prokaryote-based system that could aid with human skin disease is imaginable. For example, a sunscreen that changes colors when the DNA damage may be too intense for a sunbather as a warning, or a sunscreen that becomes more protective as the possibility of genetic damage increased. Further research should focus on how to best implement the biofilm producing output module to protect human DNA.
<center>

{|
|-
|[[Image:Toggleswitch.JPG]]
|[[Image:UVinducedbiofilm.JPG]]
|-
|'''Fig. 4.''' The RecA / traA toggleswitch mechanism. (Kobayashi - Permission Pending)
|'''Fig. 5.''' Crystal violet absorbance measures biofilm formation. (Kobayashi - Permission Pending)
|}
</center>
Finally, synthetic biology has made its mark on the field of medicine by creating drugs that are less expensive to produce than those made by pharmaceutical companies. Jay Keasling’s project involving the antimalarial drug artemisinin is the best example. Yeast was engineered to increase normal farnesyl pyrophosphate (FPP) production, convert FPP to amorphadiene, and oxidizing amorphadiene to artemisinic acid, which can then easily be separated from the yeast cells and converted to artemisinin in the laboratory (Fig. 6). This innovation allows the production of artemisinin at lower prices than those currently on the market, and without regard to environmental constraints (Ro 942). On a completely different tangent, Collins’s lab engineered T7 bacteriophages to deliver biofilm-degrading enzymes such as dispersin B after infecting and replicating in an ''E. coli'' biofilm. Dispersin B works to degrade an adhesin that is critical for biofilm formation. Phages that released dispersin B were found to be 99.997% effective at removing biofilm, which is 4.5 orders of magnitude higher than non-enzymatic phages (Lu 11197) (Fig. 7). The specificity of phages for a certain type of bacteria make them a viable vector to be used in human treatments, which may range from removing dental plaques to cleaning inserted medical devices. Using synthetic biology to engineer an antimalarial drug in yeast or a biofilm-destroying enzyme in bacteriophages greatly increases the possible contributions to the field of medicine.

<center>

{|
|-
|[[Image:Artemesinin.JPG]]
|[[Image:BiofilmDegradation.JPG]]
|-
|'''Fig. 6.''' All the steps in the box indicate steps that were optimized in yeast for a maximum artemisinic acid output. (Ro - Permission Pending)
|'''Fig. 7.''' The amount of biofilm was decreased when the dispersin B enzymes were incorporated into T7 bacteriophages. (Lu - Permission Pending)
|}
</center>

Medical applications of synthetic biology are wide-ranging and eminently applicable to daily life. Synthetic biology based cancer treatments that utilize ''E.coli'' engineered to sense and attack tissues with high cell density and low oxygen availability are promising and need to be tested in vivo. Designing vectors out of attenuated bacteria may be a safe, effective method for disease treatment. Using biofilms to sense sun damage potential may decrease the likelihood of skin cancer. Finally, using engineered cells to produce medicines in a cost-effective, environmentally-friendly way may revolutionize the pharmaceutical industry. Investing more research efforts in the field of synthetic biology may turn out to be an investment in medical technology.

'''Works Cited'''

-[http://www.bio.davidson.edu/courses/synthetic/papers/2006_anderson.pdf Anderson JC, Clarke EJ, Arkin, AP, and Voigt CA (2006). Environmentally controlled invasion of cancer cells by engineered bacteria. Journal of Molecular Biology 355:619-27.]

-[http://www.nature.com/gt/journal/v11/n15/abs/3302281a.html Critchley RJ, Jezzard S, Radford KJ, Goussard S, Lemoine NR, et al. (2004). Potential therapeutic applications of recombinant, invasive ''E. coli''. Gene Therapy 11:1224-33.]

-[http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&uid=15203915&cmd=showdetailview&indexed=google Garmory HS, Leary SEC, Griffin, KF, Williamson D, Brown, KA, and Titball RW (2003). The use of live attenuated bacteria as a delivery system for heterologous antigens. Journal of Drug Targeting 11:471-79.]

-[http://www.pnas.org/cgi/reprint/101/22/8414 Kobayashi H, et al. (2004). Programmable cells: Interfacing natural and engineered gene networks. PNAS 101: 8414-19.]

-[http://www.bio.davidson.edu/courses/synthetic/papers/Phage_BioFilms.pdf Lu, TK, and Collins JJ (2007). Dispersing biofilms with engineered enzymatic bacteriophage. PNAS 104: 11197-11202.]

-[http://www.nature.com/cgt/journal/v10/n10/abs/7700634a.html Nemunaitis J, Cunningham C, Senzer N, Kuhn J, Cramm J, Litz C, et al. (2003). Pilot trial of genetically modified, attenuated ''Salmonella'' expressing cytosine deaminase gene in refractory cancer patients. Cancer Gene Therapy 10:737-744.]

-[http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6W85-49FGKHC-N&_user=10&_coverDate=09%2F30%2F2003&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=d4529b45974bfe7d9ddab1fc865f088b Pawelek JM, Low KB, and Bermudes D (2003). Bacteria as tumour-targeting vectors. Lancet Oncology 4:548-56.]

-[http://www.bio.davidson.edu/courses/synthetic/papers/Keasling_malaria.pdf Ro D, et al. (2006). Production of the antimalarial drug precursor artemisinic acid in engineered yeast. Nature 440:940-43.]

Medical Applications of Synthetic Biology - Samantha Simpson

2007-11-23T21:33:01Z

Sasimpson:

'''Project Proposal'''

My project will be on medical applications of synthetic biology. I will reference Anderson's paper on utilizing quorum-sensing and hypoxia-responsive genes coupled with invasin from ''Yersinia tuberculosis'' to invade cancer-causing cells. Coupled with Critchley's work, which describes a bacterial system that invades eukaryotic cells and delivers proteins coded for in the bacteria's genome, one could possibly create 'search and destroy' ''E.coli'' that can locate, invade, and kill tumor cells. Garmory's paper also highlights the possibility of using bacteria as a drug-delivering system, specifically vaccine vectors. Kobayashi describes cells that produce a protective biofilm layer after exposure to DNA-damaging agents. Ro engineers yeast to create an anti-malarial drug that can be created at a lower cost than the previous standard production process. These papers, and others that have a focus on direct medical applications such as novel cancer therapies, vaccination technology, biological protection, and the creation of new medicines, will be referenced to create a cohesive overview of applications of synthetic biology to the medical field.

'''Paper'''

Synthetic biology is a rapidly emerging field that strives to re-design existing biological systems and components or fabricate novel biological systems and components. The end result of experimentation in synthetic biology is to help scientists understand a naturally occurring genetic pathway more fully or to create a new, useful pathway. Recent research efforts have focused on engineering bacteria to create a new form of biofuel or to make promoters of varying efficiencies for more exact gene expression. Other research efforts focus on engineering yeast to make antimalarial drugs or engineering ''E. coli'' to recognize and invade tumor-like cells, both of which are medical applications of synthetic biology. Advancements such as novel cancer therapies, vaccination technology, biological protection, and the creation of new medicines highlight synthetic biology’s potential to create breakthroughs in medical science. This paper will examine various medical applications of synthetic biology and further development that needs to be done before current research can be used in hospitals and doctors’ offices.

Synthetic biologists consider finding a cure for cancer a reachable goal. Previous to the development of synthetic biology as a field, it was known that three types of bacteria, ''Bifodobacterium'', ''Clostridium'', and ''Salmonella'' all preferentially infect the dense cells of tumors (Pawelek 548). All three types of bacteria are associated with reducing tumor size in patients who are infected with them, and it was noted that all three bacteria act anaerobically. Capitalizing on the idea that tumors create an unusually dense, anaerobic mass of cells, Christopher Voigt’s lab engineered bacteria to identify and invade cancer cells. They utilized the ''lux'' quorum sensing from ''Vibrio fischeri'' to identify cells growing at high densities (Fig. 1) and the ''fdhF'' promoter to identify cells growing in anaerobic conditions (Fig. 2) (Anderson 619). They coupled this identification mechanism with the invasin output module from ''Yersinia pseudotuberculosis'' so the ''E. coli'' could invade specific cells. Next, scientists hope to insert a gene that would destroy the invaded cells, making the complete cycle a search, invade, and destroy loop that would only be sensitive to cancer cells. One promising destroy mechanism has been implemented in humans via intratumoral injection, based on ''Salmonella'' that contains the ''E. coli'' cytosine deaminase gene that converts 5-fluorocytosine to 5-fluorouracil, a chemotherapy drug (Nemunaitis 737). Unfortunately, while the tumors did produce 5-fluorouracil (5-FU), they did not produce it in high enough yields to cause regression of the cancer. Another experiment flooded mouse cells with 6-methylpurine-2’deoxyribose (6-MPDR), which was converted to the toxin 6-methyl purine (MeP) by the enzyme purine nucleoside phosphorylase (PNP), which is naturally found in ''E. coli'' (Critchley 1226) (Fig. 3). The problem with this is that all cells in the body might be exposed to the toxin MeP. In addition to the search and destroy mechanism, scientists hope to increase selectivity by combining the hypoxia-sensing and density-sensing units so that ''E. coli'' will only enter eukaryotic cells that exhibit both characteristics. This would make it more likely that invasin-enhanced ''E. coli'' would only invade cancer cells rather than muscle tissue that had low oxygen due to exercise (Anderson 624). To create a fully functional, synthetic biology-based approach to cancer therapy, scientists need to develop bacteria that discriminately invades cancer cells and destroys them. Most of the pieces are there – scientists have engineered ''E. coli'' that selectively invades hypoxic cells or dense cells and ''Salmonella'' that can destroy tumors in vivo – they just need to be put together effectively.

<center>

{|
|-
|[[Image:InvasinDensity.JPG]]
|[[Image:InvasinAnaerobic2.JPG]]
|[[Image:Tumor.JPG]]
|-
|'''Fig. 1.''' The red line represents when the invasin output module was paired with the inducible promoter ''lux'' which senses high cell density, the black line is constitutively on invasin. (Anderson - Permission Pending)
|'''Fig. 2.''' The invasin output module was paired with the inducible promoter ''fdhF'' which senses anaerobic conditions. (Anderson - Permission Pending)
|'''Fig. 3.''' Tumor size after injections. Cancer cells were injected into mice, and 5 days later injections with PBS, 6-MPDR, and invasin-enhanced ''E-coli'' began. Tumor cells recieving injections of invasin-enhanced ''E.coli'' and 6-MPDR grew the slowest, but did not get smaller. (Critchley - Permission Pending)
|}
</center>

Using bacteria as a vaccine vector has similar issues of specificity and implementation in a human system. Bacteria was first thought to be a good vector because they would not degrade at mucosal surfaces, they would absorb better and survive the low pH in the gastrointestinal tract (Garmory 471). A particular strain of ''Salmonella'' with one or more deletions in the shikimate pathway, which creates a precursor for phenylalanine and tyrosine, is a promising vector because it gets into the body easily but does not harmfully effect the tissue and is gone within a week or two of introduction. Scientists engineered this strain to carry the ''Yersinia pestis'' V antigen, and studied its ability to protect mice from ''Y. pestis''. Unfortunately, the inoculation was not extremely successful and only 6 of 20 mice showed a strong response (Garmory 473). Garmory and colleagues cited several reasons for this: the copy number of the plasmid containing the V antigen might not have been high enough; the attenuated ''Salmonella'' may not have been able to reach cells that would be targeted by ''Y. pestis''; and the inability of the cell to secrete the antigen. Many changes need to be made to live vector vaccine design before it is an effective method of treating or preventing disease.

Another method of disease prevention with a synthetic biology approach is related to skin cancer. ''E. coli'' engineered to sense single-stranded DNA after DNA damage was coupled with a mechanism to produce a biofilm around the cell. The biofilm output module was tested with DNA damage done by mitomycin C, a DNA crosslinking agent, or UV irradiation, a known cause of skin cancer. Single-stranded DNA activates RecA, which in turn represses the CI repressor protein, thus allowing the transcription of the PL promoter (Kobayashi 8416). The PL promoter is in charge of the ''traA'' gene, which controls biofilm formation (Fig. 4). The biofilm protection, when part of a toggle switch mechanism, lasted indefinitely when pulsed with UV light at 8 J/m2 for only 2 seconds (Kobayashi 8417) (Fig. 5). The biofilm-production mechanism has not been engineered in eukaryotic cells, but a prokaryote-based system that could aid with human skin disease is imaginable. For example, a sunscreen that changes colors when the DNA damage may be too intense for a sunbather as a warning, or a sunscreen that becomes more protective as the possibility of genetic damage increased. Further research should focus on how to best implement the biofilm producing output module to protect human DNA.
<center>

{|
|-
|[[Image:Toggleswitch.JPG]]
|[[Image:UVinducedbiofilm.JPG]]
|-
|'''Fig. 4.''' The RecA / traA toggleswitch mechanism. (Kobayashi - Permission Pending)
|'''Fig. 5.''' Crystal violet absorbance measures biofilm formation. (Kobayashi - Permission Pending)
|}
</center>
Finally, synthetic biology has made its mark on the field of medicine by creating drugs that are less expensive to produce than those made by pharmaceutical companies. Jay Keasling’s project involving the antimalarial drug artemisinin is the best example. Yeast was engineered to increase normal farnesyl pyrophosphate (FPP) production, convert FPP to amorphadiene, and oxidizing amorphadiene to artemisinic acid, which can then easily be separated from the yeast cells and converted to artemisinin in the laboratory (Fig. 6). This innovation allows the production of artemisinin at lower prices than those currently on the market, and without regard to environmental constraints (Ro 942). On a completely different tangent, Collins’s lab engineered T7 bacteriophages to deliver biofilm-degrading enzymes such as dispersin B after infecting and replicating in an ''E. coli'' biofilm. Dispersin B works to degrade an adhesin that is critical for biofilm formation. Phages that released dispersin B were found to be 99.997% effective at removing biofilm, which is 4.5 orders of magnitude higher than non-enzymatic phages (Lu 11197) (Fig. 7). The specificity of phages for a certain type of bacteria make them a viable vector to be used in human treatments, which may range from removing dental plaques to cleaning inserted medical devices. Using synthetic biology to engineer an antimalarial drug in yeast or a biofilm-destroying enzyme in bacteriophages greatly increases the possible contributions to the field of medicine.

<center>

{|
|-
|[[Image:Artemesinin.JPG]]
|[[Image:BiofilmDegradation.JPG]]
|-
|'''Fig. 6.''' All the steps in the box indicate steps that were optimized in yeast for a maximum artemisinic acid output. (Ro - Permission Pending)
|'''Fig. 7.''' The amount of biofilm was decreased when the dispersin B enzymes were incorporated into T7 bacteriophages. (Lu - Permission Pending)
|}
</center>

Medical applications of synthetic biology are wide-ranging and eminently applicable to daily life. Synthetic biology based cancer treatments that utilize ''E.coli'' engineered to sense and attack tissues with high cell density and low oxygen availability are promising and need to be tested in vivo. Designing vectors out of attenuated bacteria may be a safe, effective method for disease treatment. Using biofilms to sense sun damage potential may decrease the likelihood of skin cancer. Finally, using engineered cells to produce medicines in a cost-effective, environmentally-friendly way may revolutionize the pharmaceutical industry. Investing more research efforts in the field of synthetic biology may turn out to be an investment in medical technology.

'''Works Cited'''

-[http://www.bio.davidson.edu/courses/synthetic/papers/2006_anderson.pdf Anderson JC, Clarke EJ, Arkin, AP, and Voigt CA (2006). Environmentally controlled invasion of cancer cells by engineered bacteria. Journal of Molecular Biology 355:619-27.]

-[http://www.nature.com/gt/journal/v11/n15/abs/3302281a.html Critchley RJ, Jezzard S, Radford KJ, Goussard S, Lemoine NR, et al. (2004). Potential therapeutic applications of recombinant, invasive ''E. coli''. Gene Therapy 11:1224-33.]

-[http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&uid=15203915&cmd=showdetailview&indexed=google Garmory HS, Leary SEC, Griffin, KF, Williamson D, Brown, KA, and Titball RW (2003). The use of live attenuated bacteria as a delivery system for heterologous antigens. Journal of Drug Targeting 11:471-79.]

-Kobayashi H, et al. (2004). Programmable cells: Interfacing natural and engineered gene networks. PNAS 101: 8414-19.

-[http://www.bio.davidson.edu/courses/synthetic/papers/Phage_BioFilms.pdf Lu, TK, and Collins JJ (2007). Dispersing biofilms with engineered enzymatic bacteriophage. PNAS 104: 11197-11202.]

-Nemunaitis J, Cunningham C, Senzer N, Kuhn J, Cramm J, Litz C, et al. (2003). Pilot trial of genetically modified, attenuated ''Salmonella'' expressing cytosine deaminase gene in refractory cancer patients. Cancer Gene Therapy 10:737-744.

-Pawelek JM, Low KB, and Bermudes D (2003). Bacteria as tumour-targeting vectors. Lancet Oncology 4:548-56.

-[http://www.bio.davidson.edu/courses/synthetic/papers/Keasling_malaria.pdf Ro D, et al. (2006). Production of the antimalarial drug precursor artemisinic acid in engineered yeast. Nature 440:940-43.]

Medical Applications of Synthetic Biology - Samantha Simpson

2007-11-23T21:31:16Z

Sasimpson:

'''Project Proposal'''

My project will be on medical applications of synthetic biology. I will reference Anderson's paper on utilizing quorum-sensing and hypoxia-responsive genes coupled with invasin from ''Yersinia tuberculosis'' to invade cancer-causing cells. Coupled with Critchley's work, which describes a bacterial system that invades eukaryotic cells and delivers proteins coded for in the bacteria's genome, one could possibly create 'search and destroy' ''E.coli'' that can locate, invade, and kill tumor cells. Garmory's paper also highlights the possibility of using bacteria as a drug-delivering system, specifically vaccine vectors. Kobayashi describes cells that produce a protective biofilm layer after exposure to DNA-damaging agents. Ro engineers yeast to create an anti-malarial drug that can be created at a lower cost than the previous standard production process. These papers, and others that have a focus on direct medical applications such as novel cancer therapies, vaccination technology, biological protection, and the creation of new medicines, will be referenced to create a cohesive overview of applications of synthetic biology to the medical field.

'''Paper'''

Synthetic biology is a rapidly emerging field that strives to re-design existing biological systems and components or fabricate novel biological systems and components. The end result of experimentation in synthetic biology is to help scientists understand a naturally occurring genetic pathway more fully or to create a new, useful pathway. Recent research efforts have focused on engineering bacteria to create a new form of biofuel or to make promoters of varying efficiencies for more exact gene expression. Other research efforts focus on engineering yeast to make antimalarial drugs or engineering ''E. coli'' to recognize and invade tumor-like cells, both of which are medical applications of synthetic biology. Advancements such as novel cancer therapies, vaccination technology, biological protection, and the creation of new medicines highlight synthetic biology’s potential to create breakthroughs in medical science. This paper will examine various medical applications of synthetic biology and further development that needs to be done before current research can be used in hospitals and doctors’ offices.

Synthetic biologists consider finding a cure for cancer a reachable goal. Previous to the development of synthetic biology as a field, it was known that three types of bacteria, ''Bifodobacterium'', ''Clostridium'', and ''Salmonella'' all preferentially infect the dense cells of tumors (Pawelek 548). All three types of bacteria are associated with reducing tumor size in patients who are infected with them, and it was noted that all three bacteria act anaerobically. Capitalizing on the idea that tumors create an unusually dense, anaerobic mass of cells, Christopher Voigt’s lab engineered bacteria to identify and invade cancer cells. They utilized the ''lux'' quorum sensing from ''Vibrio fischeri'' to identify cells growing at high densities (Fig. 1) and the ''fdhF'' promoter to identify cells growing in anaerobic conditions (Fig. 2) (Anderson 619). They coupled this identification mechanism with the invasin output module from ''Yersinia pseudotuberculosis'' so the ''E. coli'' could invade specific cells. Next, scientists hope to insert a gene that would destroy the invaded cells, making the complete cycle a search, invade, and destroy loop that would only be sensitive to cancer cells. One promising destroy mechanism has been implemented in humans via intratumoral injection, based on ''Salmonella'' that contains the ''E. coli'' cytosine deaminase gene that converts 5-fluorocytosine to 5-fluorouracil, a chemotherapy drug (Nemunaitis 737). Unfortunately, while the tumors did produce 5-fluorouracil (5-FU), they did not produce it in high enough yields to cause regression of the cancer. Another experiment flooded mouse cells with 6-methylpurine-2’deoxyribose (6-MPDR), which was converted to the toxin 6-methyl purine (MeP) by the enzyme purine nucleoside phosphorylase (PNP), which is naturally found in ''E. coli'' (Critchley 1226) (Fig. 3). The problem with this is that all cells in the body might be exposed to the toxin MeP. In addition to the search and destroy mechanism, scientists hope to increase selectivity by combining the hypoxia-sensing and density-sensing units so that ''E. coli'' will only enter eukaryotic cells that exhibit both characteristics. This would make it more likely that invasin-enhanced ''E. coli'' would only invade cancer cells rather than muscle tissue that had low oxygen due to exercise (Anderson 624). To create a fully functional, synthetic biology-based approach to cancer therapy, scientists need to develop bacteria that discriminately invades cancer cells and destroys them. Most of the pieces are there – scientists have engineered ''E. coli'' that selectively invades hypoxic cells or dense cells and ''Salmonella'' that can destroy tumors in vivo – they just need to be put together effectively.

<center>

{|
|-
|[[Image:InvasinDensity.JPG]]
|[[Image:InvasinAnaerobic2.JPG]]
|[[Image:Tumor.JPG]]
|-
|'''Fig. 1.''' The red line represents when the invasin output module was paired with the inducible promoter ''lux'' which senses high cell density, the black line is constitutively on invasin. (Anderson - Permission Pending)
|'''Fig. 2.''' The invasin output module was paired with the inducible promoter ''fdhF'' which senses anaerobic conditions. (Anderson - Permission Pending)
|'''Fig. 3.''' Tumor size after injections. Cancer cells were injected into mice, and 5 days later injections with PBS, 6-MPDR, and invasin-enhanced ''E-coli'' began. Tumor cells recieving injections of invasin-enhanced ''E.coli'' and 6-MPDR grew the slowest, but did not get smaller. (Critchley - Permission Pending)
|}
</center>

Using bacteria as a vaccine vector has similar issues of specificity and implementation in a human system. Bacteria was first thought to be a good vector because they would not degrade at mucosal surfaces, they would absorb better and survive the low pH in the gastrointestinal tract (Garmory 471). A particular strain of ''Salmonella'' with one or more deletions in the shikimate pathway, which creates a precursor for phenylalanine and tyrosine, is a promising vector because it gets into the body easily but does not harmfully effect the tissue and is gone within a week or two of introduction. Scientists engineered this strain to carry the ''Yersinia pestis'' V antigen, and studied its ability to protect mice from ''Y. pestis''. Unfortunately, the inoculation was not extremely successful and only 6 of 20 mice showed a strong response (Garmory 473). Garmory and colleagues cited several reasons for this: the copy number of the plasmid containing the V antigen might not have been high enough; the attenuated ''Salmonella'' may not have been able to reach cells that would be targeted by ''Y. pestis''; and the inability of the cell to secrete the antigen. Many changes need to be made to live vector vaccine design before it is an effective method of treating or preventing disease.

Another method of disease prevention with a synthetic biology approach is related to skin cancer. ''E. coli'' engineered to sense single-stranded DNA after DNA damage was coupled with a mechanism to produce a biofilm around the cell. The biofilm output module was tested with DNA damage done by mitomycin C, a DNA crosslinking agent, or UV irradiation, a known cause of skin cancer. Single-stranded DNA activates RecA, which in turn represses the CI repressor protein, thus allowing the transcription of the PL promoter (Kobayashi 8416). The PL promoter is in charge of the ''traA'' gene, which controls biofilm formation (Fig. 4). The biofilm protection, when part of a toggle switch mechanism, lasted indefinitely when pulsed with UV light at 8 J/m2 for only 2 seconds (Kobayashi 8417) (Fig. 5). The biofilm-production mechanism has not been engineered in eukaryotic cells, but a prokaryote-based system that could aid with human skin disease is imaginable. For example, a sunscreen that changes colors when the DNA damage may be too intense for a sunbather as a warning, or a sunscreen that becomes more protective as the possibility of genetic damage increased. Further research should focus on how to best implement the biofilm producing output module to protect human DNA.
<center>

{|
|-
|[[Image:Toggleswitch.JPG]]
|[[Image:UVinducedbiofilm.JPG]]
|-
|'''Fig. 4.''' The RecA / traA toggleswitch mechanism. (Kobayashi - Permission Pending)
|'''Fig. 5.''' Crystal violet absorbance measures biofilm formation. (Kobayashi - Permission Pending)
|}
</center>
Finally, synthetic biology has made its mark on the field of medicine by creating drugs that are less expensive to produce than those made by pharmaceutical companies. Jay Keasling’s project involving the antimalarial drug artemisinin is the best example. Yeast was engineered to increase normal farnesyl pyrophosphate (FPP) production, convert FPP to amorphadiene, and oxidizing amorphadiene to artemisinic acid, which can then easily be separated from the yeast cells and converted to artemisinin in the laboratory (Fig. 6). This innovation allows the production of artemisinin at lower prices than those currently on the market, and without regard to environmental constraints (Ro 942). On a completely different tangent, Collins’s lab engineered T7 bacteriophages to deliver biofilm-degrading enzymes such as dispersin B after infecting and replicating in an ''E. coli'' biofilm. Dispersin B works to degrade an adhesin that is critical for biofilm formation. Phages that released dispersin B were found to be 99.997% effective at removing biofilm, which is 4.5 orders of magnitude higher than non-enzymatic phages (Lu 11197) (Fig. 7). The specificity of phages for a certain type of bacteria make them a viable vector to be used in human treatments, which may range from removing dental plaques to cleaning inserted medical devices. Using synthetic biology to engineer an antimalarial drug in yeast or a biofilm-destroying enzyme in bacteriophages greatly increases the possible contributions to the field of medicine.

<center>

{|
|-
|[[Image:Artemesinin.JPG]]
|[[Image:BiofilmDegradation.JPG]]
|-
|'''Fig. 6.''' All the steps in the box indicate steps that were optimized in yeast for a maximum artemisinic acid output. (Ro - Permission Pending)
|'''Fig. 7.''' The amount of biofilm was decreased when the dispersin B enzymes were incorporated into T7 bacteriophages. (Lu - Permission Pending)
|}
</center>

Medical applications of synthetic biology are wide-ranging and eminently applicable to daily life. Synthetic biology based cancer treatments that utilize ''E.coli'' engineered to sense and attack tissues with high cell density and low oxygen availability are promising and need to be tested in vivo. Designing vectors out of attenuated bacteria may be a safe, effective method for disease treatment. Using biofilms to sense sun damage potential may decrease the likelihood of skin cancer. Finally, using engineered cells to produce medicines in a cost-effective, environmentally-friendly way may revolutionize the pharmaceutical industry. Investing more research efforts in the field of synthetic biology may turn out to be an investment in medical technology.

'''Works Cited'''

-[http://www.bio.davidson.edu/courses/synthetic/papers/2006_anderson.pdf Anderson JC, Clarke EJ, Arkin, AP, and Voigt CA (2006). Environmentally controlled invasion of cancer cells by engineered bacteria. Journal of Molecular Biology 355:619-27.]

-[http://www.nature.com/gt/journal/v11/n15/abs/3302281a.html Critchley RJ, Jezzard S, Radford KJ, Goussard S, Lemoine NR, et al. (2004). Potential therapeutic applications of recombinant, invasive ''E. coli''. Gene Therapy 11:1224-33.]

-Garmory HS, Leary SEC, Griffin, KF, Williamson D, Brown, KA, and Titball RW (2003). The use of live attenuated bacteria as a delivery system for heterologous antigens. Journal of Drug Targeting 11:471-79.

-Kobayashi H, et al. (2004). Programmable cells: Interfacing natural and engineered gene networks. PNAS 101: 8414-19.

-[http://www.bio.davidson.edu/courses/synthetic/papers/Phage_BioFilms.pdf Lu, TK, and Collins JJ (2007). Dispersing biofilms with engineered enzymatic bacteriophage. PNAS 104: 11197-11202.]

-Nemunaitis J, Cunningham C, Senzer N, Kuhn J, Cramm J, Litz C, et al. (2003). Pilot trial of genetically modified, attenuated ''Salmonella'' expressing cytosine deaminase gene in refractory cancer patients. Cancer Gene Therapy 10:737-744.

-Pawelek JM, Low KB, and Bermudes D (2003). Bacteria as tumour-targeting vectors. Lancet Oncology 4:548-56.

-[http://www.bio.davidson.edu/courses/synthetic/papers/Keasling_malaria.pdf Ro D, et al. (2006). Production of the antimalarial drug precursor artemisinic acid in engineered yeast. Nature 440:940-43.]

Medical Applications of Synthetic Biology - Samantha Simpson

2007-11-23T21:29:41Z

Sasimpson:

'''Project Proposal'''

My project will be on medical applications of synthetic biology. I will reference Anderson's paper on utilizing quorum-sensing and hypoxia-responsive genes coupled with invasin from ''Yersinia tuberculosis'' to invade cancer-causing cells. Coupled with Critchley's work, which describes a bacterial system that invades eukaryotic cells and delivers proteins coded for in the bacteria's genome, one could possibly create 'search and destroy' ''E.coli'' that can locate, invade, and kill tumor cells. Garmory's paper also highlights the possibility of using bacteria as a drug-delivering system, specifically vaccine vectors. Kobayashi describes cells that produce a protective biofilm layer after exposure to DNA-damaging agents. Ro engineers yeast to create an anti-malarial drug that can be created at a lower cost than the previous standard production process. These papers, and others that have a focus on direct medical applications such as novel cancer therapies, vaccination technology, biological protection, and the creation of new medicines, will be referenced to create a cohesive overview of applications of synthetic biology to the medical field.

'''Paper'''

Synthetic biology is a rapidly emerging field that strives to re-design existing biological systems and components or fabricate novel biological systems and components. The end result of experimentation in synthetic biology is to help scientists understand a naturally occurring genetic pathway more fully or to create a new, useful pathway. Recent research efforts have focused on engineering bacteria to create a new form of biofuel or to make promoters of varying efficiencies for more exact gene expression. Other research efforts focus on engineering yeast to make antimalarial drugs or engineering ''E. coli'' to recognize and invade tumor-like cells, both of which are medical applications of synthetic biology. Advancements such as novel cancer therapies, vaccination technology, biological protection, and the creation of new medicines highlight synthetic biology’s potential to create breakthroughs in medical science. This paper will examine various medical applications of synthetic biology and further development that needs to be done before current research can be used in hospitals and doctors’ offices.

Synthetic biologists consider finding a cure for cancer a reachable goal. Previous to the development of synthetic biology as a field, it was known that three types of bacteria, ''Bifodobacterium'', ''Clostridium'', and ''Salmonella'' all preferentially infect the dense cells of tumors (Pawelek 548). All three types of bacteria are associated with reducing tumor size in patients who are infected with them, and it was noted that all three bacteria act anaerobically. Capitalizing on the idea that tumors create an unusually dense, anaerobic mass of cells, Christopher Voigt’s lab engineered bacteria to identify and invade cancer cells. They utilized the ''lux'' quorum sensing from ''Vibrio fischeri'' to identify cells growing at high densities (Fig. 1) and the ''fdhF'' promoter to identify cells growing in anaerobic conditions (Fig. 2) (Anderson 619). They coupled this identification mechanism with the invasin output module from ''Yersinia pseudotuberculosis'' so the ''E. coli'' could invade specific cells. Next, scientists hope to insert a gene that would destroy the invaded cells, making the complete cycle a search, invade, and destroy loop that would only be sensitive to cancer cells. One promising destroy mechanism has been implemented in humans via intratumoral injection, based on ''Salmonella'' that contains the ''E. coli'' cytosine deaminase gene that converts 5-fluorocytosine to 5-fluorouracil, a chemotherapy drug (Nemunaitis 737). Unfortunately, while the tumors did produce 5-fluorouracil (5-FU), they did not produce it in high enough yields to cause regression of the cancer. Another experiment flooded mouse cells with 6-methylpurine-2’deoxyribose (6-MPDR), which was converted to the toxin 6-methyl purine (MeP) by the enzyme purine nucleoside phosphorylase (PNP), which is naturally found in ''E. coli'' (Critchley 1226) (Fig. 3). The problem with this is that all cells in the body might be exposed to the toxin MeP. In addition to the search and destroy mechanism, scientists hope to increase selectivity by combining the hypoxia-sensing and density-sensing units so that ''E. coli'' will only enter eukaryotic cells that exhibit both characteristics. This would make it more likely that invasin-enhanced ''E. coli'' would only invade cancer cells rather than muscle tissue that had low oxygen due to exercise (Anderson 624). To create a fully functional, synthetic biology-based approach to cancer therapy, scientists need to develop bacteria that discriminately invades cancer cells and destroys them. Most of the pieces are there – scientists have engineered ''E. coli'' that selectively invades hypoxic cells or dense cells and ''Salmonella'' that can destroy tumors in vivo – they just need to be put together effectively.

<center>

{|
|-
|[[Image:InvasinDensity.JPG]]
|[[Image:InvasinAnaerobic2.JPG]]
|[[Image:Tumor.JPG]]
|-
|'''Fig. 1.''' The red line represents when the invasin output module was paired with the inducible promoter ''lux'' which senses high cell density, the black line is constitutively on invasin. (Anderson - Permission Pending)
|'''Fig. 2.''' The invasin output module was paired with the inducible promoter ''fdhF'' which senses anaerobic conditions. (Anderson - Permission Pending)
|'''Fig. 3.''' Tumor size after injections. Cancer cells were injected into mice, and 5 days later injections with PBS, 6-MPDR, and invasin-enhanced ''E-coli'' began. Tumor cells recieving injections of invasin-enhanced ''E.coli'' and 6-MPDR grew the slowest, but did not get smaller. (Critchley - Permission Pending)
|}
</center>

Using bacteria as a vaccine vector has similar issues of specificity and implementation in a human system. Bacteria was first thought to be a good vector because they would not degrade at mucosal surfaces, they would absorb better and survive the low pH in the gastrointestinal tract (Garmory 471). A particular strain of ''Salmonella'' with one or more deletions in the shikimate pathway, which creates a precursor for phenylalanine and tyrosine, is a promising vector because it gets into the body easily but does not harmfully effect the tissue and is gone within a week or two of introduction. Scientists engineered this strain to carry the ''Yersinia pestis'' V antigen, and studied its ability to protect mice from ''Y. pestis''. Unfortunately, the inoculation was not extremely successful and only 6 of 20 mice showed a strong response (Garmory 473). Garmory and colleagues cited several reasons for this: the copy number of the plasmid containing the V antigen might not have been high enough; the attenuated ''Salmonella'' may not have been able to reach cells that would be targeted by ''Y. pestis''; and the inability of the cell to secrete the antigen. Many changes need to be made to live vector vaccine design before it is an effective method of treating or preventing disease.

Another method of disease prevention with a synthetic biology approach is related to skin cancer. ''E. coli'' engineered to sense single-stranded DNA after DNA damage was coupled with a mechanism to produce a biofilm around the cell. The biofilm output module was tested with DNA damage done by mitomycin C, a DNA crosslinking agent, or UV irradiation, a known cause of skin cancer. Single-stranded DNA activates RecA, which in turn represses the CI repressor protein, thus allowing the transcription of the PL promoter (Kobayashi 8416). The PL promoter is in charge of the ''traA'' gene, which controls biofilm formation (Fig. 4). The biofilm protection, when part of a toggle switch mechanism, lasted indefinitely when pulsed with UV light at 8 J/m2 for only 2 seconds (Kobayashi 8417) (Fig. 5). The biofilm-production mechanism has not been engineered in eukaryotic cells, but a prokaryote-based system that could aid with human skin disease is imaginable. For example, a sunscreen that changes colors when the DNA damage may be too intense for a sunbather as a warning, or a sunscreen that becomes more protective as the possibility of genetic damage increased. Further research should focus on how to best implement the biofilm producing output module to protect human DNA.
<center>

{|
|-
|[[Image:Toggleswitch.JPG]]
|[[Image:UVinducedbiofilm.JPG]]
|-
|'''Fig. 4.''' The RecA / traA toggleswitch mechanism. (Kobayashi - Permission Pending)
|'''Fig. 5.''' Crystal violet absorbance measures biofilm formation. (Kobayashi - Permission Pending)
|}
</center>
Finally, synthetic biology has made its mark on the field of medicine by creating drugs that are less expensive to produce than those made by pharmaceutical companies. Jay Keasling’s project involving the antimalarial drug artemisinin is the best example. Yeast was engineered to increase normal farnesyl pyrophosphate (FPP) production, convert FPP to amorphadiene, and oxidizing amorphadiene to artemisinic acid, which can then easily be separated from the yeast cells and converted to artemisinin in the laboratory (Fig. 6). This innovation allows the production of artemisinin at lower prices than those currently on the market, and without regard to environmental constraints (Ro 942). On a completely different tangent, Collins’s lab engineered T7 bacteriophages to deliver biofilm-degrading enzymes such as dispersin B after infecting and replicating in an ''E. coli'' biofilm. Dispersin B works to degrade an adhesin that is critical for biofilm formation. Phages that released dispersin B were found to be 99.997% effective at removing biofilm, which is 4.5 orders of magnitude higher than non-enzymatic phages (Lu 11197) (Fig. 7). The specificity of phages for a certain type of bacteria make them a viable vector to be used in human treatments, which may range from removing dental plaques to cleaning inserted medical devices. Using synthetic biology to engineer an antimalarial drug in yeast or a biofilm-destroying enzyme in bacteriophages greatly increases the possible contributions to the field of medicine.

<center>

{|
|-
|[[Image:Artemesinin.JPG]]
|[[Image:BiofilmDegradation.JPG]]
|-
|'''Fig. 6.''' All the steps in the box indicate steps that were optimized in yeast for a maximum artemisinic acid output. (Ro - Permission Pending)
|'''Fig. 7.''' The amount of biofilm was decreased when the dispersin B enzymes were incorporated into T7 bacteriophages. (Lu - Permission Pending)
|}
</center>

Medical applications of synthetic biology are wide-ranging and eminently applicable to daily life. Synthetic biology based cancer treatments that utilize ''E.coli'' engineered to sense and attack tissues with high cell density and low oxygen availability are promising and need to be tested in vivo. Designing vectors out of attenuated bacteria may be a safe, effective method for disease treatment. Using biofilms to sense sun damage potential may decrease the likelihood of skin cancer. Finally, using engineered cells to produce medicines in a cost-effective, environmentally-friendly way may revolutionize the pharmaceutical industry. Investing more research efforts in the field of synthetic biology may turn out to be an investment in medical technology.

'''Works Cited'''

-[http://www.bio.davidson.edu/courses/synthetic/papers/2006_anderson.pdf Anderson JC, Clarke EJ, Arkin, AP, and Voigt CA (2006). Environmentally controlled invasion of cancer cells by engineered bacteria. Journal of Molecular Biology 355:619-27.]

-Critchley RJ, Jezzard S, Radford KJ, Goussard S, Lemoine NR, et al. (2004). Potential therapeutic applications of recombinant, invasive ''E. coli''. Gene Therapy 11:1224-33.

-Garmory HS, Leary SEC, Griffin, KF, Williamson D, Brown, KA, and Titball RW (2003). The use of live attenuated bacteria as a delivery system for heterologous antigens. Journal of Drug Targeting 11:471-79.

-Kobayashi H, et al. (2004). Programmable cells: Interfacing natural and engineered gene networks. PNAS 101: 8414-19.

-[http://www.bio.davidson.edu/courses/synthetic/papers/Phage_BioFilms.pdf Lu, TK, and Collins JJ (2007). Dispersing biofilms with engineered enzymatic bacteriophage. PNAS 104: 11197-11202.]

-Nemunaitis J, Cunningham C, Senzer N, Kuhn J, Cramm J, Litz C, et al. (2003). Pilot trial of genetically modified, attenuated ''Salmonella'' expressing cytosine deaminase gene in refractory cancer patients. Cancer Gene Therapy 10:737-744.

-Pawelek JM, Low KB, and Bermudes D (2003). Bacteria as tumour-targeting vectors. Lancet Oncology 4:548-56.

-[http://www.bio.davidson.edu/courses/synthetic/papers/Keasling_malaria.pdf Ro D, et al. (2006). Production of the antimalarial drug precursor artemisinic acid in engineered yeast. Nature 440:940-43.]

Medical Applications of Synthetic Biology - Samantha Simpson

2007-11-23T21:28:28Z

Sasimpson:

'''Project Proposal'''

My project will be on medical applications of synthetic biology. I will reference Anderson's paper on utilizing quorum-sensing and hypoxia-responsive genes coupled with invasin from ''Yersinia tuberculosis'' to invade cancer-causing cells. Coupled with Critchley's work, which describes a bacterial system that invades eukaryotic cells and delivers proteins coded for in the bacteria's genome, one could possibly create 'search and destroy' ''E.coli'' that can locate, invade, and kill tumor cells. Garmory's paper also highlights the possibility of using bacteria as a drug-delivering system, specifically vaccine vectors. Kobayashi describes cells that produce a protective biofilm layer after exposure to DNA-damaging agents. Ro engineers yeast to create an anti-malarial drug that can be created at a lower cost than the previous standard production process. These papers, and others that have a focus on direct medical applications such as novel cancer therapies, vaccination technology, biological protection, and the creation of new medicines, will be referenced to create a cohesive overview of applications of synthetic biology to the medical field.

'''Paper'''

Synthetic biology is a rapidly emerging field that strives to re-design existing biological systems and components or fabricate novel biological systems and components. The end result of experimentation in synthetic biology is to help scientists understand a naturally occurring genetic pathway more fully or to create a new, useful pathway. Recent research efforts have focused on engineering bacteria to create a new form of biofuel or to make promoters of varying efficiencies for more exact gene expression. Other research efforts focus on engineering yeast to make antimalarial drugs or engineering ''E. coli'' to recognize and invade tumor-like cells, both of which are medical applications of synthetic biology. Advancements such as novel cancer therapies, vaccination technology, biological protection, and the creation of new medicines highlight synthetic biology’s potential to create breakthroughs in medical science. This paper will examine various medical applications of synthetic biology and further development that needs to be done before current research can be used in hospitals and doctors’ offices.

Synthetic biologists consider finding a cure for cancer a reachable goal. Previous to the development of synthetic biology as a field, it was known that three types of bacteria, ''Bifodobacterium'', ''Clostridium'', and ''Salmonella'' all preferentially infect the dense cells of tumors (Pawelek 548). All three types of bacteria are associated with reducing tumor size in patients who are infected with them, and it was noted that all three bacteria act anaerobically. Capitalizing on the idea that tumors create an unusually dense, anaerobic mass of cells, Christopher Voigt’s lab engineered bacteria to identify and invade cancer cells. They utilized the ''lux'' quorum sensing from ''Vibrio fischeri'' to identify cells growing at high densities (Fig. 1) and the ''fdhF'' promoter to identify cells growing in anaerobic conditions (Fig. 2) (Anderson 619). They coupled this identification mechanism with the invasin output module from ''Yersinia pseudotuberculosis'' so the ''E. coli'' could invade specific cells. Next, scientists hope to insert a gene that would destroy the invaded cells, making the complete cycle a search, invade, and destroy loop that would only be sensitive to cancer cells. One promising destroy mechanism has been implemented in humans via intratumoral injection, based on ''Salmonella'' that contains the ''E. coli'' cytosine deaminase gene that converts 5-fluorocytosine to 5-fluorouracil, a chemotherapy drug (Nemunaitis 737). Unfortunately, while the tumors did produce 5-fluorouracil (5-FU), they did not produce it in high enough yields to cause regression of the cancer. Another experiment flooded mouse cells with 6-methylpurine-2’deoxyribose (6-MPDR), which was converted to the toxin 6-methyl purine (MeP) by the enzyme purine nucleoside phosphorylase (PNP), which is naturally found in ''E. coli'' (Critchley 1226) (Fig. 3). The problem with this is that all cells in the body might be exposed to the toxin MeP. In addition to the search and destroy mechanism, scientists hope to increase selectivity by combining the hypoxia-sensing and density-sensing units so that ''E. coli'' will only enter eukaryotic cells that exhibit both characteristics. This would make it more likely that invasin-enhanced ''E. coli'' would only invade cancer cells rather than muscle tissue that had low oxygen due to exercise (Anderson 624). To create a fully functional, synthetic biology-based approach to cancer therapy, scientists need to develop bacteria that discriminately invades cancer cells and destroys them. Most of the pieces are there – scientists have engineered ''E. coli'' that selectively invades hypoxic cells or dense cells and ''Salmonella'' that can destroy tumors in vivo – they just need to be put together effectively.

<center>

{|
|-
|[[Image:InvasinDensity.JPG]]
|[[Image:InvasinAnaerobic2.JPG]]
|[[Image:Tumor.JPG]]
|-
|'''Fig. 1.''' The red line represents when the invasin output module was paired with the inducible promoter ''lux'' which senses high cell density, the black line is constitutively on invasin. (Anderson - Permission Pending)
|'''Fig. 2.''' The invasin output module was paired with the inducible promoter ''fdhF'' which senses anaerobic conditions. (Anderson - Permission Pending)
|'''Fig. 3.''' Tumor size after injections. Cancer cells were injected into mice, and 5 days later injections with PBS, 6-MPDR, and invasin-enhanced ''E-coli'' began. Tumor cells recieving injections of invasin-enhanced ''E.coli'' and 6-MPDR grew the slowest, but did not get smaller. (Critchley - Permission Pending)
|}
</center>

Using bacteria as a vaccine vector has similar issues of specificity and implementation in a human system. Bacteria was first thought to be a good vector because they would not degrade at mucosal surfaces, they would absorb better and survive the low pH in the gastrointestinal tract (Garmory 471). A particular strain of ''Salmonella'' with one or more deletions in the shikimate pathway, which creates a precursor for phenylalanine and tyrosine, is a promising vector because it gets into the body easily but does not harmfully effect the tissue and is gone within a week or two of introduction. Scientists engineered this strain to carry the ''Yersinia pestis'' V antigen, and studied its ability to protect mice from ''Y. pestis''. Unfortunately, the inoculation was not extremely successful and only 6 of 20 mice showed a strong response (Garmory 473). Garmory and colleagues cited several reasons for this: the copy number of the plasmid containing the V antigen might not have been high enough; the attenuated ''Salmonella'' may not have been able to reach cells that would be targeted by ''Y. pestis''; and the inability of the cell to secrete the antigen. Many changes need to be made to live vector vaccine design before it is an effective method of treating or preventing disease.

Another method of disease prevention with a synthetic biology approach is related to skin cancer. ''E. coli'' engineered to sense single-stranded DNA after DNA damage was coupled with a mechanism to produce a biofilm around the cell. The biofilm output module was tested with DNA damage done by mitomycin C, a DNA crosslinking agent, or UV irradiation, a known cause of skin cancer. Single-stranded DNA activates RecA, which in turn represses the CI repressor protein, thus allowing the transcription of the PL promoter (Kobayashi 8416). The PL promoter is in charge of the ''traA'' gene, which controls biofilm formation (Fig. 4). The biofilm protection, when part of a toggle switch mechanism, lasted indefinitely when pulsed with UV light at 8 J/m2 for only 2 seconds (Kobayashi 8417) (Fig. 5). The biofilm-production mechanism has not been engineered in eukaryotic cells, but a prokaryote-based system that could aid with human skin disease is imaginable. For example, a sunscreen that changes colors when the DNA damage may be too intense for a sunbather as a warning, or a sunscreen that becomes more protective as the possibility of genetic damage increased. Further research should focus on how to best implement the biofilm producing output module to protect human DNA.
<center>

{|
|-
|[[Image:Toggleswitch.JPG]]
|[[Image:UVinducedbiofilm.JPG]]
|-
|'''Fig. 4.''' The RecA / traA toggleswitch mechanism. (Kobayashi - Permission Pending)
|'''Fig. 5.''' Crystal violet absorbance measures biofilm formation. (Kobayashi - Permission Pending)
|}
</center>
Finally, synthetic biology has made its mark on the field of medicine by creating drugs that are less expensive to produce than those made by pharmaceutical companies. Jay Keasling’s project involving the antimalarial drug artemisinin is the best example. Yeast was engineered to increase normal farnesyl pyrophosphate (FPP) production, convert FPP to amorphadiene, and oxidizing amorphadiene to artemisinic acid, which can then easily be separated from the yeast cells and converted to artemisinin in the laboratory (Fig. 6). This innovation allows the production of artemisinin at lower prices than those currently on the market, and without regard to environmental constraints (Ro 942). On a completely different tangent, Collins’s lab engineered T7 bacteriophages to deliver biofilm-degrading enzymes such as dispersin B after infecting and replicating in an ''E. coli'' biofilm. Dispersin B works to degrade an adhesin that is critical for biofilm formation. Phages that released dispersin B were found to be 99.997% effective at removing biofilm, which is 4.5 orders of magnitude higher than non-enzymatic phages (Lu 11197) (Fig. 7). The specificity of phages for a certain type of bacteria make them a viable vector to be used in human treatments, which may range from removing dental plaques to cleaning inserted medical devices. Using synthetic biology to engineer an antimalarial drug in yeast or a biofilm-destroying enzyme in bacteriophages greatly increases the possible contributions to the field of medicine.

<center>

{|
|-
|[[Image:Artemesinin.JPG]]
|[[Image:BiofilmDegradation.JPG]]
|-
|'''Fig. 6.''' All the steps in the box indicate steps that were optimized in yeast for a maximum artemisinic acid output. (Ro - Permission Pending)
|'''Fig. 7.''' The amount of biofilm was decreased when the dispersin B enzymes were incorporated into T7 bacteriophages. (Lu - Permission Pending)
|}
</center>

Medical applications of synthetic biology are wide-ranging and eminently applicable to daily life. Synthetic biology based cancer treatments that utilize ''E.coli'' engineered to sense and attack tissues with high cell density and low oxygen availability are promising and need to be tested in vivo. Designing vectors out of attenuated bacteria may be a safe, effective method for disease treatment. Using biofilms to sense sun damage potential may decrease the likelihood of skin cancer. Finally, using engineered cells to produce medicines in a cost-effective, environmentally-friendly way may revolutionize the pharmaceutical industry. Investing more research efforts in the field of synthetic biology may turn out to be an investment in medical technology.

'''Works Cited'''

-[http://www.bio.davidson.edu/courses/synthetic/papers/2006_anderson.pdf Anderson JC, Clarke EJ, Arkin, AP, and Voigt CA (2006). Environmentally controlled invasion of cancer cells by engineered bacteria. Journal of Molecular Biology 355:619-27.]

-Critchley RJ, Jezzard S, Radford KJ, Goussard S, Lemoine NR, et al. (2004). Potential therapeutic applications of recombinant, invasive ''E. coli''. Gene Therapy 11:1224-33.

-Garmory HS, Leary SEC, Griffin, KF, Williamson D, Brown, KA, and Titball RW (2003). The use of live attenuated bacteria as a delivery system for heterologous antigens. Journal of Drug Targeting 11:471-79.

-Kobayashi H, et al. (2004). Programmable cells: Interfacing natural and engineered gene networks. PNAS 101: 8414-19.

-[http://www.bio.davidson.edu/courses/synthetic/papers/Phage_BioFilms.pdf Lu, TK, and Collins JJ (2007). Dispersing biofilms with engineered enzymatic bacteriophage. PNAS 104: 11197-11202.]

-Nemunaitis J, Cunningham C, Senzer N, Kuhn J, Cramm J, Litz C, et al. (2003). Pilot trial of genetically modified, attenuated ''Salmonella'' expressing cytosine deaminase gene in refractory cancer patients. Cancer Gene Therapy 10:737-744.

-Pawelek JM, Low KB, and Bermudes D (2003). Bacteria as tumour-targeting vectors. Lancet Oncology 4:548-56.

-Ro D, et al. (2006). Production of the antimalarial drug precursor artemisinic acid in engineered yeast. Nature 440:940-43.

Medical Applications of Synthetic Biology - Samantha Simpson

2007-11-23T21:26:57Z

Sasimpson:

'''Project Proposal'''

My project will be on medical applications of synthetic biology. I will reference Anderson's paper on utilizing quorum-sensing and hypoxia-responsive genes coupled with invasin from ''Yersinia tuberculosis'' to invade cancer-causing cells. Coupled with Critchley's work, which describes a bacterial system that invades eukaryotic cells and delivers proteins coded for in the bacteria's genome, one could possibly create 'search and destroy' ''E.coli'' that can locate, invade, and kill tumor cells. Garmory's paper also highlights the possibility of using bacteria as a drug-delivering system, specifically vaccine vectors. Kobayashi describes cells that produce a protective biofilm layer after exposure to DNA-damaging agents. Ro engineers yeast to create an anti-malarial drug that can be created at a lower cost than the previous standard production process. These papers, and others that have a focus on direct medical applications such as novel cancer therapies, vaccination technology, biological protection, and the creation of new medicines, will be referenced to create a cohesive overview of applications of synthetic biology to the medical field.

'''Paper'''

Synthetic biology is a rapidly emerging field that strives to re-design existing biological systems and components or fabricate novel biological systems and components. The end result of experimentation in synthetic biology is to help scientists understand a naturally occurring genetic pathway more fully or to create a new, useful pathway. Recent research efforts have focused on engineering bacteria to create a new form of biofuel or to make promoters of varying efficiencies for more exact gene expression. Other research efforts focus on engineering yeast to make antimalarial drugs or engineering ''E. coli'' to recognize and invade tumor-like cells, both of which are medical applications of synthetic biology. Advancements such as novel cancer therapies, vaccination technology, biological protection, and the creation of new medicines highlight synthetic biology’s potential to create breakthroughs in medical science. This paper will examine various medical applications of synthetic biology and further development that needs to be done before current research can be used in hospitals and doctors’ offices.

Synthetic biologists consider finding a cure for cancer a reachable goal. Previous to the development of synthetic biology as a field, it was known that three types of bacteria, ''Bifodobacterium'', ''Clostridium'', and ''Salmonella'' all preferentially infect the dense cells of tumors (Pawelek 548). All three types of bacteria are associated with reducing tumor size in patients who are infected with them, and it was noted that all three bacteria act anaerobically. Capitalizing on the idea that tumors create an unusually dense, anaerobic mass of cells, Christopher Voigt’s lab engineered bacteria to identify and invade cancer cells. They utilized the ''lux'' quorum sensing from ''Vibrio fischeri'' to identify cells growing at high densities (Fig. 1) and the ''fdhF'' promoter to identify cells growing in anaerobic conditions (Fig. 2) (Anderson 619). They coupled this identification mechanism with the invasin output module from ''Yersinia pseudotuberculosis'' so the ''E. coli'' could invade specific cells. Next, scientists hope to insert a gene that would destroy the invaded cells, making the complete cycle a search, invade, and destroy loop that would only be sensitive to cancer cells. One promising destroy mechanism has been implemented in humans via intratumoral injection, based on ''Salmonella'' that contains the ''E. coli'' cytosine deaminase gene that converts 5-fluorocytosine to 5-fluorouracil, a chemotherapy drug (Nemunaitis 737). Unfortunately, while the tumors did produce 5-fluorouracil (5-FU), they did not produce it in high enough yields to cause regression of the cancer. Another experiment flooded mouse cells with 6-methylpurine-2’deoxyribose (6-MPDR), which was converted to the toxin 6-methyl purine (MeP) by the enzyme purine nucleoside phosphorylase (PNP), which is naturally found in ''E. coli'' (Critchley 1226) (Fig. 3). The problem with this is that all cells in the body might be exposed to the toxin MeP. In addition to the search and destroy mechanism, scientists hope to increase selectivity by combining the hypoxia-sensing and density-sensing units so that ''E. coli'' will only enter eukaryotic cells that exhibit both characteristics. This would make it more likely that invasin-enhanced ''E. coli'' would only invade cancer cells rather than muscle tissue that had low oxygen due to exercise (Anderson 624). To create a fully functional, synthetic biology-based approach to cancer therapy, scientists need to develop bacteria that discriminately invades cancer cells and destroys them. Most of the pieces are there – scientists have engineered ''E. coli'' that selectively invades hypoxic cells or dense cells and ''Salmonella'' that can destroy tumors in vivo – they just need to be put together effectively.

<center>

{|
|-
|[[Image:InvasinDensity.JPG]]
|[[Image:InvasinAnaerobic2.JPG]]
|[[Image:Tumor.JPG]]
|-
|'''Fig. 1.''' The red line represents when the invasin output module was paired with the inducible promoter ''lux'' which senses high cell density, the black line is constitutively on invasin. (Anderson - Permission Pending)
|'''Fig. 2.''' The invasin output module was paired with the inducible promoter ''fdhF'' which senses anaerobic conditions. (Anderson - Permission Pending)
|'''Fig. 3.''' Tumor size after injections. Cancer cells were injected into mice, and 5 days later injections with PBS, 6-MPDR, and invasin-enhanced ''E-coli'' began. Tumor cells recieving injections of invasin-enhanced ''E.coli'' and 6-MPDR grew the slowest, but did not get smaller. (Critchley - Permission Pending)
|}
</center>

Using bacteria as a vaccine vector has similar issues of specificity and implementation in a human system. Bacteria was first thought to be a good vector because they would not degrade at mucosal surfaces, they would absorb better and survive the low pH in the gastrointestinal tract (Garmory 471). A particular strain of ''Salmonella'' with one or more deletions in the shikimate pathway, which creates a precursor for phenylalanine and tyrosine, is a promising vector because it gets into the body easily but does not harmfully effect the tissue and is gone within a week or two of introduction. Scientists engineered this strain to carry the ''Yersinia pestis'' V antigen, and studied its ability to protect mice from ''Y. pestis''. Unfortunately, the inoculation was not extremely successful and only 6 of 20 mice showed a strong response (Garmory 473). Garmory and colleagues cited several reasons for this: the copy number of the plasmid containing the V antigen might not have been high enough; the attenuated ''Salmonella'' may not have been able to reach cells that would be targeted by ''Y. pestis''; and the inability of the cell to secrete the antigen. Many changes need to be made to live vector vaccine design before it is an effective method of treating or preventing disease.

Another method of disease prevention with a synthetic biology approach is related to skin cancer. ''E. coli'' engineered to sense single-stranded DNA after DNA damage was coupled with a mechanism to produce a biofilm around the cell. The biofilm output module was tested with DNA damage done by mitomycin C, a DNA crosslinking agent, or UV irradiation, a known cause of skin cancer. Single-stranded DNA activates RecA, which in turn represses the CI repressor protein, thus allowing the transcription of the PL promoter (Kobayashi 8416). The PL promoter is in charge of the ''traA'' gene, which controls biofilm formation (Fig. 4). The biofilm protection, when part of a toggle switch mechanism, lasted indefinitely when pulsed with UV light at 8 J/m2 for only 2 seconds (Kobayashi 8417) (Fig. 5). The biofilm-production mechanism has not been engineered in eukaryotic cells, but a prokaryote-based system that could aid with human skin disease is imaginable. For example, a sunscreen that changes colors when the DNA damage may be too intense for a sunbather as a warning, or a sunscreen that becomes more protective as the possibility of genetic damage increased. Further research should focus on how to best implement the biofilm producing output module to protect human DNA.
<center>

{|
|-
|[[Image:Toggleswitch.JPG]]
|[[Image:UVinducedbiofilm.JPG]]
|-
|'''Fig. 4.''' The RecA / traA toggleswitch mechanism. (Kobayashi - Permission Pending)
|'''Fig. 5.''' Crystal violet absorbance measures biofilm formation. (Kobayashi - Permission Pending)
|}
</center>
Finally, synthetic biology has made its mark on the field of medicine by creating drugs that are less expensive to produce than those made by pharmaceutical companies. Jay Keasling’s project involving the antimalarial drug artemisinin is the best example. Yeast was engineered to increase normal farnesyl pyrophosphate (FPP) production, convert FPP to amorphadiene, and oxidizing amorphadiene to artemisinic acid, which can then easily be separated from the yeast cells and converted to artemisinin in the laboratory (Fig. 6). This innovation allows the production of artemisinin at lower prices than those currently on the market, and without regard to environmental constraints (Ro 942). On a completely different tangent, Collins’s lab engineered T7 bacteriophages to deliver biofilm-degrading enzymes such as dispersin B after infecting and replicating in an ''E. coli'' biofilm. Dispersin B works to degrade an adhesin that is critical for biofilm formation. Phages that released dispersin B were found to be 99.997% effective at removing biofilm, which is 4.5 orders of magnitude higher than non-enzymatic phages (Lu 11197) (Fig. 7). The specificity of phages for a certain type of bacteria make them a viable vector to be used in human treatments, which may range from removing dental plaques to cleaning inserted medical devices. Using synthetic biology to engineer an antimalarial drug in yeast or a biofilm-destroying enzyme in bacteriophages greatly increases the possible contributions to the field of medicine.

<center>

{|
|-
|[[Image:Artemesinin.JPG]]
|[[Image:BiofilmDegradation.JPG]]
|-
|'''Fig. 6.''' All the steps in the box indicate steps that were optimized in yeast for a maximum artemisinic acid output. (Ro - Permission Pending)
|'''Fig. 7.''' The amount of biofilm was decreased when the dispersin B enzymes were incorporated into T7 bacteriophages. (Lu - Permission Pending)
|}
</center>

Medical applications of synthetic biology are wide-ranging and eminently applicable to daily life. Synthetic biology based cancer treatments that utilize ''E.coli'' engineered to sense and attack tissues with high cell density and low oxygen availability are promising and need to be tested in vivo. Designing vectors out of attenuated bacteria may be a safe, effective method for disease treatment. Using biofilms to sense sun damage potential may decrease the likelihood of skin cancer. Finally, using engineered cells to produce medicines in a cost-effective, environmentally-friendly way may revolutionize the pharmaceutical industry. Investing more research efforts in the field of synthetic biology may turn out to be an investment in medical technology.

'''Works Cited'''

-[http://www.bio.davidson.edu/courses/synthetic/papers/2006_anderson.pdf Anderson JC, Clarke EJ, Arkin, AP, and Voigt CA (2006). Environmentally controlled invasion of cancer cells by engineered bacteria. Journal of Molecular Biology 355:619-27.]

-Critchley RJ, Jezzard S, Radford KJ, Goussard S, Lemoine NR, et al. (2004). Potential therapeutic applications of recombinant, invasive ''E. coli''. Gene Therapy 11:1224-33.

-Garmory HS, Leary SEC, Griffin, KF, Williamson D, Brown, KA, and Titball RW (2003). The use of live attenuated bacteria as a delivery system for heterologous antigens. Journal of Drug Targeting 11:471-79.

-Kobayashi H, et al. (2004). Programmable cells: Interfacing natural and engineered gene networks. PNAS 101: 8414-19.

-Lu, TK, and Collins JJ (2007). Dispersing biofilms with engineered enzymatic bacteriophage. PNAS 104: 11197-11202.

-Nemunaitis J, Cunningham C, Senzer N, Kuhn J, Cramm J, Litz C, et al. (2003). Pilot trial of genetically modified, attenuated ''Salmonella'' expressing cytosine deaminase gene in refractory cancer patients. Cancer Gene Therapy 10:737-744.

-Pawelek JM, Low KB, and Bermudes D (2003). Bacteria as tumour-targeting vectors. Lancet Oncology 4:548-56.

-Ro D, et al. (2006). Production of the antimalarial drug precursor artemisinic acid in engineered yeast. Nature 440:940-43.

Medical Applications of Synthetic Biology - Samantha Simpson

2007-11-23T21:25:19Z

Sasimpson:

'''Project Proposal'''

My project will be on medical applications of synthetic biology. I will reference Anderson's paper on utilizing quorum-sensing and hypoxia-responsive genes coupled with invasin from ''Yersinia tuberculosis'' to invade cancer-causing cells. Coupled with Critchley's work, which describes a bacterial system that invades eukaryotic cells and delivers proteins coded for in the bacteria's genome, one could possibly create 'search and destroy' ''E.coli'' that can locate, invade, and kill tumor cells. Garmory's paper also highlights the possibility of using bacteria as a drug-delivering system, specifically vaccine vectors. Kobayashi describes cells that produce a protective biofilm layer after exposure to DNA-damaging agents. Ro engineers yeast to create an anti-malarial drug that can be created at a lower cost than the previous standard production process. These papers, and others that have a focus on direct medical applications such as novel cancer therapies, vaccination technology, biological protection, and the creation of new medicines, will be referenced to create a cohesive overview of applications of synthetic biology to the medical field.

'''Paper'''

Synthetic biology is a rapidly emerging field that strives to re-design existing biological systems and components or fabricate novel biological systems and components. The end result of experimentation in synthetic biology is to help scientists understand a naturally occurring genetic pathway more fully or to create a new, useful pathway. Recent research efforts have focused on engineering bacteria to create a new form of biofuel or to make promoters of varying efficiencies for more exact gene expression. Other research efforts focus on engineering yeast to make antimalarial drugs or engineering ''E. coli'' to recognize and invade tumor-like cells, both of which are medical applications of synthetic biology. Advancements such as novel cancer therapies, vaccination technology, biological protection, and the creation of new medicines highlight synthetic biology’s potential to create breakthroughs in medical science. This paper will examine various medical applications of synthetic biology and further development that needs to be done before current research can be used in hospitals and doctors’ offices.

Synthetic biologists consider finding a cure for cancer a reachable goal. Previous to the development of synthetic biology as a field, it was known that three types of bacteria, ''Bifodobacterium'', ''Clostridium'', and ''Salmonella'' all preferentially infect the dense cells of tumors (Pawelek 548). All three types of bacteria are associated with reducing tumor size in patients who are infected with them, and it was noted that all three bacteria act anaerobically. Capitalizing on the idea that tumors create an unusually dense, anaerobic mass of cells, Christopher Voigt’s lab engineered bacteria to identify and invade cancer cells. They utilized the ''lux'' quorum sensing from ''Vibrio fischeri'' to identify cells growing at high densities (Fig. 1) and the ''fdhF'' promoter to identify cells growing in anaerobic conditions (Fig. 2) (Anderson 619). They coupled this identification mechanism with the invasin output module from ''Yersinia pseudotuberculosis'' so the ''E. coli'' could invade specific cells. Next, scientists hope to insert a gene that would destroy the invaded cells, making the complete cycle a search, invade, and destroy loop that would only be sensitive to cancer cells. One promising destroy mechanism has been implemented in humans via intratumoral injection, based on ''Salmonella'' that contains the ''E. coli'' cytosine deaminase gene that converts 5-fluorocytosine to 5-fluorouracil, a chemotherapy drug (Nemunaitis 737). Unfortunately, while the tumors did produce 5-fluorouracil (5-FU), they did not produce it in high enough yields to cause regression of the cancer. Another experiment flooded mouse cells with 6-methylpurine-2’deoxyribose (6-MPDR), which was converted to the toxin 6-methyl purine (MeP) by the enzyme purine nucleoside phosphorylase (PNP), which is naturally found in ''E. coli'' (Critchley 1226) (Fig. 3). The problem with this is that all cells in the body might be exposed to the toxin MeP. In addition to the search and destroy mechanism, scientists hope to increase selectivity by combining the hypoxia-sensing and density-sensing units so that ''E. coli'' will only enter eukaryotic cells that exhibit both characteristics. This would make it more likely that invasin-enhanced ''E. coli'' would only invade cancer cells rather than muscle tissue that had low oxygen due to exercise (Anderson 624). To create a fully functional, synthetic biology-based approach to cancer therapy, scientists need to develop bacteria that discriminately invades cancer cells and destroys them. Most of the pieces are there – scientists have engineered ''E. coli'' that selectively invades hypoxic cells or dense cells and ''Salmonella'' that can destroy tumors in vivo – they just need to be put together effectively.

<center>

{|
|-
|[[Image:InvasinDensity.JPG]]
|[[Image:InvasinAnaerobic2.JPG]]
|[[Image:Tumor.JPG]]
|-
|'''Fig. 1.''' The red line represents when the invasin output module was paired with the inducible promoter ''lux'' which senses high cell density, the black line is constitutively on invasin. (Anderson - Permission Pending)
|'''Fig. 2.''' The invasin output module was paired with the inducible promoter ''fdhF'' which senses anaerobic conditions. (Anderson - Permission Pending)
|'''Fig. 3.''' Tumor size after injections. Cancer cells were injected into mice, and 5 days later injections with PBS, 6-MPDR, and invasin-enhanced ''E-coli'' began. Tumor cells recieving injections of invasin-enhanced ''E.coli'' and 6-MPDR grew the slowest, but did not get smaller. (Critchley - Permission Pending)
|}
</center>

Using bacteria as a vaccine vector has similar issues of specificity and implementation in a human system. Bacteria was first thought to be a good vector because they would not degrade at mucosal surfaces, they would absorb better and survive the low pH in the gastrointestinal tract (Garmory 471). A particular strain of ''Salmonella'' with one or more deletions in the shikimate pathway, which creates a precursor for phenylalanine and tyrosine, is a promising vector because it gets into the body easily but does not harmfully effect the tissue and is gone within a week or two of introduction. Scientists engineered this strain to carry the ''Yersinia pestis'' V antigen, and studied its ability to protect mice from ''Y. pestis''. Unfortunately, the inoculation was not extremely successful and only 6 of 20 mice showed a strong response (Garmory 473). Garmory and colleagues cited several reasons for this: the copy number of the plasmid containing the V antigen might not have been high enough; the attenuated ''Salmonella'' may not have been able to reach cells that would be targeted by ''Y. pestis''; and the inability of the cell to secrete the antigen. Many changes need to be made to live vector vaccine design before it is an effective method of treating or preventing disease.

Another method of disease prevention with a synthetic biology approach is related to skin cancer. ''E. coli'' engineered to sense single-stranded DNA after DNA damage was coupled with a mechanism to produce a biofilm around the cell. The biofilm output module was tested with DNA damage done by mitomycin C, a DNA crosslinking agent, or UV irradiation, a known cause of skin cancer. Single-stranded DNA activates RecA, which in turn represses the CI repressor protein, thus allowing the transcription of the PL promoter (Kobayashi 8416). The PL promoter is in charge of the ''traA'' gene, which controls biofilm formation (Fig. 4). The biofilm protection, when part of a toggle switch mechanism, lasted indefinitely when pulsed with UV light at 8 J/m2 for only 2 seconds (Kobayashi 8417) (Fig. 5). The biofilm-production mechanism has not been engineered in eukaryotic cells, but a prokaryote-based system that could aid with human skin disease is imaginable. For example, a sunscreen that changes colors when the DNA damage may be too intense for a sunbather as a warning, or a sunscreen that becomes more protective as the possibility of genetic damage increased. Further research should focus on how to best implement the biofilm producing output module to protect human DNA.
<center>

{|
|-
|[[Image:Toggleswitch.JPG]]
|[[Image:UVinducedbiofilm.JPG]]
|-
|'''Fig. 4.''' The RecA / traA toggleswitch mechanism. (Kobayashi - Permission Pending)
|'''Fig. 5.''' Crystal violet absorbance measures biofilm formation. (Kobayashi - Permission Pending)
|}
</center>
Finally, synthetic biology has made its mark on the field of medicine by creating drugs that are less expensive to produce than those made by pharmaceutical companies. Jay Keasling’s project involving the antimalarial drug artemisinin is the best example. Yeast was engineered to increase normal farnesyl pyrophosphate (FPP) production, convert FPP to amorphadiene, and oxidizing amorphadiene to artemisinic acid, which can then easily be separated from the yeast cells and converted to artemisinin in the laboratory (Fig. 6). This innovation allows the production of artemisinin at lower prices than those currently on the market, and without regard to environmental constraints (Ro 942). On a completely different tangent, Collins’s lab engineered T7 bacteriophages to deliver biofilm-degrading enzymes such as dispersin B after infecting and replicating in an ''E. coli'' biofilm. Dispersin B works to degrade an adhesin that is critical for biofilm formation. Phages that released dispersin B were found to be 99.997% effective at removing biofilm, which is 4.5 orders of magnitude higher than non-enzymatic phages (Lu 11197) (Fig. 7). The specificity of phages for a certain type of bacteria make them a viable vector to be used in human treatments, which may range from removing dental plaques to cleaning inserted medical devices. Using synthetic biology to engineer an antimalarial drug in yeast or a biofilm-destroying enzyme in bacteriophages greatly increases the possible contributions to the field of medicine.

<center>

{|
|-
|[[Image:Artemesinin.JPG]]
|[[Image:BiofilmDegradation.JPG]]
|-
|'''Fig. 6.''' All the steps in the box indicate steps that were optimized in yeast for a maximum artemisinic acid output. (Ro - Permission Pending)
|'''Fig. 7.''' The amount of biofilm was decreased when the dispersin B enzymes were incorporated into T7 bacteriophages. (Lu - Permission Pending)
|}
</center>

Medical applications of synthetic biology are wide-ranging and eminently applicable to daily life. Synthetic biology based cancer treatments that utilize ''E.coli'' engineered to sense and attack tissues with high cell density and low oxygen availability are promising and need to be tested in vivo. Designing vectors out of attenuated bacteria may be a safe, effective method for disease treatment. Using biofilms to sense sun damage potential may decrease the likelihood of skin cancer. Finally, using engineered cells to produce medicines in a cost-effective, environmentally-friendly way may revolutionize the pharmaceutical industry. Investing more research efforts in the field of synthetic biology may turn out to be an investment in medical technology.

'''Works Cited'''

-Anderson JC, Clarke EJ, Arkin, AP, and Voigt CA (2006). Environmentally controlled invasion of cancer cells by engineered bacteria. Journal of Molecular Biology 355:619-27.

-Critchley RJ, Jezzard S, Radford KJ, Goussard S, Lemoine NR, et al. (2004). Potential therapeutic applications of recombinant, invasive ''E. coli''. Gene Therapy 11:1224-33.

-Garmory HS, Leary SEC, Griffin, KF, Williamson D, Brown, KA, and Titball RW (2003). The use of live attenuated bacteria as a delivery system for heterologous antigens. Journal of Drug Targeting 11:471-79.

-Kobayashi H, et al. (2004). Programmable cells: Interfacing natural and engineered gene networks. PNAS 101: 8414-19.

-Lu, TK, and Collins JJ (2007). Dispersing biofilms with engineered enzymatic bacteriophage. PNAS 104: 11197-11202.

-Nemunaitis J, Cunningham C, Senzer N, Kuhn J, Cramm J, Litz C, et al. (2003). Pilot trial of genetically modified, attenuated ''Salmonella'' expressing cytosine deaminase gene in refractory cancer patients. Cancer Gene Therapy 10:737-744.

-Pawelek JM, Low KB, and Bermudes D (2003). Bacteria as tumour-targeting vectors. Lancet Oncology 4:548-56.

-Ro D, et al. (2006). Production of the antimalarial drug precursor artemisinic acid in engineered yeast. Nature 440:940-43.

Davidson College Synthetic Biology Seminar (Fall 2007)

2007-11-23T21:23:18Z

Sasimpson:

== Davidson College Synthetic Biology Seminar, Fall 2007 ==
Click [http://www.bio.davidson.edu/Courses/Synthetic/synthetic_Seminar.html here] to access the class webpage

'''Synthetic Biology: A Brief Introduction'''

In 1978, the Nobel Prize in Medicine went to Werner Arber, Daniel Nathans, and Hamilton O. Smith for discovering restriction enzymes (www.nobelprize.org), which launched recombinant DNA technology. Scientists were able to splice open segments of DNA at specific restriction sites and add or take away different DNA segments, seeing how these DNA changes affected cell function. Recombinant DNA technology marks the beginning of the field of synthetic biology.

Using restriction enzymes and recombinant DNA, scientists focused on areas as diverse as biofuels, cellular memory, and directed evolution. Synthetic biology now captures a wide range of disciplines that all hold one thing in common: engineering genomes using preexisting and new biological systems and components. Mathematical modeling informs the design of the synthetic system, device, or circuit, which is implemented in wet lab experiments. Results from experimentation enhance the model. The ultimate goal of synthetic biology, therefore, is not only building novel biological systems, but creating a better understanding of existing ones.

'''Our Papers'''

Students in the synthetic biology seminar each chose a topic in synthetic biology on which to write a paper. With our selections, we do not claim to cover all aspects of synthetic biology but instead to provide an overview on subjects we found interesting.

[[Stochasticity in Gene Expression- Mike Waters]] <br>
My paper will cover a characterization, implications, and ways to manipulate stochastic processes during gene expression.

[[Post-transcriptional Regulation Technologies - Erin Zwack]]

Using regulatory RNA, gene expression can now be controlled at the stage after transcription but before translation.

[[CellularMemory:Main Page | Synthetic Cellular Memory - Will DeLoache]]

Synthetic cellular memory refers to the engineering of living organisms to produce "a protracted response to a transient stimulus" ([http://gcat.davidson.edu/GcatWiki/index.php/CellularMemory:References Ajo-Franklin, 2007]). The construction of such rationally designed memory mechanisms in living organisms provides a more thorough understanding of naturally occurring gene networks. In the future, modular cellular memory networks will likely be a key component of many synthetic biological designs, ranging from biocomputing to engineered cell differentiation.

[[Medical Applications of Synthetic Biology - Samantha Simpson]]

Medical applications of synthetic biology range from treating cancer, creating low-cost medication, protecting from DNA damage, and using biological vectors as vaccines. My paper explores these current collaborations between medicine and synthetic biology, and the challenges and benefits to come in the future.

[[Logic Gates - Emma Garren]]

Logic gates are computing units that perform a logical function on one or more inputs to produce a single output. Synthetic biologists use various cellular regulation mechanisms (transcription, translation, etc.) to create modular gene expression devices that can be combined in order to engineer cells that perform increasingly complex tasks.

[[Laura Voss - Synthetic Biology Seminar | Promoters and Reporters in Synthetic Biology - Laura Voss]]

Key to the construction of gene circuits and biosensors are promoter and reporter genes, which control how a cell's genes are transcribed when the cell's environment changes. In addition to using promoters and reporters as available to build cellular machines, synthetic biologists can also alter, redesign, or engineer these genetic components in order to refine biological design.

[[Applications of Ribozymes in Synthetic Systems - Danielle Jordan]]

Ribozymes, or RNA enzymes, serve an important role in cellular function both by acting as carriers of genetic infomation and as catalysts for chemical reactions. These enzymes, which represent important ways of regulating genes, that have yet to be fully discovered. Synthetic biology attempts to understand these complex interactions by creating artificial ribozymes and placing them into existing systems. This modular method of gene regulation could open new ways of solving existing promoter and reporter interactions.

[[Directed Evolution and Synthetic Biology - Hunter Stone]]

Directed evolution is a method of cellular engineering that uses Darwinian selection to evolve proteins and RNA with desirable properties not found in nature. The reliance of this method on the randomness of mutation and nature's selective properties sharply contrasts to the logical modeling and reasoning associated with traditional synthetic methods. Some might say that the fundamental lack of planning involved with directed evolution means it is constitutionally different than synthetic biology. Regardless, the method has been shown to be effective in achieving desired results in a number of cases, and could prove instrumental in the optimization of synthetically-designed constructs.

Davidson College Synthetic Biology Seminar (Fall 2007)

2007-11-23T21:21:06Z

Sasimpson:

== Davidson College Synthetic Biology Seminar, Fall 2007 ==
Click [http://www.bio.davidson.edu/Courses/Synthetic/synthetic_Seminar.html here] to access the class webpage

'''Synthetic Biology: A Brief Introduction'''

In 1978, the Nobel Prize in Medicine went to Werner Arber, Daniel Nathans, and Hamilton O. Smith for discovering restriction enzymes (www.nobelprize.org), which launched recombinant DNA technology. Scientists were able to splice open segments of DNA at specific restriction sites and add or take away different DNA segments, seeing how these DNA changes affected cell function. Recombinant DNA technology marks the beginning of the field of synthetic biology.

Using restriction enzymes and recombinant DNA, scientists focused on areas as diverse as biofuels, cellular memory, and directed evolution. Synthetic biology now captures a wide range of disciplines that all hold one thing in common: engineering genomes using preexisting and new biological systems and components. Mathematical modeling informs the design of the synthetic system, device, or circuit, which is implemented in wet lab experiments. Results from experimentation enhance the model. The ultimate goal of synthetic biology, therefore, is not only building novel biological systems, but creating a better understanding of existing ones.

List of topics covered with links to them

Other interesting images and/or information.

[[Stochasticity in Gene Expression- Mike Waters]] <br>
My paper will cover a characterization, implications, and ways to manipulate stochastic processes during gene expression.

[[Post-transcriptional Regulation Technologies - Erin Zwack]]

Using regulatory RNA, gene expression can now be controlled at the stage after transcription but before translation.

[[CellularMemory:Main Page | Synthetic Cellular Memory - Will DeLoache]]

Synthetic cellular memory refers to the engineering of living organisms to produce "a protracted response to a transient stimulus" ([http://gcat.davidson.edu/GcatWiki/index.php/CellularMemory:References Ajo-Franklin, 2007]). The construction of such rationally designed memory mechanisms in living organisms provides a more thorough understanding of naturally occurring gene networks. In the future, modular cellular memory networks will likely be a key component of many synthetic biological designs, ranging from biocomputing to engineered cell differentiation.

[[Medical Applications of Synthetic Biology - Samantha Simpson]]

Medical applications of synthetic biology range from treating cancer, creating low-cost medication, protecting from DNA damage, and using biological vectors as vaccines. My paper explores these current collaborations between medicine and synthetic biology, and the challenges and benefits to come in the future.

[[Logic Gates - Emma Garren]]

Logic gates are computing units that perform a logical function on one or more inputs to produce a single output. Synthetic biologists use various cellular regulation mechanisms (transcription, translation, etc.) to create modular gene expression devices that can be combined in order to engineer cells that perform increasingly complex tasks.

[[Laura Voss - Synthetic Biology Seminar | Promoters and Reporters in Synthetic Biology - Laura Voss]]

Key to the construction of gene circuits and biosensors are promoter and reporter genes, which control how a cell's genes are transcribed when the cell's environment changes. In addition to using promoters and reporters as available to build cellular machines, synthetic biologists can also alter, redesign, or engineer these genetic components in order to refine biological design.

[[Applications of Ribozymes in Synthetic Systems - Danielle Jordan]]

Ribozymes, or RNA enzymes, serve an important role in cellular function both by acting as carriers of genetic infomation and as catalysts for chemical reactions. These enzymes, which represent important ways of regulating genes, that have yet to be fully discovered. Synthetic biology attempts to understand these complex interactions by creating artificial ribozymes and placing them into existing systems. This modular method of gene regulation could open new ways of solving existing promoter and reporter interactions.

[[Directed Evolution and Synthetic Biology - Hunter Stone]]

Directed evolution is a method of cellular engineering that uses Darwinian selection to evolve proteins and RNA with desirable properties not found in nature. The reliance of this method on the randomness of mutation and nature's selective properties sharply contrasts to the logical modeling and reasoning associated with traditional synthetic methods. Some might say that the fundamental lack of planning involved with directed evolution means it is constitutionally different than synthetic biology. Regardless, the method has been shown to be effective in achieving desired results in a number of cases, and could prove instrumental in the optimization of synthetically-designed constructs.

Davidson College Synthetic Biology Seminar (Fall 2007)

2007-11-23T21:20:44Z

Sasimpson:

== Davidson College Synthetic Biology Seminar, Fall 2007 ==
Click [http://www.bio.davidson.edu/Courses/Synthetic/synthetic_Seminar.html here] to access the class webpage

Synthetic Biology: A Brief Introduction

In 1978, the Nobel Prize in Medicine went to Werner Arber, Daniel Nathans, and Hamilton O. Smith for discovering restriction enzymes (www.nobelprize.org), which launched recombinant DNA technology. Scientists were able to splice open segments of DNA at specific restriction sites and add or take away different DNA segments, seeing how these DNA changes affected cell function. Recombinant DNA technology marks the beginning of the field of synthetic biology.

Using restriction enzymes and recombinant DNA, scientists focused on areas as diverse as biofuels, cellular memory, and directed evolution. Synthetic biology now captures a wide range of disciplines that all hold one thing in common: engineering genomes using preexisting and new biological systems and components. Mathematical modeling informs the design of the synthetic system, device, or circuit, which is implemented in wet lab experiments. Results from experimentation enhance the model. The ultimate goal of synthetic biology, therefore, is not only building novel biological systems, but creating a better understanding of existing ones.

List of topics covered with links to them

Other interesting images and/or information.

[[Stochasticity in Gene Expression- Mike Waters]] <br>
My paper will cover a characterization, implications, and ways to manipulate stochastic processes during gene expression.

[[Post-transcriptional Regulation Technologies - Erin Zwack]]

Using regulatory RNA, gene expression can now be controlled at the stage after transcription but before translation.

[[CellularMemory:Main Page | Synthetic Cellular Memory - Will DeLoache]]

Synthetic cellular memory refers to the engineering of living organisms to produce "a protracted response to a transient stimulus" ([http://gcat.davidson.edu/GcatWiki/index.php/CellularMemory:References Ajo-Franklin, 2007]). The construction of such rationally designed memory mechanisms in living organisms provides a more thorough understanding of naturally occurring gene networks. In the future, modular cellular memory networks will likely be a key component of many synthetic biological designs, ranging from biocomputing to engineered cell differentiation.

[[Medical Applications of Synthetic Biology - Samantha Simpson]]

Medical applications of synthetic biology range from treating cancer, creating low-cost medication, protecting from DNA damage, and using biological vectors as vaccines. My paper explores these current collaborations between medicine and synthetic biology, and the challenges and benefits to come in the future.

[[Logic Gates - Emma Garren]]

Logic gates are computing units that perform a logical function on one or more inputs to produce a single output. Synthetic biologists use various cellular regulation mechanisms (transcription, translation, etc.) to create modular gene expression devices that can be combined in order to engineer cells that perform increasingly complex tasks.

[[Laura Voss - Synthetic Biology Seminar | Promoters and Reporters in Synthetic Biology - Laura Voss]]

Key to the construction of gene circuits and biosensors are promoter and reporter genes, which control how a cell's genes are transcribed when the cell's environment changes. In addition to using promoters and reporters as available to build cellular machines, synthetic biologists can also alter, redesign, or engineer these genetic components in order to refine biological design.

[[Applications of Ribozymes in Synthetic Systems - Danielle Jordan]]

Ribozymes, or RNA enzymes, serve an important role in cellular function both by acting as carriers of genetic infomation and as catalysts for chemical reactions. These enzymes, which represent important ways of regulating genes, that have yet to be fully discovered. Synthetic biology attempts to understand these complex interactions by creating artificial ribozymes and placing them into existing systems. This modular method of gene regulation could open new ways of solving existing promoter and reporter interactions.

[[Directed Evolution and Synthetic Biology - Hunter Stone]]

Directed evolution is a method of cellular engineering that uses Darwinian selection to evolve proteins and RNA with desirable properties not found in nature. The reliance of this method on the randomness of mutation and nature's selective properties sharply contrasts to the logical modeling and reasoning associated with traditional synthetic methods. Some might say that the fundamental lack of planning involved with directed evolution means it is constitutionally different than synthetic biology. Regardless, the method has been shown to be effective in achieving desired results in a number of cases, and could prove instrumental in the optimization of synthetically-designed constructs.

Davidson College Synthetic Biology Seminar (Fall 2007)

2007-11-19T14:00:39Z

Sasimpson:

== Davidson College Synthetic Biology Seminar, Fall 2007 ==
Click [http://www.bio.davidson.edu/Courses/Synthetic/synthetic_Seminar.html here] to access the class webpage

Summarize what synthetic biology is

Synthetic Biology is the re-design of existing biological systems and components and the fabrication of novel biological systems and components.

List of topics covered with links to them

Other interesting images and/or information.

[[Mike Waters Synthetic]]

[[Erin Zwack Synthetic Biology Seminar]]

[[CellularMemory:Main Page | Will DeLoache - Synthetic Cellular Memory]]

[[Medical Applications of Synthetic Biology - Samantha Simpson]]

Medical applications of synthetic biology range from treating cancer, creating low-cost medication, protecting from DNA damage, and using biological vectors as vaccines. My paper explores these current collaborations between medicine and synthetic biology, and the challenges and benefits to come in the future.

[[Logic Gates - Emma Garren]]

[[Laura Voss - Synthetic Biology Seminar]]

[[Danielle Jordan - Synthetic Biology Seminar]]

[[Hunter Stone - Synthetic Biology Seminar]]

Davidson College Synthetic Biology Seminar (Fall 2007)

2007-11-19T14:00:26Z

Sasimpson:

== Davidson College Synthetic Biology Seminar, Fall 2007 ==
Click [http://www.bio.davidson.edu/Courses/Synthetic/synthetic_Seminar.html here] to access the class webpage

Summarize what synthetic biology is

Synthetic Biology is the re-design of existing biological systems and components and the fabrication of novel biological systems and components.

List of topics covered with links to them

Other interesting images and/or information.

[[Mike Waters Synthetic]]

[[Erin Zwack Synthetic Biology Seminar]]

[[CellularMemory:Main Page | Will DeLoache - Synthetic Cellular Memory]]

[[Medical Applications of Synthetic Biology - Samantha Simpson]]
Medical applications of synthetic biology range from treating cancer, creating low-cost medication, protecting from DNA damage, and using biological vectors as vaccines. My paper explores these current collaborations between medicine and synthetic biology, and the challenges and benefits to come in the future.

[[Logic Gates - Emma Garren]]

[[Laura Voss - Synthetic Biology Seminar]]

[[Danielle Jordan - Synthetic Biology Seminar]]

[[Hunter Stone - Synthetic Biology Seminar]]

Davidson College Synthetic Biology Seminar (Fall 2007)

2007-11-19T13:57:00Z

Sasimpson:

== Davidson College Synthetic Biology Seminar, Fall 2007 ==
Click [http://www.bio.davidson.edu/Courses/Synthetic/synthetic_Seminar.html here] to access the class webpage

Summarize what synthetic biology is

Synthetic Biology is the re-design of existing biological systems and components and the fabrication of novel biological systems and components.

List of topics covered with links to them

Other interesting images and/or information.

[[Mike Waters Synthetic]]

[[Erin Zwack Synthetic Biology Seminar]]

[[CellularMemory:Main Page | Will DeLoache - Synthetic Cellular Memory]]

[[Medical Applications of Synthetic Biology - Samantha Simpson]]

[[Logic Gates - Emma Garren]]

[[Laura Voss - Synthetic Biology Seminar]]

[[Danielle Jordan - Synthetic Biology Seminar]]

[[Hunter Stone - Synthetic Biology Seminar]]

Medical Applications of Synthetic Biology - Samantha Simpson

2007-11-19T13:56:14Z

Sasimpson: Samantha Simpson - Medical Applications of Synthetic Biology moved to Medical Applications of Synthetic Biology - Samantha Simpson

'''Project Proposal'''

My project will be on medical applications of synthetic biology. I will reference Anderson's paper on utilizing quorum-sensing and hypoxia-responsive genes coupled with invasin from ''Yersinia tuberculosis'' to invade cancer-causing cells. Coupled with Critchley's work, which describes a bacterial system that invades eukaryotic cells and delivers proteins coded for in the bacteria's genome, one could possibly create 'search and destroy' ''E.coli'' that can locate, invade, and kill tumor cells. Garmory's paper also highlights the possibility of using bacteria as a drug-delivering system, specifically vaccine vectors. Kobayashi describes cells that produce a protective biofilm layer after exposure to DNA-damaging agents. Ro engineers yeast to create an anti-malarial drug that can be created at a lower cost than the previous standard production process. These papers, and others that have a focus on direct medical applications such as novel cancer therapies, vaccination technology, biological protection, and the creation of new medicines, will be referenced to create a cohesive overview of applications of synthetic biology to the medical field.

'''Paper'''

Synthetic biology is a rapidly emerging field that strives to re-design existing biological systems and components or fabricate novel biological systems and components. The end result of experimentation in synthetic biology is to help scientists understand a naturally occurring genetic pathway more fully or to create a new, useful pathway. Recent research efforts have focused on engineering bacteria to create a new form of biofuel or to make promoters of varying efficiencies for more exact gene expression. Other research efforts focus on engineering yeast to make antimalarial drugs or engineering ''E. coli'' to recognize and invade tumor-like cells, both of which are medical applications of synthetic biology. Advancements such as novel cancer therapies, vaccination technology, biological protection, and the creation of new medicines highlight synthetic biology’s potential to create breakthroughs in medical science. This paper will examine various medical applications of synthetic biology and further development that needs to be done before current research can be used in hospitals and doctors’ offices.

Synthetic biologists consider finding a cure for cancer a reachable goal. Previous to the development of synthetic biology as a field, it was known that three types of bacteria, ''Bifodobacterium'', ''Clostridium'', and ''Salmonella'' all preferentially infect the dense cells of tumors (Pawelek 548). All three types of bacteria are associated with reducing tumor size in patients who are infected with them, and it was noted that all three bacteria act anaerobically. Capitalizing on the idea that tumors create an unusually dense, anaerobic mass of cells, Christopher Voigt’s lab engineered bacteria to identify and invade cancer cells. They utilized the ''lux'' quorum sensing from ''Vibrio fischeri'' to identify cells growing at high densities (Fig. 1) and the ''fdhF'' promoter to identify cells growing in anaerobic conditions (Fig. 2) (Anderson 619). They coupled this identification mechanism with the invasin output module from ''Yersinia pseudotuberculosis'' so the ''E. coli'' could invade specific cells. Next, scientists hope to insert a gene that would destroy the invaded cells, making the complete cycle a search, invade, and destroy loop that would only be sensitive to cancer cells. One promising destroy mechanism has been implemented in humans via intratumoral injection, based on ''Salmonella'' that contains the ''E. coli'' cytosine deaminase gene that converts 5-fluorocytosine to 5-fluorouracil, a chemotherapy drug (Nemunaitis 737). Unfortunately, while the tumors did produce 5-fluorouracil (5-FU), they did not produce it in high enough yields to cause regression of the cancer. Another experiment flooded mouse cells with 6-methylpurine-2’deoxyribose (6-MPDR), which was converted to the toxin 6-methyl purine (MeP) by the enzyme purine nucleoside phosphorylase (PNP), which is naturally found in ''E. coli'' (Critchley 1226) (Fig. 3). The problem with this is that all cells in the body might be exposed to the toxin MeP. In addition to the search and destroy mechanism, scientists hope to increase selectivity by combining the hypoxia-sensing and density-sensing units so that ''E. coli'' will only enter eukaryotic cells that exhibit both characteristics. This would make it more likely that invasin-enhanced ''E. coli'' would only invade cancer cells rather than muscle tissue that had low oxygen due to exercise (Anderson 624). To create a fully functional, synthetic biology-based approach to cancer therapy, scientists need to develop bacteria that discriminately invades cancer cells and destroys them. Most of the pieces are there – scientists have engineered ''E. coli'' that selectively invades hypoxic cells or dense cells and ''Salmonella'' that can destroy tumors in vivo – they just need to be put together effectively.

<center>

{|
|-
|[[Image:InvasinDensity.JPG]]
|[[Image:InvasinAnaerobic2.JPG]]
|[[Image:Tumor.JPG]]
|-
|'''Fig. 1.''' The red line represents when the invasin output module was paired with the inducible promoter ''lux'' which senses high cell density, the black line is constitutively on invasin. (Anderson)
|'''Fig. 2.''' The invasin output module was paired with the inducible promoter ''fdhF'' which senses anaerobic conditions. (Anderson)
|'''Fig. 3.''' Tumor size after injections. Cancer cells were injected into mice, and 5 days later injections with PBS, 6-MPDR, and invasin-enhanced ''E-coli'' began. Tumor cells recieving injections of invasin-enhanced ''E.coli'' and 6-MPDR grew the slowest, but did not get smaller. (Critchley)
|}
</center>

Using bacteria as a vaccine vector has similar issues of specificity and implementation in a human system. Bacteria was first thought to be a good vector because they would not degrade at mucosal surfaces, they would absorb better and survive the low pH in the gastrointestinal tract (Garmory 471). A particular strain of ''Salmonella'' with one or more deletions in the shikimate pathway, which creates a precursor for phenylalanine and tyrosine, is a promising vector because it gets into the body easily but does not harmfully effect the tissue and is gone within a week or two of introduction. Scientists engineered this strain to carry the ''Yersinia pestis'' V antigen, and studied its ability to protect mice from ''Y. pestis''. Unfortunately, the inoculation was not extremely successful and only 6 of 20 mice showed a strong response (Garmory 473). Garmory and colleagues cited several reasons for this: the copy number of the plasmid containing the V antigen might not have been high enough; the attenuated ''Salmonella'' may not have been able to reach cells that would be targeted by ''Y. pestis''; and the inability of the cell to secrete the antigen. Many changes need to be made to live vector vaccine design before it is an effective method of treating or preventing disease.

Another method of disease prevention with a synthetic biology approach is related to skin cancer. ''E. coli'' engineered to sense single-stranded DNA after DNA damage was coupled with a mechanism to produce a biofilm around the cell. The biofilm output module was tested with DNA damage done by mitomycin C, a DNA crosslinking agent, or UV irradiation, a known cause of skin cancer. Single-stranded DNA activates RecA, which in turn represses the CI repressor protein, thus allowing the transcription of the PL promoter (Kobayashi 8416). The PL promoter is in charge of the ''traA'' gene, which controls biofilm formation (Fig. 4). The biofilm protection, when part of a toggle switch mechanism, lasted indefinitely when pulsed with UV light at 8 J/m2 for only 2 seconds (Kobayashi 8417) (Fig. 5). The biofilm-production mechanism has not been engineered in eukaryotic cells, but a prokaryote-based system that could aid with human skin disease is imaginable. For example, a sunscreen that changes colors when the DNA damage may be too intense for a sunbather as a warning, or a sunscreen that becomes more protective as the possibility of genetic damage increased. Further research should focus on how to best implement the biofilm producing output module to protect human DNA.
<center>

{|
|-
|[[Image:Toggleswitch.JPG]]
|[[Image:UVinducedbiofilm.JPG]]
|-
|'''Fig. 4.''' The RecA / traA toggleswitch mechanism. (Kobayashi)
|'''Fig. 5.''' Crystal violet absorbance measures biofilm formation. (Kobayashi)
|}
</center>
Finally, synthetic biology has made its mark on the field of medicine by creating drugs that are less expensive to produce than those made by pharmaceutical companies. Jay Keasling’s project involving the antimalarial drug artemisinin is the best example. Yeast was engineered to increase normal farnesyl pyrophosphate (FPP) production, convert FPP to amorphadiene, and oxidizing amorphadiene to artemisinic acid, which can then easily be separated from the yeast cells and converted to artemisinin in the laboratory (Fig. 6). This innovation allows the production of artemisinin at lower prices than those currently on the market, and without regard to environmental constraints (Ro 942). On a completely different tangent, Collins’s lab engineered T7 bacteriophages to deliver biofilm-degrading enzymes such as dispersin B after infecting and replicating in an ''E. coli'' biofilm. Dispersin B works to degrade an adhesin that is critical for biofilm formation. Phages that released dispersin B were found to be 99.997% effective at removing biofilm, which is 4.5 orders of magnitude higher than non-enzymatic phages (Lu 11197) (Fig. 7). The specificity of phages for a certain type of bacteria make them a viable vector to be used in human treatments, which may range from removing dental plaques to cleaning inserted medical devices. Using synthetic biology to engineer an antimalarial drug in yeast or a biofilm-destroying enzyme in bacteriophages greatly increases the possible contributions to the field of medicine.

<center>

{|
|-
|[[Image:Artemesinin.JPG]]
|[[Image:BiofilmDegradation.JPG]]
|-
|'''Fig. 6.''' All the steps in the box indicate steps that were optimized in yeast for a maximum artemisinic acid output. (Ro)
|'''Fig. 7.''' The amount of biofilm was decreased when the dispersin B enzymes were incorporated into T7 bacteriophages. (Lu)
|}
</center>

Medical applications of synthetic biology are wide-ranging and eminently applicable to daily life. Synthetic biology based cancer treatments that utilize ''E.coli'' engineered to sense and attack tissues with high cell density and low oxygen availability are promising and need to be tested in vivo. Designing vectors out of attenuated bacteria may be a safe, effective method for disease treatment. Using biofilms to sense sun damage potential may decrease the likelihood of skin cancer. Finally, using engineered cells to produce medicines in a cost-effective, environmentally-friendly way may revolutionize the pharmaceutical industry. Investing more research efforts in the field of synthetic biology may turn out to be an investment in medical technology.

'''Works Cited'''

-Anderson JC, Clarke EJ, Arkin, AP, and Voigt CA (2006). Environmentally controlled invasion of cancer cells by engineered bacteria. Journal of Molecular Biology 355:619-27.

-Critchley RJ, Jezzard S, Radford KJ, Goussard S, Lemoine NR, et al. (2004). Potential therapeutic applications of recombinant, invasive ''E. coli''. Gene Therapy 11:1224-33.

-Garmory HS, Leary SEC, Griffin, KF, Williamson D, Brown, KA, and Titball RW (2003). The use of live attenuated bacteria as a delivery system for heterologous antigens. Journal of Drug Targeting 11:471-79.

-Kobayashi H, et al. (2004). Programmable cells: Interfacing natural and engineered gene networks. PNAS 101: 8414-19.

-Lu, TK, and Collins JJ (2007). Dispersing biofilms with engineered enzymatic bacteriophage. PNAS 104: 11197-11202.

-Nemunaitis J, Cunningham C, Senzer N, Kuhn J, Cramm J, Litz C, et al. (2003). Pilot trial of genetically modified, attenuated ''Salmonella'' expressing cytosine deaminase gene in refractory cancer patients. Cancer Gene Therapy 10:737-744.

-Pawelek JM, Low KB, and Bermudes D (2003). Bacteria as tumour-targeting vectors. Lancet Oncology 4:548-56.

-Ro D, et al. (2006). Production of the antimalarial drug precursor artemisinic acid in engineered yeast. Nature 440:940-43.

Samantha Simpson - Medical Applications of Synthetic Biology

2007-11-19T13:56:14Z

Sasimpson: Samantha Simpson - Medical Applications of Synthetic Biology moved to Medical Applications of Synthetic Biology - Samantha Simpson

#redirect [[Medical Applications of Synthetic Biology - Samantha Simpson]]

Medical Applications of Synthetic Biology - Samantha Simpson

2007-11-13T04:13:22Z

Sasimpson:

'''Project Proposal'''

My project will be on medical applications of synthetic biology. I will reference Anderson's paper on utilizing quorum-sensing and hypoxia-responsive genes coupled with invasin from ''Yersinia tuberculosis'' to invade cancer-causing cells. Coupled with Critchley's work, which describes a bacterial system that invades eukaryotic cells and delivers proteins coded for in the bacteria's genome, one could possibly create 'search and destroy' ''E.coli'' that can locate, invade, and kill tumor cells. Garmory's paper also highlights the possibility of using bacteria as a drug-delivering system, specifically vaccine vectors. Kobayashi describes cells that produce a protective biofilm layer after exposure to DNA-damaging agents. Ro engineers yeast to create an anti-malarial drug that can be created at a lower cost than the previous standard production process. These papers, and others that have a focus on direct medical applications such as novel cancer therapies, vaccination technology, biological protection, and the creation of new medicines, will be referenced to create a cohesive overview of applications of synthetic biology to the medical field.

'''Paper'''

Synthetic biology is a rapidly emerging field that strives to re-design existing biological systems and components or fabricate novel biological systems and components. The end result of experimentation in synthetic biology is to help scientists understand a naturally occurring genetic pathway more fully or to create a new, useful pathway. Recent research efforts have focused on engineering bacteria to create a new form of biofuel or to make promoters of varying efficiencies for more exact gene expression. Other research efforts focus on engineering yeast to make antimalarial drugs or engineering ''E. coli'' to recognize and invade tumor-like cells, both of which are medical applications of synthetic biology. Advancements such as novel cancer therapies, vaccination technology, biological protection, and the creation of new medicines highlight synthetic biology’s potential to create breakthroughs in medical science. This paper will examine various medical applications of synthetic biology and further development that needs to be done before current research can be used in hospitals and doctors’ offices.

Synthetic biologists consider finding a cure for cancer a reachable goal. Previous to the development of synthetic biology as a field, it was known that three types of bacteria, ''Bifodobacterium'', ''Clostridium'', and ''Salmonella'' all preferentially infect the dense cells of tumors (Pawelek 548). All three types of bacteria are associated with reducing tumor size in patients who are infected with them, and it was noted that all three bacteria act anaerobically. Capitalizing on the idea that tumors create an unusually dense, anaerobic mass of cells, Christopher Voigt’s lab engineered bacteria to identify and invade cancer cells. They utilized the ''lux'' quorum sensing from ''Vibrio fischeri'' to identify cells growing at high densities (Fig. 1) and the ''fdhF'' promoter to identify cells growing in anaerobic conditions (Fig. 2) (Anderson 619). They coupled this identification mechanism with the invasin output module from ''Yersinia pseudotuberculosis'' so the ''E. coli'' could invade specific cells. Next, scientists hope to insert a gene that would destroy the invaded cells, making the complete cycle a search, invade, and destroy loop that would only be sensitive to cancer cells. One promising destroy mechanism has been implemented in humans via intratumoral injection, based on ''Salmonella'' that contains the ''E. coli'' cytosine deaminase gene that converts 5-fluorocytosine to 5-fluorouracil, a chemotherapy drug (Nemunaitis 737). Unfortunately, while the tumors did produce 5-fluorouracil (5-FU), they did not produce it in high enough yields to cause regression of the cancer. Another experiment flooded mouse cells with 6-methylpurine-2’deoxyribose (6-MPDR), which was converted to the toxin 6-methyl purine (MeP) by the enzyme purine nucleoside phosphorylase (PNP), which is naturally found in ''E. coli'' (Critchley 1226) (Fig. 3). The problem with this is that all cells in the body might be exposed to the toxin MeP. In addition to the search and destroy mechanism, scientists hope to increase selectivity by combining the hypoxia-sensing and density-sensing units so that ''E. coli'' will only enter eukaryotic cells that exhibit both characteristics. This would make it more likely that invasin-enhanced ''E. coli'' would only invade cancer cells rather than muscle tissue that had low oxygen due to exercise (Anderson 624). To create a fully functional, synthetic biology-based approach to cancer therapy, scientists need to develop bacteria that discriminately invades cancer cells and destroys them. Most of the pieces are there – scientists have engineered ''E. coli'' that selectively invades hypoxic cells or dense cells and ''Salmonella'' that can destroy tumors in vivo – they just need to be put together effectively.

<center>

{|
|-
|[[Image:InvasinDensity.JPG]]
|[[Image:InvasinAnaerobic2.JPG]]
|[[Image:Tumor.JPG]]
|-
|'''Fig. 1.''' The red line represents when the invasin output module was paired with the inducible promoter ''lux'' which senses high cell density, the black line is constitutively on invasin. (Anderson)
|'''Fig. 2.''' The invasin output module was paired with the inducible promoter ''fdhF'' which senses anaerobic conditions. (Anderson)
|'''Fig. 3.''' Tumor size after injections. Cancer cells were injected into mice, and 5 days later injections with PBS, 6-MPDR, and invasin-enhanced ''E-coli'' began. Tumor cells recieving injections of invasin-enhanced ''E.coli'' and 6-MPDR grew the slowest, but did not get smaller. (Critchley)
|}
</center>

Using bacteria as a vaccine vector has similar issues of specificity and implementation in a human system. Bacteria was first thought to be a good vector because they would not degrade at mucosal surfaces, they would absorb better and survive the low pH in the gastrointestinal tract (Garmory 471). A particular strain of ''Salmonella'' with one or more deletions in the shikimate pathway, which creates a precursor for phenylalanine and tyrosine, is a promising vector because it gets into the body easily but does not harmfully effect the tissue and is gone within a week or two of introduction. Scientists engineered this strain to carry the ''Yersinia pestis'' V antigen, and studied its ability to protect mice from ''Y. pestis''. Unfortunately, the inoculation was not extremely successful and only 6 of 20 mice showed a strong response (Garmory 473). Garmory and colleagues cited several reasons for this: the copy number of the plasmid containing the V antigen might not have been high enough; the attenuated ''Salmonella'' may not have been able to reach cells that would be targeted by ''Y. pestis''; and the inability of the cell to secrete the antigen. Many changes need to be made to live vector vaccine design before it is an effective method of treating or preventing disease.

Another method of disease prevention with a synthetic biology approach is related to skin cancer. ''E. coli'' engineered to sense single-stranded DNA after DNA damage was coupled with a mechanism to produce a biofilm around the cell. The biofilm output module was tested with DNA damage done by mitomycin C, a DNA crosslinking agent, or UV irradiation, a known cause of skin cancer. Single-stranded DNA activates RecA, which in turn represses the CI repressor protein, thus allowing the transcription of the PL promoter (Kobayashi 8416). The PL promoter is in charge of the ''traA'' gene, which controls biofilm formation (Fig. 4). The biofilm protection, when part of a toggle switch mechanism, lasted indefinitely when pulsed with UV light at 8 J/m2 for only 2 seconds (Kobayashi 8417) (Fig. 5). The biofilm-production mechanism has not been engineered in eukaryotic cells, but a prokaryote-based system that could aid with human skin disease is imaginable. For example, a sunscreen that changes colors when the DNA damage may be too intense for a sunbather as a warning, or a sunscreen that becomes more protective as the possibility of genetic damage increased. Further research should focus on how to best implement the biofilm producing output module to protect human DNA.
<center>

{|
|-
|[[Image:Toggleswitch.JPG]]
|[[Image:UVinducedbiofilm.JPG]]
|-
|'''Fig. 4.''' The RecA / traA toggleswitch mechanism. (Kobayashi)
|'''Fig. 5.''' Crystal violet absorbance measures biofilm formation. (Kobayashi)
|}
</center>
Finally, synthetic biology has made its mark on the field of medicine by creating drugs that are less expensive to produce than those made by pharmaceutical companies. Jay Keasling’s project involving the antimalarial drug artemisinin is the best example. Yeast was engineered to increase normal farnesyl pyrophosphate (FPP) production, convert FPP to amorphadiene, and oxidizing amorphadiene to artemisinic acid, which can then easily be separated from the yeast cells and converted to artemisinin in the laboratory (Fig. 6). This innovation allows the production of artemisinin at lower prices than those currently on the market, and without regard to environmental constraints (Ro 942). On a completely different tangent, Collins’s lab engineered T7 bacteriophages to deliver biofilm-degrading enzymes such as dispersin B after infecting and replicating in an ''E. coli'' biofilm. Dispersin B works to degrade an adhesin that is critical for biofilm formation. Phages that released dispersin B were found to be 99.997% effective at removing biofilm, which is 4.5 orders of magnitude higher than non-enzymatic phages (Lu 11197) (Fig. 7). The specificity of phages for a certain type of bacteria make them a viable vector to be used in human treatments, which may range from removing dental plaques to cleaning inserted medical devices. Using synthetic biology to engineer an antimalarial drug in yeast or a biofilm-destroying enzyme in bacteriophages greatly increases the possible contributions to the field of medicine.

<center>

{|
|-
|[[Image:Artemesinin.JPG]]
|[[Image:BiofilmDegradation.JPG]]
|-
|'''Fig. 6.''' All the steps in the box indicate steps that were optimized in yeast for a maximum artemisinic acid output. (Ro)
|'''Fig. 7.''' The amount of biofilm was decreased when the dispersin B enzymes were incorporated into T7 bacteriophages. (Lu)
|}
</center>

Medical applications of synthetic biology are wide-ranging and eminently applicable to daily life. Synthetic biology based cancer treatments that utilize ''E.coli'' engineered to sense and attack tissues with high cell density and low oxygen availability are promising and need to be tested in vivo. Designing vectors out of attenuated bacteria may be a safe, effective method for disease treatment. Using biofilms to sense sun damage potential may decrease the likelihood of skin cancer. Finally, using engineered cells to produce medicines in a cost-effective, environmentally-friendly way may revolutionize the pharmaceutical industry. Investing more research efforts in the field of synthetic biology may turn out to be an investment in medical technology.

'''Works Cited'''

-Anderson JC, Clarke EJ, Arkin, AP, and Voigt CA (2006). Environmentally controlled invasion of cancer cells by engineered bacteria. Journal of Molecular Biology 355:619-27.

-Critchley RJ, Jezzard S, Radford KJ, Goussard S, Lemoine NR, et al. (2004). Potential therapeutic applications of recombinant, invasive ''E. coli''. Gene Therapy 11:1224-33.

-Garmory HS, Leary SEC, Griffin, KF, Williamson D, Brown, KA, and Titball RW (2003). The use of live attenuated bacteria as a delivery system for heterologous antigens. Journal of Drug Targeting 11:471-79.

-Kobayashi H, et al. (2004). Programmable cells: Interfacing natural and engineered gene networks. PNAS 101: 8414-19.

-Lu, TK, and Collins JJ (2007). Dispersing biofilms with engineered enzymatic bacteriophage. PNAS 104: 11197-11202.

-Nemunaitis J, Cunningham C, Senzer N, Kuhn J, Cramm J, Litz C, et al. (2003). Pilot trial of genetically modified, attenuated ''Salmonella'' expressing cytosine deaminase gene in refractory cancer patients. Cancer Gene Therapy 10:737-744.

-Pawelek JM, Low KB, and Bermudes D (2003). Bacteria as tumour-targeting vectors. Lancet Oncology 4:548-56.

-Ro D, et al. (2006). Production of the antimalarial drug precursor artemisinic acid in engineered yeast. Nature 440:940-43.

Medical Applications of Synthetic Biology - Samantha Simpson

2007-11-13T04:08:01Z

Sasimpson:

'''Project Proposal'''

My project will be on medical applications of synthetic biology. I will reference Anderson's paper on utilizing quorum-sensing and hypoxia-responsive genes coupled with invasin from ''Yersinia tuberculosis'' to invade cancer-causing cells. Coupled with Critchley's work, which describes a bacterial system that invades eukaryotic cells and delivers proteins coded for in the bacteria's genome, one could possibly create 'search and destroy' ''E.coli'' that can locate, invade, and kill tumor cells. Garmory's paper also highlights the possibility of using bacteria as a drug-delivering system, specifically vaccine vectors. Kobayashi describes cells that produce a protective biofilm layer after exposure to DNA-damaging agents. Ro engineers yeast to create an anti-malarial drug that can be created at a lower cost than the previous standard production process. These papers, and others that have a focus on direct medical applications such as novel cancer therapies, vaccination technology, biological protection, and the creation of new medicines, will be referenced to create a cohesive overview of applications of synthetic biology to the medical field.

'''Paper'''

Synthetic biology is a rapidly emerging field that strives to re-design existing biological systems and components or fabricate novel biological systems and components. The end result of experimentation in synthetic biology is to help scientists understand a naturally occurring genetic pathway more fully or to create a new, useful pathway. Recent research efforts have focused on engineering bacteria to create a new form of biofuel or to make promoters of varying efficiencies for more exact gene expression. Other research efforts focus on engineering yeast to make antimalarial drugs or engineering ''E. coli'' to recognize and invade tumor-like cells, both of which are medical applications of synthetic biology. Advancements such as novel cancer therapies, vaccination technology, biological protection, and the creation of new medicines highlight synthetic biology’s potential to create breakthroughs in medical science. This paper will examine various medical applications of synthetic biology and further development that needs to be done before current research can be used in hospitals and doctors’ offices.

Synthetic biologists consider finding a cure for cancer a reachable goal. Previous to the development of synthetic biology as a field, it was known that three types of bacteria, ''Bifodobacterium'', ''Clostridium'', and ''Salmonella'' all preferentially infect the dense cells of tumors (Pawelek 548). All three types of bacteria are associated with reducing tumor size in patients who are infected with them, and it was noted that all three bacteria act anaerobically. Capitalizing on the idea that tumors create an unusually dense, anaerobic mass of cells, Christopher Voigt’s lab engineered bacteria to identify and invade cancer cells. They utilized the ''lux'' quorum sensing from ''Vibrio fischeri'' to identify cells growing at high densities (Fig. 1) and the ''fdhF'' promoter to identify cells growing in anaerobic conditions (Fig. 2) (Anderson 619). They coupled this identification mechanism with the invasin output module from ''Yersinia pseudotuberculosis'' so the ''E. coli'' could invade specific cells. Next, scientists hope to insert a gene that would destroy the invaded cells, making the complete cycle a search, invade, and destroy loop that would only be sensitive to cancer cells. One promising destroy mechanism has been implemented in humans via intratumoral injection, based on ''Salmonella'' that contains the ''E. coli'' cytosine deaminase gene that converts 5-fluorocytosine to 5-fluorouracil, a chemotherapy drug (Nemunaitis 737). Unfortunately, while the tumors did produce 5-fluorouracil (5-FU), they did not produce it in high enough yields to cause regression of the cancer. Another experiment flooded mouse cells with 6-methylpurine-2’deoxyribose (6-MPDR), which was converted to the toxin 6-methyl purine (MeP) by the enzyme purine nucleoside phosphorylase (PNP), which is naturally found in ''E. coli'' (Critchley 1226) (Fig. 3). The problem with this is that all cells in the body might be exposed to the toxin MeP. In addition to the search and destroy mechanism, scientists hope to increase selectivity by combining the hypoxia-sensing and density-sensing units so that ''E. coli'' will only enter eukaryotic cells that exhibit both characteristics. This would make it more likely that invasin-enhanced ''E. coli'' would only invade cancer cells rather than muscle tissue that had low oxygen due to exercise (Anderson 624). To create a fully functional, synthetic biology-based approach to cancer therapy, scientists need to develop bacteria that discriminately invades cancer cells and destroys them. Most of the pieces are there – scientists have engineered ''E. coli'' that selectively invades hypoxic cells or dense cells and ''Salmonella'' that can destroy tumors in vivo – they just need to be put together effectively.

<center>

{|
|-
|[[Image:InvasinDensity.JPG]]
|[[Image:InvasinAnaerobic2.JPG]]
|[[Image:Tumor.JPG]]
|-
|'''Fig. 1.''' The red line represents when the invasin output module was paired with the inducible promoter ''lux'' which senses high cell density, the black line is constitutively on invasin. (Anderson)
|'''Fig. 2.''' The invasin output module was paired with the inducible promoter ''fdhF'' which senses anaerobic conditions. (Anderson)
|'''Fig. 3.''' Tumor size after injections. Cancer cells were injected into mice, and 5 days later injections with PBS, 6-MPDR, and invasin-enhanced ''E-coli'' began. Tumor cells recieving injections of invasin-enhanced ''E.coli'' and 6-MPDR grew the slowest, but did not get smaller. (Critchley)
|}
</center>

Using bacteria as a vaccine vector has similar issues of specificity and implementation in a human system. Bacteria was first thought to be a good vector because they would not degrade at mucosal surfaces, they would absorb better and survive the low pH in the gastrointestinal tract (Garmory 471). A particular strain of ''Salmonella'' with one or more deletions in the shikimate pathway, which creates a precursor for phenylalanine and tyrosine, is a promising vector because it gets into the body easily but does not harmfully effect the tissue and is gone within a week or two of introduction. Scientists engineered this strain to carry the ''Yersinia pestis'' V antigen, and studied its ability to protect mice from ''Y. pestis''. Unfortunately, the inoculation was not extremely successful and only 6 of 20 mice showed a strong response (Garmory 473). Garmory and colleagues cited several reasons for this: the copy number of the plasmid containing the V antigen might not have been high enough; the attenuated ''Salmonella'' may not have been able to reach cells that would be targeted by ''Y. pestis''; and the inability of the cell to secrete the antigen. Many changes need to be made to live vector vaccine design before it is an effective method of treating or preventing disease.

Another method of disease prevention with a synthetic biology approach is related to skin cancer. ''E. coli'' engineered to sense single-stranded DNA after DNA damage was coupled with a mechanism to produce a biofilm around the cell. The biofilm output module was tested with DNA damage done by mitomycin C, a DNA crosslinking agent, or UV irradiation, a known cause of skin cancer. Single-stranded DNA activates RecA, which in turn represses the CI repressor protein, thus allowing the transcription of the PL promoter (Kobayashi 8416). The PL promoter is in charge of the ''traA'' gene, which controls biofilm formation. The biofilm protection, when part of a toggle switch mechanism, lasted indefinitely when pulsed with UV light at 8 J/m2 for only 2 seconds (Kobayashi 8417) (Fig. 4). The biofilm-production mechanism has not been engineered in eukaryotic cells, but a prokaryote-based system that could aid with human skin disease is imaginable. For example, a sunscreen that changes colors when the DNA damage may be too intense for a sunbather as a warning, or a sunscreen that becomes more protective as the possibility of genetic damage increased. Further research should focus on how to best implement the biofilm producing output module to protect human DNA.
<center>

{|
|-
|[[Image:Toggleswitch.JPG]]
|[[Image:UVinducedbiofilm.JPG]]
|-
|'''Fig. 4.''' The RecA / traA toggleswitch mechanism. (Kobayashi)
|'''Fig. 5.''' Crystal violet absorbance measures biofilm formation. (Kobayashi)
|}
</center>
Finally, synthetic biology has made its mark on the field of medicine by creating drugs that are less expensive to produce than those made by pharmaceutical companies. Jay Keasling’s project involving the antimalarial drug artemisinin is the best example. Yeast was engineered to increase normal farnesyl pyrophosphate (FPP) production, convert FPP to amorphadiene, and oxidizing amorphadiene to artemisinic acid, which can then easily be separated from the yeast cells and converted to artemisinin in the laboratory (Fig. 5). This innovation allows the production of artemisinin at lower prices than those currently on the market, and without regard to environmental constraints (Ro 942). On a completely different tangent, Collins’s lab engineered T7 bacteriophages to deliver biofilm-degrading enzymes such as dispersin B after infecting and replicating in an ''E. coli'' biofilm. Dispersin B works to degrade an adhesin that is critical for biofilm formation. Phages that released dispersin B were found to be 99.997% effective at removing biofilm, which is 4.5 orders of magnitude higher than non-enzymatic phages (Lu 11197) (Fig. 6). The specificity of phages for a certain type of bacteria make them a viable vector to be used in human treatments, which may range from removing dental plaques to cleaning inserted medical devices. Using synthetic biology to engineer an antimalarial drug in yeast or a biofilm-destroying enzyme in bacteriophages greatly increases the possible contributions to the field of medicine.

<center>

{|
|-
|[[Image:Artemesinin.JPG]]
|[[Image:BiofilmDegradation.JPG]]
|-
|'''Fig. 5.''' All the steps in the box indicate steps that were optimized in yeast for a maximum artemisinic acid output. (Ro)
|'''Fig. 6.''' The amount of biofilm was decreased when the dispersin B enzymes were incorporated into T7 bacteriophages. (Lu)
|}
</center>

Medical applications of synthetic biology are wide-ranging and eminently applicable to daily life. Synthetic biology based cancer treatments that utilize E.coli engineered to sense and attack tissues with high cell density and low oxygen availability are promising and need to be tested in vivo. Designing vectors out of attenuated bacteria may be a safe, effective method for disease treatment. Using biofilms to sense sun damage potential may decrease the likelihood of skin cancer. Finally, using engineered cells to produce medicines in a cost-effective, environmentally-friendly way may revolutionize the pharmaceutical industry. Investing more research efforts in the field of synthetic biology may turn out to be an investment in medical technology.

'''Works Cited'''

Anderson JC, Clarke EJ, Arkin, AP, and Voigt CA (2006). Environmentally controlled invasion of cancer cells by engineered bacteria. Journal of Molecular Biology 355:619-27.

Critchley RJ, Jezzard S, Radford KJ, Goussard S, Lemoine NR, et al. (2004). Potential therapeutic applications of recombinant, invasive ''E. coli''. Gene Therapy 11:1224-33.

Garmory HS, Leary SEC, Griffin, KF, Williamson D, Brown, KA, and Titball RW (2003). The use of live attenuated bacteria as a delivery system for heterologous antigens. Journal of Drug Targeting 11:471-79.

Kobayashi H, et al. (2004). Programmable cells: Interfacing natural and engineered gene networks. PNAS 101: 8414-19.

Lu, TK, and Collins JJ (2007). Dispersing biofilms with engineered enzymatic bacteriophage. PNAS 104: 11197-11202.

Nemunaitis J, Cunningham C, Senzer N, Kuhn J, Cramm J, Litz C, et al. (2003). Pilot trial of genetically modified, attenuated ''Salmonella'' expressing cytosine deaminase gene in refractory cancer patients. Cancer Gene Therapy 10:737-744.

Pawelek JM, Low KB, and Bermudes D (2003). Bacteria as tumour-targeting vectors. Lancet Oncology 4:548-56.

Ro D, et al. (2006). Production of the antimalarial drug precursor artemisinic acid in engineered yeast. Nature 440:940-43.

File:Toggleswitch.JPG

2007-11-13T04:05:59Z

Sasimpson:

Medical Applications of Synthetic Biology - Samantha Simpson

2007-11-13T04:05:40Z

Sasimpson:

'''Project Proposal'''

My project will be on medical applications of synthetic biology. I will reference Anderson's paper on utilizing quorum-sensing and hypoxia-responsive genes coupled with invasin from ''Yersinia tuberculosis'' to invade cancer-causing cells. Coupled with Critchley's work, which describes a bacterial system that invades eukaryotic cells and delivers proteins coded for in the bacteria's genome, one could possibly create 'search and destroy' ''E.coli'' that can locate, invade, and kill tumor cells. Garmory's paper also highlights the possibility of using bacteria as a drug-delivering system, specifically vaccine vectors. Kobayashi describes cells that produce a protective biofilm layer after exposure to DNA-damaging agents. Ro engineers yeast to create an anti-malarial drug that can be created at a lower cost than the previous standard production process. These papers, and others that have a focus on direct medical applications such as novel cancer therapies, vaccination technology, biological protection, and the creation of new medicines, will be referenced to create a cohesive overview of applications of synthetic biology to the medical field.

'''Paper'''

Synthetic biology is a rapidly emerging field that strives to re-design existing biological systems and components or fabricate novel biological systems and components. The end result of experimentation in synthetic biology is to help scientists understand a naturally occurring genetic pathway more fully or to create a new, useful pathway. Recent research efforts have focused on engineering bacteria to create a new form of biofuel or to make promoters of varying efficiencies for more exact gene expression. Other research efforts focus on engineering yeast to make antimalarial drugs or engineering ''E. coli'' to recognize and invade tumor-like cells, both of which are medical applications of synthetic biology. Advancements such as novel cancer therapies, vaccination technology, biological protection, and the creation of new medicines highlight synthetic biology’s potential to create breakthroughs in medical science. This paper will examine various medical applications of synthetic biology and further development that needs to be done before current research can be used in hospitals and doctors’ offices.

Synthetic biologists consider finding a cure for cancer a reachable goal. Previous to the development of synthetic biology as a field, it was known that three types of bacteria, ''Bifodobacterium'', ''Clostridium'', and ''Salmonella'' all preferentially infect the dense cells of tumors (Pawelek 548). All three types of bacteria are associated with reducing tumor size in patients who are infected with them, and it was noted that all three bacteria act anaerobically. Capitalizing on the idea that tumors create an unusually dense, anaerobic mass of cells, Christopher Voigt’s lab engineered bacteria to identify and invade cancer cells. They utilized the ''lux'' quorum sensing from ''Vibrio fischeri'' to identify cells growing at high densities (Fig. 1) and the ''fdhF'' promoter to identify cells growing in anaerobic conditions (Fig. 2) (Anderson 619). They coupled this identification mechanism with the invasin output module from ''Yersinia pseudotuberculosis'' so the ''E. coli'' could invade specific cells. Next, scientists hope to insert a gene that would destroy the invaded cells, making the complete cycle a search, invade, and destroy loop that would only be sensitive to cancer cells. One promising destroy mechanism has been implemented in humans via intratumoral injection, based on ''Salmonella'' that contains the ''E. coli'' cytosine deaminase gene that converts 5-fluorocytosine to 5-fluorouracil, a chemotherapy drug (Nemunaitis 737). Unfortunately, while the tumors did produce 5-fluorouracil (5-FU), they did not produce it in high enough yields to cause regression of the cancer. Another experiment flooded mouse cells with 6-methylpurine-2’deoxyribose (6-MPDR), which was converted to the toxin 6-methyl purine (MeP) by the enzyme purine nucleoside phosphorylase (PNP), which is naturally found in ''E. coli'' (Critchley 1226) (Fig. 3). The problem with this is that all cells in the body might be exposed to the toxin MeP. In addition to the search and destroy mechanism, scientists hope to increase selectivity by combining the hypoxia-sensing and density-sensing units so that ''E. coli'' will only enter eukaryotic cells that exhibit both characteristics. This would make it more likely that invasin-enhanced ''E. coli'' would only invade cancer cells rather than muscle tissue that had low oxygen due to exercise (Anderson 624). To create a fully functional, synthetic biology-based approach to cancer therapy, scientists need to develop bacteria that discriminately invades cancer cells and destroys them. Most of the pieces are there – scientists have engineered ''E. coli'' that selectively invades hypoxic cells or dense cells and ''Salmonella'' that can destroy tumors in vivo – they just need to be put together effectively.

<center>

{|
|-
|[[Image:InvasinDensity.JPG]]
|[[Image:InvasinAnaerobic2.JPG]]
|[[Image:Tumor.JPG]]
|-
|'''Fig. 1.''' The red line represents when the invasin output module was paired with the inducible promoter ''lux'' which senses high cell density, the black line is constitutively on invasin. (Anderson)
|'''Fig. 2.''' The invasin output module was paired with the inducible promoter ''fdhF'' which senses anaerobic conditions. (Anderson)
|'''Fig. 3.''' Tumor size after injections. Cancer cells were injected into mice, and 5 days later injections with PBS, 6-MPDR, and invasin-enhanced ''E-coli'' began. Tumor cells recieving injections of invasin-enhanced ''E.coli'' and 6-MPDR grew the slowest, but did not get smaller. (Critchley)
|}
</center>

Using bacteria as a vaccine vector has similar issues of specificity and implementation in a human system. Bacteria was first thought to be a good vector because they would not degrade at mucosal surfaces, they would absorb better and survive the low pH in the gastrointestinal tract (Garmory 471). A particular strain of ''Salmonella'' with one or more deletions in the shikimate pathway, which creates a precursor for phenylalanine and tyrosine, is a promising vector because it gets into the body easily but does not harmfully effect the tissue and is gone within a week or two of introduction. Scientists engineered this strain to carry the ''Yersinia pestis'' V antigen, and studied its ability to protect mice from ''Y. pestis''. Unfortunately, the inoculation was not extremely successful and only 6 of 20 mice showed a strong response (Garmory 473). Garmory and colleagues cited several reasons for this: the copy number of the plasmid containing the V antigen might not have been high enough; the attenuated ''Salmonella'' may not have been able to reach cells that would be targeted by ''Y. pestis''; and the inability of the cell to secrete the antigen. Many changes need to be made to live vector vaccine design before it is an effective method of treating or preventing disease.

Another method of disease prevention with a synthetic biology approach is related to skin cancer. ''E. coli'' engineered to sense single-stranded DNA after DNA damage was coupled with a mechanism to produce a biofilm around the cell. The biofilm output module was tested with DNA damage done by mitomycin C, a DNA crosslinking agent, or UV irradiation, a known cause of skin cancer. Single-stranded DNA activates RecA, which in turn represses the CI repressor protein, thus allowing the transcription of the PL promoter (Kobayashi 8416). The PL promoter is in charge of the ''traA'' gene, which controls biofilm formation. The biofilm protection, when part of a toggle switch mechanism, lasted indefinitely when pulsed with UV light at 8 J/m2 for only 2 seconds (Kobayashi 8417) (Fig. 4). The biofilm-production mechanism has not been engineered in eukaryotic cells, but a prokaryote-based system that could aid with human skin disease is imaginable. For example, a sunscreen that changes colors when the DNA damage may be too intense for a sunbather as a warning, or a sunscreen that becomes more protective as the possibility of genetic damage increased. Further research should focus on how to best implement the biofilm producing output module to protect human DNA.
<center>
[[Image:UVinducedbiofilm.JPG]]

'''Fig. 4.''' Crystal violet absorbance measures biofilm formation. (Kobayashi)

</center>
Finally, synthetic biology has made its mark on the field of medicine by creating drugs that are less expensive to produce than those made by pharmaceutical companies. Jay Keasling’s project involving the antimalarial drug artemisinin is the best example. Yeast was engineered to increase normal farnesyl pyrophosphate (FPP) production, convert FPP to amorphadiene, and oxidizing amorphadiene to artemisinic acid, which can then easily be separated from the yeast cells and converted to artemisinin in the laboratory (Fig. 5). This innovation allows the production of artemisinin at lower prices than those currently on the market, and without regard to environmental constraints (Ro 942). On a completely different tangent, Collins’s lab engineered T7 bacteriophages to deliver biofilm-degrading enzymes such as dispersin B after infecting and replicating in an ''E. coli'' biofilm. Dispersin B works to degrade an adhesin that is critical for biofilm formation. Phages that released dispersin B were found to be 99.997% effective at removing biofilm, which is 4.5 orders of magnitude higher than non-enzymatic phages (Lu 11197) (Fig. 6). The specificity of phages for a certain type of bacteria make them a viable vector to be used in human treatments, which may range from removing dental plaques to cleaning inserted medical devices. Using synthetic biology to engineer an antimalarial drug in yeast or a biofilm-destroying enzyme in bacteriophages greatly increases the possible contributions to the field of medicine.

<center>

{|
|-
|[[Image:Artemesinin.JPG]]
|[[Image:BiofilmDegradation.JPG]]
|-
|'''Fig. 5.''' All the steps in the box indicate steps that were optimized in yeast for a maximum artemisinic acid output. (Ro)
|'''Fig. 6.''' The amount of biofilm was decreased when the dispersin B enzymes were incorporated into T7 bacteriophages. (Lu)
|}
</center>

Medical applications of synthetic biology are wide-ranging and eminently applicable to daily life. Synthetic biology based cancer treatments that utilize E.coli engineered to sense and attack tissues with high cell density and low oxygen availability are promising and need to be tested in vivo. Designing vectors out of attenuated bacteria may be a safe, effective method for disease treatment. Using biofilms to sense sun damage potential may decrease the likelihood of skin cancer. Finally, using engineered cells to produce medicines in a cost-effective, environmentally-friendly way may revolutionize the pharmaceutical industry. Investing more research efforts in the field of synthetic biology may turn out to be an investment in medical technology.

'''Works Cited'''

Anderson JC, Clarke EJ, Arkin, AP, and Voigt CA (2006). Environmentally controlled invasion of cancer cells by engineered bacteria. Journal of Molecular Biology 355:619-27.

Critchley RJ, Jezzard S, Radford KJ, Goussard S, Lemoine NR, et al. (2004). Potential therapeutic applications of recombinant, invasive ''E. coli''. Gene Therapy 11:1224-33.

Garmory HS, Leary SEC, Griffin, KF, Williamson D, Brown, KA, and Titball RW (2003). The use of live attenuated bacteria as a delivery system for heterologous antigens. Journal of Drug Targeting 11:471-79.

Kobayashi H, et al. (2004). Programmable cells: Interfacing natural and engineered gene networks. PNAS 101: 8414-19.

Lu, TK, and Collins JJ (2007). Dispersing biofilms with engineered enzymatic bacteriophage. PNAS 104: 11197-11202.

Nemunaitis J, Cunningham C, Senzer N, Kuhn J, Cramm J, Litz C, et al. (2003). Pilot trial of genetically modified, attenuated ''Salmonella'' expressing cytosine deaminase gene in refractory cancer patients. Cancer Gene Therapy 10:737-744.

Pawelek JM, Low KB, and Bermudes D (2003). Bacteria as tumour-targeting vectors. Lancet Oncology 4:548-56.

Ro D, et al. (2006). Production of the antimalarial drug precursor artemisinic acid in engineered yeast. Nature 440:940-43.

Medical Applications of Synthetic Biology - Samantha Simpson

2007-11-13T03:38:39Z

Sasimpson:

'''Project Proposal'''

My project will be on medical applications of synthetic biology. I will reference Anderson's paper on utilizing quorum-sensing and hypoxia-responsive genes coupled with invasin from ''Yersinia tuberculosis'' to invade cancer-causing cells. Coupled with Critchley's work, which describes a bacterial system that invades eukaryotic cells and delivers proteins coded for in the bacteria's genome, one could possibly create 'search and destroy' ''E.coli'' that can locate, invade, and kill tumor cells. Garmory's paper also highlights the possibility of using bacteria as a drug-delivering system, specifically vaccine vectors. Kobayashi describes cells that produce a protective biofilm layer after exposure to DNA-damaging agents. Ro engineers yeast to create an anti-malarial drug that can be created at a lower cost than the previous standard production process. These papers, and others that have a focus on direct medical applications such as novel cancer therapies, vaccination technology, biological protection, and the creation of new medicines, will be referenced to create a cohesive overview of applications of synthetic biology to the medical field.

'''Paper'''

Synthetic biology is a rapidly emerging field that strives to re-design existing biological systems and components or fabricate novel biological systems and components. The end result of experimentation in synthetic biology is to help scientists understand a naturally occurring genetic pathway more fully or to create a new, useful pathway. Recent research efforts have focused on engineering bacteria to create a new form of biofuel or to make promoters of varying efficiencies for more exact gene expression. Other research efforts focus on engineering yeast to make antimalarial drugs or engineering ''E. coli'' to recognize and invade tumor-like cells, both of which are medical applications of synthetic biology. Advancements such as novel cancer therapies, vaccination technology, biological protection, and the creation of new medicines highlight synthetic biology’s potential to create breakthroughs in medical science. This paper will examine various medical applications of synthetic biology and further development that needs to be done before current research can be used in hospitals and doctors’ offices.

Synthetic biologists consider finding a cure for cancer a reachable goal. Previous to the development of synthetic biology as a field, it was known that three types of bacteria, ''Bifodobacterium'', ''Clostridium'', and ''Salmonella'' all preferentially infect the dense cells of tumors (Pawelek 548). All three types of bacteria are associated with reducing tumor size in patients who are infected with them, and it was noted that all three bacteria act anaerobically. Capitalizing on the idea that tumors create an unusually dense, anaerobic mass of cells, Christopher Voigt’s lab engineered bacteria to identify and invade cancer cells. They utilized the ''lux'' quorum sensing from ''Vibrio fischeri'' to identify cells growing at high densities (Fig. 1) and the ''fdhF'' promoter to identify cells growing in anaerobic conditions (Fig. 2) (Anderson 619). They coupled this identification mechanism with the invasin output module from ''Yersinia pseudotuberculosis'' so the ''E. coli'' could invade specific cells. Next, scientists hope to insert a gene that would destroy the invaded cells, making the complete cycle a search, invade, and destroy loop that would only be sensitive to cancer cells. One promising destroy mechanism has been implemented in humans via intratumoral injection, based on ''Salmonella'' that contains the ''E. coli'' cytosine deaminase gene that converts 5-fluorocytosine to 5-fluorouracil, a chemotherapy drug (Nemunaitis 737). Unfortunately, while the tumors did produce 5-fluorouracil (5-FU), they did not produce it in high enough yields to cause regression of the cancer. Another experiment flooded mouse cells with 6-methylpurine-2’deoxyribose (6-MPDR), which was converted to the toxin 6-methyl purine (MeP) by the enzyme purine nucleoside phosphorylase (PNP), which is naturally found in ''E. coli'' (Critchley 1226) (Fig. 3). The problem with this is that all cells in the body might be exposed to the toxin MeP. In addition to the search and destroy mechanism, scientists hope to increase selectivity by combining the hypoxia-sensing and density-sensing units so that ''E. coli'' will only enter eukaryotic cells that exhibit both characteristics. This would make it more likely that invasin-enhanced ''E. coli'' would only invade cancer cells rather than muscle tissue that had low oxygen due to exercise (Anderson 624). To create a fully functional, synthetic biology-based approach to cancer therapy, scientists need to develop bacteria that discriminately invades cancer cells and destroys them. Most of the pieces are there – scientists have engineered ''E. coli'' that selectively invades hypoxic cells or dense cells and ''Salmonella'' that can destroy tumors in vivo – they just need to be put together effectively.

<center>

{|
|-
|[[Image:InvasinDensity.JPG]]
|[[Image:InvasinAnaerobic2.JPG]]
|[[Image:Tumor.JPG]]
|-
|'''Fig. 1.''' The red line represents when the invasin output module was paired with the inducible promoter ''lux'' which senses high cell density, the black line is constitutively on invasin. (Anderson)
|'''Fig. 2.''' The invasin output module was paired with the inducible promoter ''fdhF'' which senses anaerobic conditions. (Anderson)
|'''Fig. 3.''' Tumor size after injections. Cancer cells were injected into mice, and 5 days later injections with PBS, 6-MPDR, and invasin-enhanced ''E-coli'' began. Tumor cells recieving injections of invasin-enhanced ''E.coli'' and 6-MPDR grew the slowest, but did not get smaller. (Critchley)
|}
</center>

Using bacteria as a vaccine vector has similar issues of specificity and implementation in a human system. Bacteria was first thought to be a good vector because they would not degrade at mucosal surfaces, they would absorb better and survive the low pH in the gastrointestinal tract (Garmory 471). A particular strain of ''Salmonella'' with one or more deletions in the shikimate pathway, which creates a precursor for phenylalanine and tyrosine, is a promising vector because it gets into the body easily but does not harmfully effect the tissue and is gone within a week or two of introduction. Scientists engineered this strain to carry the ''Yersinia pestis'' V antigen, and studied its ability to protect mice from ''Y. pestis''. Unfortunately, the inoculation was not extremely successful and only 6 of 20 mice showed a strong response (Garmory 473). Garmory and colleagues cited several reasons for this: the copy number of the plasmid containing the V antigen might not have been high enough; the attenuated ''Salmonella'' may not have been able to reach cells that would be targeted by ''Y. pestis''; and the inability of the cell to secrete the antigen. Many changes need to be made to live vector vaccine design before it is an effective method of treating or preventing disease.

Another method of disease prevention with a synthetic biology approach is related to skin cancer. ''E. coli'' engineered to sense single-stranded DNA after DNA damage was coupled with a mechanism to produce a biofilm around the cell. The biofilm output module was tested with DNA damage done by mitomycin C, a DNA crosslinking agent, or UV irradiation, a known cause of skin cancer. The biofilm protection, when part of a toggle switch mechanism, lasted indefinitely when pulsed with UV light at 8 J/m2 for only 2 seconds (Kobayashi 8417) (Fig. 4). The biofilm-production mechanism has not been engineered in eukaryotic cells, but a prokaryote-based system that could aid with human skin disease is imaginable. For example, a sunscreen that changes colors when the DNA damage may be too intense for a sunbather as a warning, or a sunscreen that becomes more protective as the possibility of genetic damage increased. Further research should focus on how to best implement the biofilm producing output module to protect human DNA.
<center>
[[Image:UVinducedbiofilm.JPG]]

'''Fig. 4.''' Crystal violet absorbance measures biofilm formation. (Kobayashi)

</center>
Finally, synthetic biology has made its mark on the field of medicine by creating drugs that are less expensive to produce than those made by pharmaceutical companies. Jay Keasling’s project involving the antimalarial drug artemisinin is the best example. Yeast was engineered to increase normal farnesyl pyrophosphate (FPP) production, convert FPP to amorphadiene, and oxidizing amorphadiene to artemisinic acid, which can then easily be separated from the yeast cells and converted to artemisinin in the laboratory (Fig. 5). This innovation allows the production of artemisinin at lower prices than those currently on the market, and without regard to environmental constraints (Ro 942). On a completely different tangent, Collins’s lab engineered T7 bacteriophages to deliver biofilm-degrading enzymes such as dispersin B after infecting and replicating in an ''E. coli'' biofilm. Dispersin B works to degrade an adhesin that is critical for biofilm formation. Phages that released dispersin B were found to be 99.997% effective at removing biofilm, which is 4.5 orders of magnitude higher than non-enzymatic phages (Lu 11197) (Fig. 6). The specificity of phages for a certain type of bacteria make them a viable vector to be used in human treatments, which may range from removing dental plaques to cleaning inserted medical devices. Using synthetic biology to engineer an antimalarial drug in yeast or a biofilm-destroying enzyme in bacteriophages greatly increases the possible contributions to the field of medicine.

<center>

{|
|-
|[[Image:Artemesinin.JPG]]
|[[Image:BiofilmDegradation.JPG]]
|-
|'''Fig. 5.''' All the steps in the box indicate steps that were optimized in yeast for a maximum artemisinic acid output. (Ro)
|'''Fig. 6.''' The amount of biofilm was decreased when the dispersin B enzymes were incorporated into T7 bacteriophages. (Lu)
|}
</center>

Medical applications of synthetic biology are wide-ranging and eminently applicable to daily life. Synthetic biology based cancer treatments that utilize E.coli engineered to sense and attack tissues with high cell density and low oxygen availability are promising and need to be tested in vivo. Designing vectors out of attenuated bacteria may be a safe, effective method for disease treatment. Using biofilms to sense sun damage potential may decrease the likelihood of skin cancer. Finally, using engineered cells to produce medicines in a cost-effective, environmentally-friendly way may revolutionize the pharmaceutical industry. Investing more research efforts in the field of synthetic biology may turn out to be an investment in medical technology.

'''Works Cited'''

Anderson JC, Clarke EJ, Arkin, AP, and Voigt CA (2006). Environmentally controlled invasion of cancer cells by engineered bacteria. Journal of Molecular Biology 355:619-27.

Critchley RJ, Jezzard S, Radford KJ, Goussard S, Lemoine NR, et al. (2004). Potential therapeutic applications of recombinant, invasive ''E. coli''. Gene Therapy 11:1224-33.

Garmory HS, Leary SEC, Griffin, KF, Williamson D, Brown, KA, and Titball RW (2003). The use of live attenuated bacteria as a delivery system for heterologous antigens. Journal of Drug Targeting 11:471-79.

Kobayashi H, et al. (2004). Programmable cells: Interfacing natural and engineered gene networks. PNAS 101: 8414-19.

Lu, TK, and Collins JJ (2007). Dispersing biofilms with engineered enzymatic bacteriophage. PNAS 104: 11197-11202.

Nemunaitis J, Cunningham C, Senzer N, Kuhn J, Cramm J, Litz C, et al. (2003). Pilot trial of genetically modified, attenuated ''Salmonella'' expressing cytosine deaminase gene in refractory cancer patients. Cancer Gene Therapy 10:737-744.

Pawelek JM, Low KB, and Bermudes D (2003). Bacteria as tumour-targeting vectors. Lancet Oncology 4:548-56.

Ro D, et al. (2006). Production of the antimalarial drug precursor artemisinic acid in engineered yeast. Nature 440:940-43.

Medical Applications of Synthetic Biology - Samantha Simpson

2007-11-13T03:26:48Z

Sasimpson:

'''Project Proposal'''

My project will be on medical applications of synthetic biology. I will reference Anderson's paper on utilizing quorum-sensing and hypoxia-responsive genes coupled with invasin from ''Yersinia tuberculosis'' to invade cancer-causing cells. Coupled with Critchley's work, which describes a bacterial system that invades eukaryotic cells and delivers proteins coded for in the bacteria's genome, one could possibly create 'search and destroy' ''E.coli'' that can locate, invade, and kill tumor cells. Garmory's paper also highlights the possibility of using bacteria as a drug-delivering system, specifically vaccine vectors. Kobayashi describes cells that produce a protective biofilm layer after exposure to DNA-damaging agents. Ro engineers yeast to create an anti-malarial drug that can be created at a lower cost than the previous standard production process. These papers, and others that have a focus on direct medical applications such as novel cancer therapies, vaccination technology, biological protection, and the creation of new medicines, will be referenced to create a cohesive overview of applications of synthetic biology to the medical field.

'''Paper'''

Synthetic biology is a rapidly emerging field that strives to re-design existing biological systems and components or fabricate novel biological systems and components. The end result of experimentation in synthetic biology is to help scientists understand a naturally occurring genetic pathway more fully or to create a new, useful pathway. Recent research efforts have focused on engineering bacteria to create a new form of biofuel or to make promoters of varying efficiencies for more exact gene expression. Other research efforts focus on engineering yeast to make antimalarial drugs or engineering ''E. coli'' to recognize and invade tumor-like cells, both of which are medical applications of synthetic biology. Advancements such as novel cancer therapies, vaccination technology, biological protection, and the creation of new medicines highlight synthetic biology’s potential to create breakthroughs in medical science. This paper will examine various medical applications of synthetic biology and further development that needs to be done before current research can be used in hospitals and doctors’ offices.

Synthetic biologists consider finding a cure for cancer a reachable goal. Previous to the development of synthetic biology as a field, it was known that three types of bacteria, ''Bifodobacterium'', ''Clostridium'', and ''Salmonella'' all preferentially infect the dense cells of tumors (Pawelek 548). All three types of bacteria are associated with reducing tumor size in patients who are infected with them, and it was noted that all three bacteria act anaerobically. Capitalizing on the idea that tumors create an unusually dense, anaerobic mass of cells, Christopher Voigt’s lab engineered bacteria to identify and invade cancer cells. They utilized the ''lux'' quorum sensing from ''Vibrio fischeri'' to identify cells growing at high densities (Fig. 1) and the ''fdhF'' promoter to identify cells growing in anerobic conditions (Fig. 2) (Anderson 619). They coupled this identification mechanism with the invasin output module from ''Yersinia pseudotuberculosis'' so the ''E. coli'' could invade specific cells. Next, scientists hope to insert a gene that would destroy the invaded cells, making the complete cycle a search, invade, and destroy loop that would only be sensitive to cancer cells. One promising destroy mechanism has been implemented in humans via intratumoral injection, based on ''Salmonella'' that contains the ''E. coli'' cytosine deaminase gene that converts 5-fluorocytosine to 5-fluorouracil, a chemotherapy drug (Nemunaitis 737). Unfortunately, while the tumors did produce 5-fluorouracil (5-FU), they did not produce it in high enough yields to cause regression of the cancer. Another experiment flooded mouse cells with 6-methylpurine-2’deoxyribose (6-MPDR), which was converted to the toxin 6-methyl purine (MeP) by the enzyme purine nucleoside phosphorylase (PNP), which is naturally found in ''E. coli'' (Critchley 1226) (Fig. 3). The problem with this is that all cells in the body might be exposed to the toxin MeP. In addition to the search and destroy mechanism, scientists hope to increase selectivity by combining the hypoxia-sensing and density-sensing units so that ''E. coli'' will only enter eukaryotic cells that exhibit both characteristics. This would make it more likely that invasin-enhanced ''E. coli'' would only invade cancer cells rather than muscle tissue that had low oxygen due to exercise (Anderson 624). To create a fully functional, synthetic biology-based approach to cancer therapy, scientists need to develop bacteria that discriminately invades cancer cells and destroys them. Most of the pieces are there – scientists have engineered ''E. coli'' that selectively invades hypoxic cells or dense cells and ''Salmonella'' that can destroy tumors in vivo – they just need to be put together effectively.

<center>

{|
|-
|[[Image:InvasinDensity.JPG]]
|[[Image:InvasinAnaerobic2.JPG]]
|[[Image:Tumor.JPG]]
|-
|'''Fig. 1.''' The red line represents when the invasin output module was paired with the inducible promoter ''lux'' which senses high cell density, the black line is constitutively on invasin. (Anderson)
|'''Fig. 2.''' The invasin output module was paired with the inducible promoter ''fdhF'' which senses anaerobic conditions. (Anderson)
|'''Fig. 3.''' Tumor size after injections. Cancer cells were injected into mice, and 5 days later injections with PBS, 6-MPDR, and invasin-enhanced ''E-coli'' began. Tumor cells recieving injections of invasin-enhanced ''E.coli'' and 6-MPDR grew the slowest, but did not get smaller. (Critchley)
|}
</center>

Using bacteria as a vaccine vector has similar issues of specificity and implementation in a human system. Bacteria was first thought to be a good vector because they would not degrade at mucosal surfaces, they would absorb better and survive the low pH in the gastrointestinal tract (Garmory 471). A particular strain of ''Salmonella'' with one or more deletions in the shikimate pathway, which creates a precursor for phenylalanine and tyrosine, is a promising vector because it gets into the body easily but does not harmfully effect the tissue and is gone within a week or two of introduction. Scientists engineered this strain to carry the ''Yersinia pestis'' V antigen, and studied its ability to protect mice from ''Y. pestis''. Unfortunately, the inoculation was not extremely successful and only 6 of 20 mice showed a strong response (Garmory 473). Garmory and colleagues cited several reasons for this: the copy number of the plasmid containing the V antigen might not have been high enough; the attenuated ''Salmonella'' may not have been able to reach cells that would be targeted by ''Y. pestis''; and the inability of the cell to secrete the antigen. Many changes need to be made to live vector vaccine design before it is an effective method of treating or preventing disease.

Another method of disease prevention with a synthetic biology approach is related to skin cancer. ''E. coli'' engineered to sense single-stranded DNA after DNA damage was coupled with a mechanism to produce a biofilm around the cell. The biofilm output module was tested with DNA damage done by mitomycin C, a DNA crosslinking agent, or UV irradiation, a known cause of skin cancer. The biofilm protection, when part of a toggle switch mechanism, lasted indefinitely when pulsed with UV light at 8 J/m2 for only 2 seconds (Kobayashi 8417) (Fig. 4). The biofilm-production mechanism has not been engineered in eukaryotic cells, but a prokaryote-based system that could aid with human skin disease is imaginable. For example, a sunscreen that changes colors when the DNA damage may be too intense for a sunbather as a warning, or a sunscreen that becomes more protective as the possibility of genetic damage increased. Further research should focus on how to best implement the biofilm producing output module to protect human DNA.
<center>
[[Image:UVinducedbiofilm.JPG]]

'''Fig. 4.''' Crystal violet absorbance measures biofilm formation. (Kobayashi)

</center>
Finally, synthetic biology has made its mark on the field of medicine by creating drugs that are less expensive to produce than those made by pharmaceutical companies. Jay Keasling’s project involving the antimalarial drug artemisinin is the best example. Yeast was engineered to increase normal farnesyl pyrophosphate (FPP) production, convert FPP to amorphadiene, and oxidizing amorphadiene to artemisinic acid, which can then easily be separated from the yeast cells and converted to artemisinin in the laboratory (Fig. 5). This innovation allows the production of artemisinin at lower prices than those currently on the market, and without regard to environmental constraints (Ro 942). On a completely different tangent, Collins’s lab engineered T7 bacteriophages to deliver biofilm-degrading enzymes such as dispersin B after infecting and replicating in an ''E. coli'' biofilm. Dispersin B works to degrade an adhesin that is critical for biofilm formation. Phages that released dispersin B were found to be 99.997% effective at removing biofilm, which is 4.5 orders of magnitude higher than non-enzymatic phages (Lu 11197) (Fig. 6). The specificity of phages for a certain type of bacteria make them a viable vector to be used in human treatments, which may range from removing dental plaques to cleaning inserted medical devices. Using synthetic biology to engineer an antimalarial drug in yeast or a biofilm-destroying enzyme in bacteriophages greatly increases the possible contributions to the field of medicine.

<center>

{|
|-
|[[Image:Artemesinin.JPG]]
|[[Image:BiofilmDegradation.JPG]]
|-
|'''Fig. 5.''' All the steps in the box indicate steps that were optimized in yeast for a maximum artemisinic acid output. (Ro)
|'''Fig. 6.''' The amount of biofilm was decreased when the dispersin B enzymes were incorporated into T7 bacteriophages. (Lu)
|}
</center>

Medical applications of synthetic biology are wide-ranging and eminently applicable to daily life. Synthetic biology based cancer treatments that utilize E.coli engineered to sense and attack tissues with high cell density and low oxygen availability are promising and need to be tested in vivo. Designing vectors out of attenuated bacteria may be a safe, effective method for disease treatment. Using biofilms to sense sun damage potential may decrease the likelihood of skin cancer. Finally, using engineered cells to produce medicines in a cost-effective, environmentally-friendly way may revolutionize the pharmaceutical industry. Investing more research efforts in the field of synthetic biology may turn out to be an investment in medical technology.

'''Works Cited'''

Anderson JC, Clarke EJ, Arkin, AP, and Voigt CA (2006). Environmentally controlled invasion of cancer cells by engineered bacteria. Journal of Molecular Biology 355:619-27.

Critchley RJ, Jezzard S, Radford KJ, Goussard S, Lemoine NR, et al. (2004). Potential therapeutic applications of recombinant, invasive ''E. coli''. Gene Therapy 11:1224-33.

Garmory HS, Leary SEC, Griffin, KF, Williamson D, Brown, KA, and Titball RW (2003). The use of live attenuated bacteria as a delivery system for heterologous antigens. Journal of Drug Targeting 11:471-79.

Kobayashi H, et al. (2004). Programmable cells: Interfacing natural and engineered gene networks. PNAS 101: 8414-19.

Lu, TK, and Collins JJ (2007). Dispersing biofilms with engineered enzymatic bacteriophage. PNAS 104: 11197-11202.

Nemunaitis J, Cunningham C, Senzer N, Kuhn J, Cramm J, Litz C, et al. (2003). Pilot trial of genetically modified, attenuated ''Salmonella'' expressing cytosine deaminase gene in refractory cancer patients. Cancer Gene Therapy 10:737-744.

Pawelek JM, Low KB, and Bermudes D (2003). Bacteria as tumour-targeting vectors. Lancet Oncology 4:548-56.

Ro D, et al. (2006). Production of the antimalarial drug precursor artemisinic acid in engineered yeast. Nature 440:940-43.

Medical Applications of Synthetic Biology - Samantha Simpson

2007-11-13T03:17:48Z

Sasimpson:

'''Project Proposal'''

My project will be on medical applications of synthetic biology. I will reference Anderson's paper on utilizing quorum-sensing and hypoxia-responsive genes coupled with invasin from ''Yersinia tuberculosis'' to invade cancer-causing cells. Coupled with Critchley's work, which describes a bacterial system that invades eukaryotic cells and delivers proteins coded for in the bacteria's genome, one could possibly create 'search and destroy' ''E.coli'' that can locate, invade, and kill tumor cells. Garmory's paper also highlights the possibility of using bacteria as a drug-delivering system, specifically vaccine vectors. Kobayashi describes cells that produce a protective biofilm layer after exposure to DNA-damaging agents. Ro engineers yeast to create an anti-malarial drug that can be created at a lower cost than the previous standard production process. These papers, and others that have a focus on direct medical applications such as novel cancer therapies, vaccination technology, biological protection, and the creation of new medicines, will be referenced to create a cohesive overview of applications of synthetic biology to the medical field.

'''Paper'''

Synthetic biology is a rapidly emerging field that strives to re-design existing biological systems and components or fabricate novel biological systems and components. The end result of experimentation in synthetic biology is to help scientists understand a naturally occurring genetic pathway more fully or to create a new, useful pathway. Recent research efforts have focused on engineering bacteria to create a new form of biofuel or to make promoters of varying efficiencies for more exact gene expression. Other research efforts focus on engineering yeast to make antimalarial drugs or engineering ''E. coli'' to recognize and invade tumor-like cells, both of which are medical applications of synthetic biology. Advancements such as novel cancer therapies, vaccination technology, biological protection, and the creation of new medicines highlight synthetic biology’s potential to create breakthroughs in medical science. This paper will examine various medical applications of synthetic biology and further development that needs to be done before current research can be used in hospitals and doctors’ offices.

Synthetic biologists consider finding a cure for cancer a reachable goal. Previous to the development of synthetic biology as a field, it was known that three types of bacteria, ''Bifodobacterium'', ''Clostridium'', and ''Salmonella'' all preferentially infect the dense cells of tumors (Pawelek 548). All three types of bacteria are associated with reducing tumor size in patients who are infected with them, and it was noted that all three bacteria act anaerobically. Capitalizing on the idea that tumors create an unusually dense, anaerobic mass of cells, Christopher Voigt’s lab engineered bacteria to identify and invade cancer cells. They utilized the ''lux'' quorum sensing from ''Vibrio fischeri'' to identify cells growing at high densities (Fig. 1) and the ''fdhF'' promoter to identify cells growing in anerobic conditions (Fig. 2) (Anderson 619). They coupled this identification mechanism with the invasin output module from ''Yersinia pseudotuberculosis'' so the ''E. coli'' could invade specific cells. Next, scientists hope to insert a gene that would destroy the invaded cells, making the complete cycle a search, invade, and destroy loop that would only be sensitive to cancer cells. One promising destroy mechanism has been implemented in humans via intratumoral injection, based on ''Salmonella'' that contains the ''E. coli'' cytosine deaminase gene that converts 5-fluorocytosine to 5-fluorouracil, a chemotherapy drug (Nemunaitis 737). Unfortunately, while the tumors did produce 5-fluorouracil (5-FU), they did not produce it in high enough yields to cause regression of the cancer. Another experiment flooded mouse cells with 6-methylpurine-2’deoxyribose (6-MPDR), which was converted to the toxin 6-methyl purine (MeP) by the enzyme purine nucleoside phosphorylase (PNP), which is naturally found in ''E. coli''(Critchley 1226) (Fig. 3). The problem with this is that all cells in the body might be exposed to the toxin MeP. In addition to the search and destroy mechanism, scientists hope to increase selectivity by combining the hypoxia-sensing and density-sensing units so that ''E. coli'' will only enter eukaryotic cells that exhibit both characteristics. This would make it more likely that invasin-enhanced ''E. coli'' would only invade cancer cells rather than muscle tissue that had low oxygen due to exercise (Anderson 624). To create a fully functional, synthetic biology-based approach to cancer therapy, scientists need to develop bacteria that discriminately invades cancer cells and destroys them. Most of the pieces are there – scientists have engineered ''E. coli'' that selectively invades hypoxic cells or dense cells and ''Salmonella'' that can destroy tumors in vivo – they just need to be put together effectively.

<center>

{|
|-
|[[Image:InvasinDensity.JPG]]
|[[Image:InvasinAnaerobic2.JPG]]
|[[Image:Tumor.JPG]]
|-
|'''Fig. 1.''' The red line represents when the invasin output module was paired with the inducible promoter ''lux'' which senses high cell density, the black line is constitutively on invasin.
|'''Fig. 2.''' The invasin output module was paired with the inducible promoter ''fdhF'' which senses anaerobic conditions.
|'''Fig. 3.''' Tumor size after injections. Cancer cells were injected into mice, and 5 days later injections with PBS, 6-MPDR, and invasin-enhanced ''E-coli'' began. Tumor cells recieving injections of invasin-enhanced ''E.coli'' and 6-MPDR grew the slowest, but did not get smaller.
|}
</center>

Using bacteria as a vaccine vector has similar issues of specificity and implementation in a human system. Bacteria was first thought to be a good vector because they would not degrade at mucosal surfaces, they would absorb better and survive the low pH in the gastrointestinal tract (Garmory 471). A particular strain of ''Salmonella'' with one or more deletions in the shikimate pathway, which creates a precursor for phenylalanine and tyrosine, is a promising vector because it gets into the body easily but does not harmfully effect the tissue and is gone within a week or two of introduction. Scientists engineered this strain to carry the ''Yersinia pestis'' V antigen, and studied its ability to protect mice from ''Y. pestis''. Unfortunately, the inoculation was not extremely successful and only 6 of 20 mice showed a strong response (Garmory 473). Garmory and colleagues cited several reasons for this: the copy number of the plasmid containing the V antigen might not have been high enough; the attenuated ''Salmonella'' may not have been able to reach cells that would be targeted by ''Y. pestis''; and the inability of the cell to secrete the antigen. Many changes need to be made to live vector vaccine design before it is an effective method of treating or preventing disease.

Another method of disease prevention with a synthetic biology approach is related to skin cancer. ''E. coli'' engineered to sense single-stranded DNA after DNA damage was coupled with a mechanism to produce a biofilm around the cell. The biofilm output module was tested with DNA damage done by mitomycin C, a DNA crosslinking agent, or UV irradiation, a known cause of skin cancer. The biofilm protection, when part of a toggle switch mechanism, lasted indefinitely when pulsed with UV light at 8 J/m2 for only 2 seconds (Kobayashi 8417) (Fig. 4). The biofilm-production mechanism has not been engineered in eukaryotic cells, but a prokaryote-based system that could aid with human skin disease is imaginable. For example, a sunscreen that changes colors when the DNA damage may be too intense for a sunbather as a warning, or a sunscreen that becomes more protective as the possibility of genetic damage increased. Further research should focus on how to best implement the biofilm producing output module to protect human DNA.
<center>
[[Image:UVinducedbiofilm.JPG]]

'''Fig. 4.''' Crystal violet absorbance measures biofilm formation.

</center>
Finally, synthetic biology has made its mark on the field of medicine by creating drugs that are less expensive to produce than those made by pharmaceutical companies. Jay Keasling’s project involving the antimalarial drug artemisinin is the best example. Yeast was engineered to increase normal farnesyl pyrophosphate (FPP) production, convert FPP to amorphadiene, and oxidizing amorphadiene to artemisinic acid, which can then easily be separated from the yeast cells and converted to artemisinin in the laboratory (Fig. 5). This innovation allows the production of artemisinin at lower prices than those currently on the market, and without regard to environmental constraints (Ro 942). On a completely different tangent, Collins’s lab engineered T7 bacteriophages to deliver biofilm-degrading enzymes such as dispersin B after infecting and replicating in an ''E. coli'' biofilm. Dispersin B works to degrade an adhesin that is critical for biofilm formation. Phages that released dispersin B were found to be 99.997% effective at removing biofilm, which is 4.5 orders of magnitude higher than non-enzymatic phages (Lu 11197) (Fig. 6). The specificity of phages for a certain type of bacteria make them a viable vector to be used in human treatments, which may range from removing dental plaques to cleaning inserted medical devices. Using synthetic biology to engineer an antimalarial drug in yeast or a biofilm-destroying enzyme in bacteriophages greatly increases the possible contributions to the field of medicine.

<center>

{|
|-
|[[Image:Artemesinin.JPG]]
|[[Image:BiofilmDegradation.JPG]]
|-
|'''Fig. 5.''' All the steps in the box indicate steps that were optimized in yeast for a maximum artemisinic acid output.
|'''Fig. 6.''' The amount of biofilm was decreased when the dispersin B enzymes were incorporated into T7 bacteriophages.
|}
</center>

Medical applications of synthetic biology are wide-ranging and eminently applicable to daily life. Synthetic biology based cancer treatments that utilize E.coli engineered to sense and attack tissues with high cell density and low oxygen availability are promising and need to be tested in vivo. Designing vectors out of attenuated bacteria may be a safe, effective method for disease treatment. Using biofilms to sense sun damage potential may decrease the likelihood of skin cancer. Finally, using engineered cells to produce medicines in a cost-effective, environmentally-friendly way may revolutionize the pharmaceutical industry. Investing more research efforts in the field of synthetic biology may turn out to be an investment in medical technology.

'''Works Cited'''

Anderson JC, Clarke EJ, Arkin, AP, and Voigt CA (2006). Environmentally controlled invasion of cancer cells by engineered bacteria. Journal of Molecular Biology 355:619-27.

Critchley RJ, Jezzard S, Radford KJ, Goussard S, Lemoine NR, et al. (2004). Potential therapeutic applications of recombinant, invasive ''E. coli''. Gene Therapy 11:1224-33.

Garmory HS, Leary SEC, Griffin, KF, Williamson D, Brown, KA, and Titball RW (2003). The use of live attenuated bacteria as a delivery system for heterologous antigens. Journal of Drug Targeting 11:471-79.

Kobayashi H, et al. (2004). Programmable cells: Interfacing natural and engineered gene networks. PNAS 101: 8414-19.

Lu, TK, and Collins JJ (2007). Dispersing biofilms with engineered enzymatic bacteriophage. PNAS 104: 11197-11202.

Nemunaitis J, Cunningham C, Senzer N, Kuhn J, Cramm J, Litz C, et al. (2003). Pilot trial of genetically modified, attenuated ''Salmonella'' expressing cytosine deaminase gene in refractory cancer patients. Cancer Gene Therapy 10:737-744.

Pawelek JM, Low KB, and Bermudes D (2003). Bacteria as tumour-targeting vectors. Lancet Oncology 4:548-56.

Ro D, et al. (2006). Production of the antimalarial drug precursor artemisinic acid in engineered yeast. Nature 440:940-43.

File:Tumor.JPG

2007-11-13T02:57:49Z

Sasimpson:

Medical Applications of Synthetic Biology - Samantha Simpson

2007-11-13T02:42:06Z

Sasimpson:

'''Project Proposal'''

My project will be on medical applications of synthetic biology. I will reference Anderson's paper on utilizing quorum-sensing and hypoxia-responsive genes coupled with invasin from ''Yersinia tuberculosis'' to invade cancer-causing cells. Coupled with Critchley's work, which describes a bacterial system that invades eukaryotic cells and delivers proteins coded for in the bacteria's genome, one could possibly create 'search and destroy' ''E.coli'' that can locate, invade, and kill tumor cells. Garmory's paper also highlights the possibility of using bacteria as a drug-delivering system, specifically vaccine vectors. Kobayashi describes cells that produce a protective biofilm layer after exposure to DNA-damaging agents. Ro engineers yeast to create an anti-malarial drug that can be created at a lower cost than the previous standard production process. These papers, and others that have a focus on direct medical applications such as novel cancer therapies, vaccination technology, biological protection, and the creation of new medicines, will be referenced to create a cohesive overview of applications of synthetic biology to the medical field.

'''Paper'''

Synthetic biology is a rapidly emerging field that strives to re-design existing biological systems and components or fabricate novel biological systems and components. The end result of experimentation in synthetic biology is to help scientists understand a naturally occurring genetic pathway more fully or to create a new, useful pathway. Recent research efforts have focused on engineering bacteria to create a new form of biofuel or to make promoters of varying efficiencies for more exact gene expression. Other research efforts focus on engineering yeast to make antimalarial drugs or engineering ''E. coli'' to recognize and invade tumor-like cells, both of which are medical applications of synthetic biology. Advancements such as novel cancer therapies, vaccination technology, biological protection, and the creation of new medicines highlight synthetic biology’s potential to create breakthroughs in medical science. This paper will examine various medical applications of synthetic biology and further development that needs to be done before current research can be used in hospitals and doctors’ offices.

Synthetic biologists consider finding a cure for cancer a reachable goal. Previous to the development of synthetic biology as a field, it was known that three types of bacteria, ''Bifodobacterium'', ''Clostridium'', and ''Salmonella'' all preferentially infect the dense cells of tumors (Pawelek 548). All three types of bacteria are associated with reducing tumor size in patients who are infected with them, and it was noted that all three bacteria act anaerobically. Capitalizing on the idea that tumors create an unusually dense, anaerobic mass of cells, Christopher Voigt’s lab engineered bacteria to identify and invade cancer cells. They utilized the ''lux'' quorum sensing from ''Vibrio fischeri'' to identify cells growing at high densities (Fig. 1) and the ''fdhF'' promoter to identify cells growing in anerobic conditions (Fig. 2) (Anderson 619). They coupled this identification mechanism with the invasin output module from ''Yersinia pseudotuberculosis'' so the ''E. coli'' could invade specific cells. Next, scientists hope to insert a gene that would destroy the invaded cells, making the complete cycle a search, invade, and destroy loop that would only be sensitive to cancer cells. One promising destroy mechanism has been implemented in humans via intratumoral injection, based on ''Salmonella'' that contains the ''E. coli'' cytosine deaminase gene that converts 5-fluorocytosine to 5-fluorouracil, a chemotherapy drug (Nemunaitis 737). Unfortunately, while the tumors did produce 5-fluorouracil (5-FU), they did not produce it in high enough yields to cause regression of the cancer. Another experiment used invasin-enhanced ''E. coli'' to enter cells in vitro and sensitize those cells to 6-methylpurine-2’deoxyribose (6-MPDR) or 5-FU by depositing proteins that converted those compounds to toxins, and then flood the system with those compounds (Critchley 1226). The problem with this is that all cells in the body would have to be exposed to potential toxins 6-MPDR or 5-FU. In addition to the search and destroy mechanism, scientists hope to increase selectivity by combining the hypoxia-sensing and density-sensing units so that ''E. coli'' will only enter eukaryotic cells that exhibit both characteristics. This would make it more likely that invasin-enhanced ''E. coli'' would only invade cancer cells rather than muscle tissue that had low oxygen due to exercise (Anderson 624). To create a fully functional, synthetic biology-based approach to cancer therapy, scientists need to develop bacteria that discriminately invades cancer cells and destroys them. Most of the pieces are there – scientists have engineered ''E. coli'' that selectively invades hypoxic cells or dense cells and ''Salmonella'' that can destroy tumors in vivo – they just need to be put together effectively.

<center>

{|
|-
|[[Image:InvasinDensity.JPG]]
|[[Image:InvasinAnaerobic2.JPG]]
|-
|'''Fig. 1.''' The red line represents when the invasin output module was paired with the inducible promoter ''lux'' which senses high cell density, the black line is constitutively on invasin.
|'''Fig. 2.''' The invasin output module was paired with the inducible promoter ''fdhF'' which senses anaerobic conditions.
|}
</center>

Using bacteria as a vaccine vector has similar issues of specificity and implementation in a human system. Bacteria was first thought to be a good vector because they would not degrade at mucosal surfaces, they would absorb better and survive the low pH in the gastrointestinal tract (Garmory 471). A particular strain of ''Salmonella'' with one or more deletions in the shikimate pathway, which creates a precursor for phenylalanine and tyrosine, is a promising vector because it gets into the body easily but does not harmfully effect the tissue and is gone within a week or two of introduction. Scientists engineered this strain to carry the ''Yersinia pestis'' V antigen, and studied its ability to protect mice from ''Y. pestis''. Unfortunately, the inoculation was not extremely successful and only 6 of 20 mice showed a strong response (Garmory 473). Garmory and colleagues cited several reasons for this: the copy number of the plasmid containing the V antigen might not have been high enough; the attenuated ''Salmonella'' may not have been able to reach cells that would be targeted by ''Y. pestis''; and the inability of the cell to secrete the antigen. Many changes need to be made to live vector vaccine design before it is an effective method of treating or preventing disease.

Another method of disease prevention with a synthetic biology approach is related to skin cancer. ''E. coli'' engineered to sense single-stranded DNA after DNA damage was coupled with a mechanism to produce a biofilm around the cell. The biofilm output module was tested with DNA damage done by mitomycin C, a DNA crosslinking agent, or UV irradiation, a known cause of skin cancer. The biofilm protection, when part of a toggle switch mechanism, lasted indefinitely when pulsed with UV light at 8 J/m2 for only 2 seconds (Kobayashi 8417) (Fig.3). The biofilm-production mechanism has not been engineered in eukaryotic cells, but a prokaryote-based system that could aid with human skin disease is imaginable. For example, a sunscreen that changes colors when the DNA damage may be too intense for a sunbather as a warning, or a sunscreen that becomes more protective as the possibility of genetic damage increased. Further research should focus on how to best implement the biofilm producing output module to protect human DNA.
<center>
[[Image:UVinducedbiofilm.JPG]]

'''Fig. 3.''' Crystal violet absorbance measures biofilm formation.

</center>
Finally, synthetic biology has made its mark on the field of medicine by creating drugs that are less expensive to produce than those made by pharmaceutical companies. Jay Keasling’s project involving the antimalarial drug artemisinin is the best example. Yeast was engineered to increase normal farnesyl pyrophosphate (FPP) production, convert FPP to amorphadiene, and oxidizing amorphadiene to artemisinic acid, which can then easily be separated from the yeast cells and converted to artemisinin in the laboratory (Fig. 4). This innovation allows the production of artemisinin at lower prices than those currently on the market, and without regard to environmental constraints (Ro 942). On a completely different tangent, Collins’s lab engineered T7 bacteriophages to deliver biofilm-degrading enzymes such as dispersin B after infecting and replicating in an ''E. coli'' biofilm. Dispersin B works to degrade an adhesin that is critical for biofilm formation. Phages that released dispersin B were found to be 99.997% effective at removing biofilm, which is 4.5 orders of magnitude higher than non-enzymatic phages (Lu 11197) (Fig. 5). The specificity of phages for a certain type of bacteria make them a viable vector to be used in human treatments, which may range from removing dental plaques to cleaning inserted medical devices. Using synthetic biology to engineer an antimalarial drug in yeast or a biofilm-destroying enzyme in bacteriophages greatly increases the possible contributions to the field of medicine.

<center>

{|
|-
|[[Image:Artemesinin.JPG]]
|[[Image:BiofilmDegradation.JPG]]
|-
|'''Fig. 4.''' All the steps in the box indicate steps that were optimized in yeast for a maximum artemisinic acid output.
|'''Fig. 5.''' The amount of biofilm was decreased when the dispersin B enzymes were incorporated into T7 bacteriophages.
|}
</center>

Medical applications of synthetic biology are wide-ranging and eminently applicable to daily life. Synthetic biology based cancer treatments that utilize E.coli engineered to sense and attack tissues with high cell density and low oxygen availability are promising and need to be tested in vivo. Designing vectors out of attenuated bacteria may be a safe, effective method for disease treatment. Using biofilms to sense sun damage potential may decrease the likelihood of skin cancer. Finally, using engineered cells to produce medicines in a cost-effective, environmentally-friendly way may revolutionize the pharmaceutical industry. Investing more research efforts in the field of synthetic biology may turn out to be an investment in medical technology.

'''Works Cited'''

Anderson JC, Clarke EJ, Arkin, AP, and Voigt CA (2006). Environmentally controlled invasion of cancer cells by engineered bacteria. Journal of Molecular Biology 355:619-27.

Critchley RJ, Jezzard S, Radford KJ, Goussard S, Lemoine NR, et al. (2004). Potential therapeutic applications of recombinant, invasive E. coli. Gene Therapy 11:1224-33.

Garmory HS, Leary SEC, Griffin, KF, Williamson D, Brown, KA, and Titball RW (2003). The use of live attenuated bacteria as a delivery system for heterologous antigens. Journal of Drug Targeting 11:471-79.

Kobayashi H, et al. (2004). Programmable cells: Interfacing natural and engineered gene networks. PNAS 101: 8414-19.

Lu, TK, and Collins JJ (2007). Dispersing biofilms with engineered enzymatic bacteriophage. PNAS 104: 11197-11202.

Nemunaitis J, Cunningham C, Senzer N, Kuhn J, Cramm J, Litz C, et al. (2003). Pilot trial of genetically modified, attenuated Salmonella expressing cytosine deaminase gene in refractory cancer patients. Cancer Gene Therapy 10:737-744.

Pawelek JM, Low KB, and Bermudes D (2003). Bacteria as tumour-targeting vectors. Lancet Oncology 4:548-56.

Ro D, et al. (2006). Production of the antimalarial drug precursor artemisinic acid in engineered yeast. Nature 440:940-43.

Medical Applications of Synthetic Biology - Samantha Simpson

2007-11-13T02:07:42Z

Sasimpson:

'''Project Proposal'''

My project will be on medical applications of synthetic biology. I will reference Anderson's paper on utilizing quorum-sensing and hypoxia-responsive genes coupled with invasin from ''Yersinia tuberculosis'' to invade cancer-causing cells. Coupled with Critchley's work, which describes a bacterial system that invades eukaryotic cells and delivers proteins coded for in the bacteria's genome, one could possibly create 'search and destroy' ''E.coli'' that can locate, invade, and kill tumor cells. Garmory's paper also highlights the possibility of using bacteria as a drug-delivering system, specifically vaccine vectors. Kobayashi describes cells that produce a protective biofilm layer after exposure to DNA-damaging agents. Ro engineers yeast to create an anti-malarial drug that can be created at a lower cost than the previous standard production process. These papers, and others that have a focus on direct medical applications such as novel cancer therapies, vaccination technology, biological protection, and the creation of new medicines, will be referenced to create a cohesive overview of applications of synthetic biology to the medical field.

'''Paper'''

Synthetic biology is a rapidly emerging field that strives to re-design existing biological systems and components or fabricate novel biological systems and components. The end result of experimentation in synthetic biology is to help scientists understand a naturally occurring genetic pathway more fully or to create a new, useful pathway. Recent research efforts have focused on engineering bacteria to create a new form of biofuel or to make promoters of varying efficiencies for more exact gene expression. Other research efforts focus on engineering yeast to make antimalarial drugs or engineering ''E. coli'' to recognize and invade tumor-like cells, both of which are medical applications of synthetic biology. Advancements such as novel cancer therapies, vaccination technology, biological protection, and the creation of new medicines highlight synthetic biology’s potential to create breakthroughs in medical science. This paper will examine various medical applications of synthetic biology and further development that needs to be done before current research can be used in hospitals and doctors’ offices.

Synthetic biologists consider finding a cure for cancer a reachable goal. Previous to the development of synthetic biology as a field, it was known that three types of bacteria, ''Bifodobacterium'', ''Clostridium'', and ''Salmonella'' all preferentially infect the dense cells of tumors (Pawelek 548). All three types of bacteria are associated with reducing tumor size in patients who are infected with them, and it was noted that all three bacteria act anaerobically. Capitalizing on the idea that tumors create an unusually dense, anaerobic mass of cells, Christopher Voigt’s lab engineered bacteria to identify and invade cancer cells. They utilized the ''lux'' quorum sensing from ''Vibrio fischeri'' to identify cells growing at high densities (Fig. 1) and the ''fdhF'' promoter to identify cells growing in anerobic conditions (Fig. 2) (Anderson 619). They coupled this identification mechanism with the invasin output module from ''Yersinia pseudotuberculosis'' so the ''E. coli'' could invade specific cells. Next, scientists hope to insert a gene that would destroy the invaded cells, making the complete cycle a search, invade, and destroy loop that would only be sensitive to cancer cells. One promising destroy mechanism has been implemented in humans via intratumoral injection, based on ''Salmonella'' that contains the ''E. coli'' cytosine deaminase gene that converts 5-fluorocytosine to 5-fluorouracil, a chemotherapy drug (Nemunaitis 737). Unfortunately, while the tumors did produce 5-fluorouracil (5-FU), they did not produce it in high enough yields to cause regression of the cancer. Another experiment used invasin-enhanced ''E. coli'' to enter cells in vitro and sensitize those cells to 6-methylpurine-2’deoxyribose (6-MPDR) or 5-FU by depositing proteins that converted those compounds to toxins, and then flood the system with those compounds (Critchley 1226). The problem with this is that all cells in the body would have to be exposed to potential toxins 6-MPDR or 5-FU. In addition to the search and destroy mechanism, scientists hope to increase selectivity by combining the hypoxia-sensing and density-sensing units so that ''E. coli'' will only enter eukaryotic cells that exhibit both characteristics. This would make it more likely that invasin-enhanced ''E. coli'' would only invade cancer cells rather than muscle tissue that had low oxygen due to exercise (Anderson 624). To create a fully functional, synthetic biology-based approach to cancer therapy, scientists need to develop bacteria that discriminately invades cancer cells and destroys them. Most of the pieces are there – scientists have engineered ''E. coli'' that selectively invades hypoxic cells or dense cells and ''Salmonella'' that can destroy tumors in vivo – they just need to be put together effectively.

<center>

{|
|-
|[[Image:InvasinDensity.JPG]]
|[[Image:InvasinAnaerobic2.JPG]]
|-
|'''Fig. 1.''' The red line represents when the invasin output module was paired with the inducible promoter ''lux'' which senses high cell density, the black line is constitutively on invasin.
|'''Fig. 2.''' The invasin output module was paired with the inducible promoter ''fdhF'' which senses anaerobic conditions.
|}
</center>

Using bacteria as a vaccine vector has similar issues of specificity and implementation in a human system. Bacteria was first thought to be a good vector because they would not degrade at mucosal surfaces, they would absorb better and survive the low pH in the gastrointestinal tract (Garmory 471). A particular strain of ''Salmonella'' with one or more deletions in the shikimate pathway, which creates a precursor for phenylalanine and tyrosine, is a promising vector because it gets into the body easily but does not harmfully effect the tissue and is gone within a week or two of introduction. Scientists engineered this strain to carry the ''Yersinia pestis'' V antigen, and studied its ability to protect mice from ''Y. pestis''. Unfortunately, the inoculation was not extremely successful and only 6 of 20 mice showed a strong response (Garmory 473). Garmory and colleagues cited several reasons for this: the copy number of the plasmid containing the V antigen might not have been high enough; the attenuated ''Salmonella'' may not have been able to reach cells that would be targeted by ''Y. pestis''; and the inability of the cell to secrete the antigen. Many changes need to be made to live vector vaccine design before it is an effective method of treating or preventing disease.

Another method of disease prevention with a synthetic biology approach is related to skin cancer. ''E. coli'' engineered to sense single-stranded DNA after DNA damage was coupled with a mechanism to produce a biofilm around the cell. The biofilm output module was tested with DNA damage done by mitomycin C, a DNA crosslinking agent, or UV irradiation, a known cause of skin cancer. The biofilm protection, when part of a toggle switch mechanism, lasted indefinitely when pulsed with UV light at 8 J/m2 for only 2 seconds (Kobayashi 8417) (Fig.1). The biofilm-production mechanism has not been engineered in eukaryotic cells, but a prokaryote-based system that could aid with human skin disease is imaginable. For example, a sunscreen that changes colors when the DNA damage may be too intense for a sunbather as a warning, or a sunscreen that becomes more protective as the possibility of genetic damage increased. Further research should focus on how to best implement the biofilm producing output module to protect human DNA.
<center>
[[Image:UVinducedbiofilm.JPG]]

'''Fig. 1.''' Crystal violet absorbance measures biofilm formation.

</center>
Finally, synthetic biology has made its mark on the field of medicine by creating drugs that are less expensive to produce than those made by pharmaceutical companies. Jay Keasling’s project involving the antimalarial drug artemisinin is the best example. Yeast was engineered to increase normal farnesyl pyrophosphate (FPP) production, convert FPP to amorphadiene, and oxidizing amorphadiene to artemisinic acid, which can then easily be separated from the yeast cells and converted to artemisinin in the laboratory (Fig. 2). This innovation allows the production of artemisinin at lower prices than those currently on the market, and without regard to environmental constraints (Ro 942). On a completely different tangent, Collins’s lab engineered T7 bacteriophages to deliver biofilm-degrading enzymes such as dispersin B after infecting and replicating in an ''E. coli'' biofilm. Dispersin B works to degrade an adhesin that is critical for biofilm formation. Phages that released dispersin B were found to be 99.997% effective at removing biofilm, which is 4.5 orders of magnitude higher than non-enzymatic phages (Lu 11197) (Fig. 3). The specificity of phages for a certain type of bacteria make them a viable vector to be used in human treatments, which may range from removing dental plaques to cleaning inserted medical devices. Using synthetic biology to engineer an antimalarial drug in yeast or a biofilm-destroying enzyme in bacteriophages greatly increases the possible contributions to the field of medicine.

<center>

{|
|-
|[[Image:Artemesinin.JPG]]
|[[Image:BiofilmDegradation.JPG]]
|-
|'''Fig. 2.''' All the steps in the box indicate steps that were optimized in yeast for a maximum artemisinic acid output.
|'''Fig. 3.''' The amount of biofilm was decreased when the dispersin B enzymes were incorporated into T7 bacteriophages.
|}
</center>

Medical applications of synthetic biology are wide-ranging and eminently applicable to daily life. Synthetic biology based cancer treatments that utilize E.coli engineered to sense and attack tissues with high cell density and low oxygen availability are promising and need to be tested in vivo. Designing vectors out of attenuated bacteria may be a safe, effective method for disease treatment. Using biofilms to sense sun damage potential may decrease the likelihood of skin cancer. Finally, using engineered cells to produce medicines in a cost-effective, environmentally-friendly way may revolutionize the pharmaceutical industry. Investing more research efforts in the field of synthetic biology may turn out to be an investment in medical technology.

'''Works Cited'''

Anderson JC, Clarke EJ, Arkin, AP, and Voigt CA (2006). Environmentally controlled invasion of cancer cells by engineered bacteria. Journal of Molecular Biology 355:619-27.

Critchley RJ, Jezzard S, Radford KJ, Goussard S, Lemoine NR, et al. (2004). Potential therapeutic applications of recombinant, invasive E. coli. Gene Therapy 11:1224-33.

Garmory HS, Leary SEC, Griffin, KF, Williamson D, Brown, KA, and Titball RW (2003). The use of live attenuated bacteria as a delivery system for heterologous antigens. Journal of Drug Targeting 11:471-79.

Kobayashi H, et al. (2004). Programmable cells: Interfacing natural and engineered gene networks. PNAS 101: 8414-19.

Lu, TK, and Collins JJ (2007). Dispersing biofilms with engineered enzymatic bacteriophage. PNAS 104: 11197-11202.

Nemunaitis J, Cunningham C, Senzer N, Kuhn J, Cramm J, Litz C, et al. (2003). Pilot trial of genetically modified, attenuated Salmonella expressing cytosine deaminase gene in refractory cancer patients. Cancer Gene Therapy 10:737-744.

Pawelek JM, Low KB, and Bermudes D (2003). Bacteria as tumour-targeting vectors. Lancet Oncology 4:548-56.

Ro D, et al. (2006). Production of the antimalarial drug precursor artemisinic acid in engineered yeast. Nature 440:940-43.

Medical Applications of Synthetic Biology - Samantha Simpson

2007-11-13T02:06:22Z

Sasimpson:

'''Project Proposal'''

My project will be on medical applications of synthetic biology. I will reference Anderson's paper on utilizing quorum-sensing and hypoxia-responsive genes coupled with invasin from ''Yersinia tuberculosis'' to invade cancer-causing cells. Coupled with Critchley's work, which describes a bacterial system that invades eukaryotic cells and delivers proteins coded for in the bacteria's genome, one could possibly create 'search and destroy' ''E.coli'' that can locate, invade, and kill tumor cells. Garmory's paper also highlights the possibility of using bacteria as a drug-delivering system, specifically vaccine vectors. Kobayashi describes cells that produce a protective biofilm layer after exposure to DNA-damaging agents. Ro engineers yeast to create an anti-malarial drug that can be created at a lower cost than the previous standard production process. These papers, and others that have a focus on direct medical applications such as novel cancer therapies, vaccination technology, biological protection, and the creation of new medicines, will be referenced to create a cohesive overview of applications of synthetic biology to the medical field.

'''Paper'''

Synthetic biology is a rapidly emerging field that strives to re-design existing biological systems and components or fabricate novel biological systems and components. The end result of experimentation in synthetic biology is to help scientists understand a naturally occurring genetic pathway more fully or to create a new, useful pathway. Recent research efforts have focused on engineering bacteria to create a new form of biofuel or to make promoters of varying efficiencies for more exact gene expression. Other research efforts focus on engineering yeast to make antimalarial drugs or engineering ''E. coli'' to recognize and invade tumor-like cells, both of which are medical applications of synthetic biology. Advancements such as novel cancer therapies, vaccination technology, biological protection, and the creation of new medicines highlight synthetic biology’s potential to create breakthroughs in medical science. This paper will examine various medical applications of synthetic biology and further development that needs to be done before current research can be used in hospitals and doctors’ offices.

Synthetic biologists consider finding a cure for cancer a reachable goal. Previous to the development of synthetic biology as a field, it was known that three types of bacteria, ''Bifodobacterium'', ''Clostridium'', and ''Salmonella'' all preferentially infect the dense cells of tumors (Pawelek 548). All three types of bacteria are associated with reducing tumor size in patients who are infected with them, and it was noted that all three bacteria act anaerobically. Capitalizing on the idea that tumors create an unusually dense, anaerobic mass of cells, Christopher Voigt’s lab engineered bacteria to identify and invade cancer cells. They utilized the ''lux'' quorum sensing from ''Vibrio fischeri'' to identify cells growing at high densities (Fig. 1) and the ''fdhF'' promoter to identify cells growing in anerobic conditions (Fig. 2)(Anderson 619). They coupled this identification mechanism with the invasin output module from ''Yersinia pseudotuberculosis'' so the ''E. coli'' could invade specific cells. Next, scientists hope to insert a gene that would destroy the invaded cells, making the complete cycle a search, invade, and destroy loop that would only be sensitive to cancer cells. One promising destroy mechanism has been implemented in humans via intratumoral injection, based on ''Salmonella'' that contains the ''E. coli'' cytosine deaminase gene that converts 5-fluorocytosine to 5-fluorouracil, a chemotherapy drug (Nemunaitis 737). Unfortunately, while the tumors did produce 5-fluorouracil (5-FU), they did not produce it in high enough yields to cause regression of the cancer. Another experiment used invasin-enhanced ''E. coli'' to enter cells in vitro and sensitize those cells to 6-methylpurine-2’deoxyribose (6-MPDR) or 5-FU by depositing proteins that converted those compounds to toxins, and then flood the system with those compounds (Critchley 1226). The problem with this is that all cells in the body would have to be exposed to potential toxins 6-MPDR or 5-FU. In addition to the search and destroy mechanism, scientists hope to increase selectivity by combining the hypoxia-sensing and density-sensing units so that ''E. coli'' will only enter eukaryotic cells that exhibit both characteristics. This would make it more likely that invasin-enhanced ''E. coli'' would only invade cancer cells rather than muscle tissue that had low oxygen due to exercise (Anderson 624). To create a fully functional, synthetic biology-based approach to cancer therapy, scientists need to develop bacteria that discriminately invades cancer cells and destroys them. Most of the pieces are there – scientists have engineered ''E. coli'' that selectively invades hypoxic cells or dense cells and ''Salmonella'' that can destroy tumors in vivo – they just need to be put together effectively.

<center>

{|
|-
|[[Image:InvasinDensity.JPG]]
|[[Image:InvasinAnaerobic2.JPG]]
|-
|'''Fig. 1.''' The invasin output module was paired with the inducible promoter ''lux'' which senses high cell density.
|'''Fig. 2.''' The invasin output module was paired with the inducible promoter ''fdhF'' which senses anaerobic conditions.
|}
</center>

Using bacteria as a vaccine vector has similar issues of specificity and implementation in a human system. Bacteria was first thought to be a good vector because they would not degrade at mucosal surfaces, they would absorb better and survive the low pH in the gastrointestinal tract (Garmory 471). A particular strain of ''Salmonella'' with one or more deletions in the shikimate pathway, which creates a precursor for phenylalanine and tyrosine, is a promising vector because it gets into the body easily but does not harmfully effect the tissue and is gone within a week or two of introduction. Scientists engineered this strain to carry the ''Yersinia pestis'' V antigen, and studied its ability to protect mice from ''Y. pestis''. Unfortunately, the inoculation was not extremely successful and only 6 of 20 mice showed a strong response (Garmory 473). Garmory and colleagues cited several reasons for this: the copy number of the plasmid containing the V antigen might not have been high enough; the attenuated ''Salmonella'' may not have been able to reach cells that would be targeted by ''Y. pestis''; and the inability of the cell to secrete the antigen. Many changes need to be made to live vector vaccine design before it is an effective method of treating or preventing disease.

Another method of disease prevention with a synthetic biology approach is related to skin cancer. ''E. coli'' engineered to sense single-stranded DNA after DNA damage was coupled with a mechanism to produce a biofilm around the cell. The biofilm output module was tested with DNA damage done by mitomycin C, a DNA crosslinking agent, or UV irradiation, a known cause of skin cancer. The biofilm protection, when part of a toggle switch mechanism, lasted indefinitely when pulsed with UV light at 8 J/m2 for only 2 seconds (Kobayashi 8417) (Fig.1). The biofilm-production mechanism has not been engineered in eukaryotic cells, but a prokaryote-based system that could aid with human skin disease is imaginable. For example, a sunscreen that changes colors when the DNA damage may be too intense for a sunbather as a warning, or a sunscreen that becomes more protective as the possibility of genetic damage increased. Further research should focus on how to best implement the biofilm producing output module to protect human DNA.
<center>
[[Image:UVinducedbiofilm.JPG]]

'''Fig. 1.''' Crystal violet absorbance measures biofilm formation.

</center>
Finally, synthetic biology has made its mark on the field of medicine by creating drugs that are less expensive to produce than those made by pharmaceutical companies. Jay Keasling’s project involving the antimalarial drug artemisinin is the best example. Yeast was engineered to increase normal farnesyl pyrophosphate (FPP) production, convert FPP to amorphadiene, and oxidizing amorphadiene to artemisinic acid, which can then easily be separated from the yeast cells and converted to artemisinin in the laboratory (Fig. 2). This innovation allows the production of artemisinin at lower prices than those currently on the market, and without regard to environmental constraints (Ro 942). On a completely different tangent, Collins’s lab engineered T7 bacteriophages to deliver biofilm-degrading enzymes such as dispersin B after infecting and replicating in an ''E. coli'' biofilm. Dispersin B works to degrade an adhesin that is critical for biofilm formation. Phages that released dispersin B were found to be 99.997% effective at removing biofilm, which is 4.5 orders of magnitude higher than non-enzymatic phages (Lu 11197) (Fig. 3). The specificity of phages for a certain type of bacteria make them a viable vector to be used in human treatments, which may range from removing dental plaques to cleaning inserted medical devices. Using synthetic biology to engineer an antimalarial drug in yeast or a biofilm-destroying enzyme in bacteriophages greatly increases the possible contributions to the field of medicine.

<center>

{|
|-
|[[Image:Artemesinin.JPG]]
|[[Image:BiofilmDegradation.JPG]]
|-
|'''Fig. 2.''' All the steps in the box indicate steps that were optimized in yeast for a maximum artemisinic acid output.
|'''Fig. 3.''' The amount of biofilm was decreased when the dispersin B enzymes were incorporated into T7 bacteriophages.
|}
</center>

Medical applications of synthetic biology are wide-ranging and eminently applicable to daily life. Synthetic biology based cancer treatments that utilize E.coli engineered to sense and attack tissues with high cell density and low oxygen availability are promising and need to be tested in vivo. Designing vectors out of attenuated bacteria may be a safe, effective method for disease treatment. Using biofilms to sense sun damage potential may decrease the likelihood of skin cancer. Finally, using engineered cells to produce medicines in a cost-effective, environmentally-friendly way may revolutionize the pharmaceutical industry. Investing more research efforts in the field of synthetic biology may turn out to be an investment in medical technology.

'''Works Cited'''

Anderson JC, Clarke EJ, Arkin, AP, and Voigt CA (2006). Environmentally controlled invasion of cancer cells by engineered bacteria. Journal of Molecular Biology 355:619-27.

Critchley RJ, Jezzard S, Radford KJ, Goussard S, Lemoine NR, et al. (2004). Potential therapeutic applications of recombinant, invasive E. coli. Gene Therapy 11:1224-33.

Garmory HS, Leary SEC, Griffin, KF, Williamson D, Brown, KA, and Titball RW (2003). The use of live attenuated bacteria as a delivery system for heterologous antigens. Journal of Drug Targeting 11:471-79.

Kobayashi H, et al. (2004). Programmable cells: Interfacing natural and engineered gene networks. PNAS 101: 8414-19.

Lu, TK, and Collins JJ (2007). Dispersing biofilms with engineered enzymatic bacteriophage. PNAS 104: 11197-11202.

Nemunaitis J, Cunningham C, Senzer N, Kuhn J, Cramm J, Litz C, et al. (2003). Pilot trial of genetically modified, attenuated Salmonella expressing cytosine deaminase gene in refractory cancer patients. Cancer Gene Therapy 10:737-744.

Pawelek JM, Low KB, and Bermudes D (2003). Bacteria as tumour-targeting vectors. Lancet Oncology 4:548-56.

Ro D, et al. (2006). Production of the antimalarial drug precursor artemisinic acid in engineered yeast. Nature 440:940-43.

File:InvasinAnaerobic2.JPG

2007-11-13T02:04:29Z

Sasimpson:

Medical Applications of Synthetic Biology - Samantha Simpson

2007-11-13T02:03:35Z

Sasimpson:

'''Project Proposal'''

My project will be on medical applications of synthetic biology. I will reference Anderson's paper on utilizing quorum-sensing and hypoxia-responsive genes coupled with invasin from ''Yersinia tuberculosis'' to invade cancer-causing cells. Coupled with Critchley's work, which describes a bacterial system that invades eukaryotic cells and delivers proteins coded for in the bacteria's genome, one could possibly create 'search and destroy' ''E.coli'' that can locate, invade, and kill tumor cells. Garmory's paper also highlights the possibility of using bacteria as a drug-delivering system, specifically vaccine vectors. Kobayashi describes cells that produce a protective biofilm layer after exposure to DNA-damaging agents. Ro engineers yeast to create an anti-malarial drug that can be created at a lower cost than the previous standard production process. These papers, and others that have a focus on direct medical applications such as novel cancer therapies, vaccination technology, biological protection, and the creation of new medicines, will be referenced to create a cohesive overview of applications of synthetic biology to the medical field.

'''Paper'''

Synthetic biology is a rapidly emerging field that strives to re-design existing biological systems and components or fabricate novel biological systems and components. The end result of experimentation in synthetic biology is to help scientists understand a naturally occurring genetic pathway more fully or to create a new, useful pathway. Recent research efforts have focused on engineering bacteria to create a new form of biofuel or to make promoters of varying efficiencies for more exact gene expression. Other research efforts focus on engineering yeast to make antimalarial drugs or engineering ''E. coli'' to recognize and invade tumor-like cells, both of which are medical applications of synthetic biology. Advancements such as novel cancer therapies, vaccination technology, biological protection, and the creation of new medicines highlight synthetic biology’s potential to create breakthroughs in medical science. This paper will examine various medical applications of synthetic biology and further development that needs to be done before current research can be used in hospitals and doctors’ offices.

Synthetic biologists consider finding a cure for cancer a reachable goal. Previous to the development of synthetic biology as a field, it was known that three types of bacteria, ''Bifodobacterium'', ''Clostridium'', and ''Salmonella'' all preferentially infect the dense cells of tumors (Pawelek 548). All three types of bacteria are associated with reducing tumor size in patients who are infected with them, and it was noted that all three bacteria act anaerobically. Capitalizing on the idea that tumors create an unusually dense, anaerobic mass of cells, Christopher Voigt’s lab engineered bacteria to identify and invade cancer cells. They utilized the ''lux'' quorum sensing from ''Vibrio fischeri'' to identify cells growing at high densities (Fig. 1) and the ''fdhF'' promoter to identify cells growing in anerobic conditions (Fig. 2)(Anderson 619). They coupled this identification mechanism with the invasin output module from ''Yersinia pseudotuberculosis'' so the ''E. coli'' could invade specific cells. Next, scientists hope to insert a gene that would destroy the invaded cells, making the complete cycle a search, invade, and destroy loop that would only be sensitive to cancer cells. One promising destroy mechanism has been implemented in humans via intratumoral injection, based on ''Salmonella'' that contains the ''E. coli'' cytosine deaminase gene that converts 5-fluorocytosine to 5-fluorouracil, a chemotherapy drug (Nemunaitis 737). Unfortunately, while the tumors did produce 5-fluorouracil (5-FU), they did not produce it in high enough yields to cause regression of the cancer. Another experiment used invasin-enhanced ''E. coli'' to enter cells in vitro and sensitize those cells to 6-methylpurine-2’deoxyribose (6-MPDR) or 5-FU by depositing proteins that converted those compounds to toxins, and then flood the system with those compounds (Critchley 1226). The problem with this is that all cells in the body would have to be exposed to potential toxins 6-MPDR or 5-FU. In addition to the search and destroy mechanism, scientists hope to increase selectivity by combining the hypoxia-sensing and density-sensing units so that ''E. coli'' will only enter eukaryotic cells that exhibit both characteristics. This would make it more likely that invasin-enhanced ''E. coli'' would only invade cancer cells rather than muscle tissue that had low oxygen due to exercise (Anderson 624). To create a fully functional, synthetic biology-based approach to cancer therapy, scientists need to develop bacteria that discriminately invades cancer cells and destroys them. Most of the pieces are there – scientists have engineered ''E. coli'' that selectively invades hypoxic cells or dense cells and ''Salmonella'' that can destroy tumors in vivo – they just need to be put together effectively.

<center>

{|
|-
|[[Image:InvasinDensity.JPG]]
|[[Image:InvasinAnaerobic.JPG]]
|-
|'''Fig. 1.''' All the steps in the box indicate steps that were optimized in yeast for a maximum artemisinic acid output.
|'''Fig. 2.''' The amount of biofilm was decreased when the dispersin B enzymes were incorporated into T7 bacteriophages.
|}
</center>

Using bacteria as a vaccine vector has similar issues of specificity and implementation in a human system. Bacteria was first thought to be a good vector because they would not degrade at mucosal surfaces, they would absorb better and survive the low pH in the gastrointestinal tract (Garmory 471). A particular strain of ''Salmonella'' with one or more deletions in the shikimate pathway, which creates a precursor for phenylalanine and tyrosine, is a promising vector because it gets into the body easily but does not harmfully effect the tissue and is gone within a week or two of introduction. Scientists engineered this strain to carry the ''Yersinia pestis'' V antigen, and studied its ability to protect mice from ''Y. pestis''. Unfortunately, the inoculation was not extremely successful and only 6 of 20 mice showed a strong response (Garmory 473). Garmory and colleagues cited several reasons for this: the copy number of the plasmid containing the V antigen might not have been high enough; the attenuated ''Salmonella'' may not have been able to reach cells that would be targeted by ''Y. pestis''; and the inability of the cell to secrete the antigen. Many changes need to be made to live vector vaccine design before it is an effective method of treating or preventing disease.

Another method of disease prevention with a synthetic biology approach is related to skin cancer. ''E. coli'' engineered to sense single-stranded DNA after DNA damage was coupled with a mechanism to produce a biofilm around the cell. The biofilm output module was tested with DNA damage done by mitomycin C, a DNA crosslinking agent, or UV irradiation, a known cause of skin cancer. The biofilm protection, when part of a toggle switch mechanism, lasted indefinitely when pulsed with UV light at 8 J/m2 for only 2 seconds (Kobayashi 8417) (Fig.1). The biofilm-production mechanism has not been engineered in eukaryotic cells, but a prokaryote-based system that could aid with human skin disease is imaginable. For example, a sunscreen that changes colors when the DNA damage may be too intense for a sunbather as a warning, or a sunscreen that becomes more protective as the possibility of genetic damage increased. Further research should focus on how to best implement the biofilm producing output module to protect human DNA.
<center>
[[Image:UVinducedbiofilm.JPG]]

'''Fig. 1.''' Crystal violet absorbance measures biofilm formation.

</center>
Finally, synthetic biology has made its mark on the field of medicine by creating drugs that are less expensive to produce than those made by pharmaceutical companies. Jay Keasling’s project involving the antimalarial drug artemisinin is the best example. Yeast was engineered to increase normal farnesyl pyrophosphate (FPP) production, convert FPP to amorphadiene, and oxidizing amorphadiene to artemisinic acid, which can then easily be separated from the yeast cells and converted to artemisinin in the laboratory (Fig. 2). This innovation allows the production of artemisinin at lower prices than those currently on the market, and without regard to environmental constraints (Ro 942). On a completely different tangent, Collins’s lab engineered T7 bacteriophages to deliver biofilm-degrading enzymes such as dispersin B after infecting and replicating in an ''E. coli'' biofilm. Dispersin B works to degrade an adhesin that is critical for biofilm formation. Phages that released dispersin B were found to be 99.997% effective at removing biofilm, which is 4.5 orders of magnitude higher than non-enzymatic phages (Lu 11197) (Fig. 3). The specificity of phages for a certain type of bacteria make them a viable vector to be used in human treatments, which may range from removing dental plaques to cleaning inserted medical devices. Using synthetic biology to engineer an antimalarial drug in yeast or a biofilm-destroying enzyme in bacteriophages greatly increases the possible contributions to the field of medicine.

<center>

{|
|-
|[[Image:Artemesinin.JPG]]
|[[Image:BiofilmDegradation.JPG]]
|-
|'''Fig. 2.''' All the steps in the box indicate steps that were optimized in yeast for a maximum artemisinic acid output.
|'''Fig. 3.''' The amount of biofilm was decreased when the dispersin B enzymes were incorporated into T7 bacteriophages.
|}
</center>

Medical applications of synthetic biology are wide-ranging and eminently applicable to daily life. Synthetic biology based cancer treatments that utilize E.coli engineered to sense and attack tissues with high cell density and low oxygen availability are promising and need to be tested in vivo. Designing vectors out of attenuated bacteria may be a safe, effective method for disease treatment. Using biofilms to sense sun damage potential may decrease the likelihood of skin cancer. Finally, using engineered cells to produce medicines in a cost-effective, environmentally-friendly way may revolutionize the pharmaceutical industry. Investing more research efforts in the field of synthetic biology may turn out to be an investment in medical technology.

'''Works Cited'''

Anderson JC, Clarke EJ, Arkin, AP, and Voigt CA (2006). Environmentally controlled invasion of cancer cells by engineered bacteria. Journal of Molecular Biology 355:619-27.

Critchley RJ, Jezzard S, Radford KJ, Goussard S, Lemoine NR, et al. (2004). Potential therapeutic applications of recombinant, invasive E. coli. Gene Therapy 11:1224-33.

Garmory HS, Leary SEC, Griffin, KF, Williamson D, Brown, KA, and Titball RW (2003). The use of live attenuated bacteria as a delivery system for heterologous antigens. Journal of Drug Targeting 11:471-79.

Kobayashi H, et al. (2004). Programmable cells: Interfacing natural and engineered gene networks. PNAS 101: 8414-19.

Lu, TK, and Collins JJ (2007). Dispersing biofilms with engineered enzymatic bacteriophage. PNAS 104: 11197-11202.

Nemunaitis J, Cunningham C, Senzer N, Kuhn J, Cramm J, Litz C, et al. (2003). Pilot trial of genetically modified, attenuated Salmonella expressing cytosine deaminase gene in refractory cancer patients. Cancer Gene Therapy 10:737-744.

Pawelek JM, Low KB, and Bermudes D (2003). Bacteria as tumour-targeting vectors. Lancet Oncology 4:548-56.

Ro D, et al. (2006). Production of the antimalarial drug precursor artemisinic acid in engineered yeast. Nature 440:940-43.

File:InvasinAnaerobic.JPG

2007-11-13T02:00:49Z

Sasimpson:

File:InvasinDensity.JPG

2007-11-13T02:00:40Z

Sasimpson:

Medical Applications of Synthetic Biology - Samantha Simpson

2007-11-13T01:52:32Z

Sasimpson:

'''Project Proposal'''

My project will be on medical applications of synthetic biology. I will reference Anderson's paper on utilizing quorum-sensing and hypoxia-responsive genes coupled with invasin from ''Yersinia tuberculosis'' to invade cancer-causing cells. Coupled with Critchley's work, which describes a bacterial system that invades eukaryotic cells and delivers proteins coded for in the bacteria's genome, one could possibly create 'search and destroy' ''E.coli'' that can locate, invade, and kill tumor cells. Garmory's paper also highlights the possibility of using bacteria as a drug-delivering system, specifically vaccine vectors. Kobayashi describes cells that produce a protective biofilm layer after exposure to DNA-damaging agents. Ro engineers yeast to create an anti-malarial drug that can be created at a lower cost than the previous standard production process. These papers, and others that have a focus on direct medical applications such as novel cancer therapies, vaccination technology, biological protection, and the creation of new medicines, will be referenced to create a cohesive overview of applications of synthetic biology to the medical field.

'''Paper'''

Synthetic biology is a rapidly emerging field that strives to re-design existing biological systems and components or fabricate novel biological systems and components. The end result of experimentation in synthetic biology is to help scientists understand a naturally occurring genetic pathway more fully or to create a new, useful pathway. Recent research efforts have focused on engineering bacteria to create a new form of biofuel or to make promoters of varying efficiencies for more exact gene expression. Other research efforts focus on engineering yeast to make antimalarial drugs or engineering ''E. coli'' to recognize and invade tumor-like cells, both of which are medical applications of synthetic biology. Advancements such as novel cancer therapies, vaccination technology, biological protection, and the creation of new medicines highlight synthetic biology’s potential to create breakthroughs in medical science. This paper will examine various medical applications of synthetic biology and further development that needs to be done before current research can be used in hospitals and doctors’ offices.

Synthetic biologists consider finding a cure for cancer a reachable goal. Previous to the development of synthetic biology as a field, it was known that three types of bacteria, ''Bifodobacterium'', ''Clostridium'', and ''Salmonella'' all preferentially infect the dense cells of tumors (Pawelek 548). All three types of bacteria are associated with reducing tumor size in patients who are infected with them, and it was noted that all three bacteria act anaerobically. Capitalizing on the idea that tumors create an unusually dense, anaerobic mass of cells, Christopher Voigt’s lab engineered bacteria to identify and invade cancer cells. They utilized the ''lux'' quorum sensing from ''Vibrio fischeri'' to identify cells growing at high densities and the ''fdhF'' promoter to identify cells growing in anerobic conditions (Anderson 619). They coupled this identification mechanism with the invasin output module from ''Yersinia pseudotuberculosis'' so the ''E. coli'' could invade specific cells. Next, scientists hope to insert a gene that would destroy the invaded cells, making the complete cycle a search, invade, and destroy loop that would only be sensitive to cancer cells. One promising destroy mechanism has been implemented in humans via intratumoral injection, based on ''Salmonella'' that contains the ''E. coli'' cytosine deaminase gene that converts 5-fluorocytosine to 5-fluorouracil, a chemotherapy drug (Nemunaitis 737). Unfortunately, while the tumors did produce 5-fluorouracil (5-FU), they did not produce it in high enough yields to cause regression of the cancer. Another experiment used invasin-enhanced ''E. coli'' to enter cells in vitro and sensitize those cells to 6-methylpurine-2’deoxyribose (6-MPDR) or 5-FU by depositing proteins that converted those compounds to toxins, and then flood the system with those compounds (Critchley 1226). The problem with this is that all cells in the body would have to be exposed to potential toxins 6-MPDR or 5-FU. In addition to the search and destroy mechanism, scientists hope to increase selectivity by combining the hypoxia-sensing and density-sensing units so that ''E. coli'' will only enter eukaryotic cells that exhibit both characteristics. This would make it more likely that invasin-enhanced ''E. coli'' would only invade cancer cells rather than muscle tissue that had low oxygen due to exercise (Anderson 624). To create a fully functional, synthetic biology-based approach to cancer therapy, scientists need to develop bacteria that discriminately invades cancer cells and destroys them. Most of the pieces are there – scientists have engineered ''E. coli'' that selectively invades hypoxic cells or dense cells and ''Salmonella'' that can destroy tumors in vivo – they just need to be put together effectively.

Using bacteria as a vaccine vector has similar issues of specificity and implementation in a human system. Bacteria was first thought to be a good vector because they would not degrade at mucosal surfaces, they would absorb better and survive the low pH in the gastrointestinal tract (Garmory 471). A particular strain of ''Salmonella'' with one or more deletions in the shikimate pathway, which creates a precursor for phenylalanine and tyrosine, is a promising vector because it gets into the body easily but does not harmfully effect the tissue and is gone within a week or two of introduction. Scientists engineered this strain to carry the ''Yersinia pestis'' V antigen, and studied its ability to protect mice from ''Y. pestis''. Unfortunately, the inoculation was not extremely successful and only 6 of 20 mice showed a strong response (Garmory 473). Garmory and colleagues cited several reasons for this: the copy number of the plasmid containing the V antigen might not have been high enough; the attenuated ''Salmonella'' may not have been able to reach cells that would be targeted by ''Y. pestis''; and the inability of the cell to secrete the antigen. Many changes need to be made to live vector vaccine design before it is an effective method of treating or preventing disease.

Another method of disease prevention with a synthetic biology approach is related to skin cancer. ''E. coli'' engineered to sense single-stranded DNA after DNA damage was coupled with a mechanism to produce a biofilm around the cell. The biofilm output module was tested with DNA damage done by mitomycin C, a DNA crosslinking agent, or UV irradiation, a known cause of skin cancer. The biofilm protection, when part of a toggle switch mechanism, lasted indefinitely when pulsed with UV light at 8 J/m2 for only 2 seconds (Kobayashi 8417) (Fig.1). The biofilm-production mechanism has not been engineered in eukaryotic cells, but a prokaryote-based system that could aid with human skin disease is imaginable. For example, a sunscreen that changes colors when the DNA damage may be too intense for a sunbather as a warning, or a sunscreen that becomes more protective as the possibility of genetic damage increased. Further research should focus on how to best implement the biofilm producing output module to protect human DNA.
<center>
[[Image:UVinducedbiofilm.JPG]]

'''Fig. 1.''' Crystal violet absorbance measures biofilm formation.

</center>
Finally, synthetic biology has made its mark on the field of medicine by creating drugs that are less expensive to produce than those made by pharmaceutical companies. Jay Keasling’s project involving the antimalarial drug artemisinin is the best example. Yeast was engineered to increase normal farnesyl pyrophosphate (FPP) production, convert FPP to amorphadiene, and oxidizing amorphadiene to artemisinic acid, which can then easily be separated from the yeast cells and converted to artemisinin in the laboratory (Fig. 2). This innovation allows the production of artemisinin at lower prices than those currently on the market, and without regard to environmental constraints (Ro 942). On a completely different tangent, Collins’s lab engineered T7 bacteriophages to deliver biofilm-degrading enzymes such as dispersin B after infecting and replicating in an ''E. coli'' biofilm. Dispersin B works to degrade an adhesin that is critical for biofilm formation. Phages that released dispersin B were found to be 99.997% effective at removing biofilm, which is 4.5 orders of magnitude higher than non-enzymatic phages (Lu 11197) (Fig. 3). The specificity of phages for a certain type of bacteria make them a viable vector to be used in human treatments, which may range from removing dental plaques to cleaning inserted medical devices. Using synthetic biology to engineer an antimalarial drug in yeast or a biofilm-destroying enzyme in bacteriophages greatly increases the possible contributions to the field of medicine.

<center>

{|
|-
|[[Image:Artemesinin.JPG]]
|[[Image:BiofilmDegradation.JPG]]
|-
|'''Fig. 2.''' All the steps in the box indicate steps that were optimized in yeast for a maximum artemisinic acid output.
|'''Fig. 3.''' The amount of biofilm was decreased when the dispersin B enzymes were incorporated into T7 bacteriophages.
|}
</center>

Medical applications of synthetic biology are wide-ranging and eminently applicable to daily life. Synthetic biology based cancer treatments that utilize E.coli engineered to sense and attack tissues with high cell density and low oxygen availability are promising and need to be tested in vivo. Designing vectors out of attenuated bacteria may be a safe, effective method for disease treatment. Using biofilms to sense sun damage potential may decrease the likelihood of skin cancer. Finally, using engineered cells to produce medicines in a cost-effective, environmentally-friendly way may revolutionize the pharmaceutical industry. Investing more research efforts in the field of synthetic biology may turn out to be an investment in medical technology.

'''Works Cited'''

Anderson JC, Clarke EJ, Arkin, AP, and Voigt CA (2006). Environmentally controlled invasion of cancer cells by engineered bacteria. Journal of Molecular Biology 355:619-27.

Critchley RJ, Jezzard S, Radford KJ, Goussard S, Lemoine NR, et al. (2004). Potential therapeutic applications of recombinant, invasive E. coli. Gene Therapy 11:1224-33.

Garmory HS, Leary SEC, Griffin, KF, Williamson D, Brown, KA, and Titball RW (2003). The use of live attenuated bacteria as a delivery system for heterologous antigens. Journal of Drug Targeting 11:471-79.

Kobayashi H, et al. (2004). Programmable cells: Interfacing natural and engineered gene networks. PNAS 101: 8414-19.

Lu, TK, and Collins JJ (2007). Dispersing biofilms with engineered enzymatic bacteriophage. PNAS 104: 11197-11202.

Nemunaitis J, Cunningham C, Senzer N, Kuhn J, Cramm J, Litz C, et al. (2003). Pilot trial of genetically modified, attenuated Salmonella expressing cytosine deaminase gene in refractory cancer patients. Cancer Gene Therapy 10:737-744.

Pawelek JM, Low KB, and Bermudes D (2003). Bacteria as tumour-targeting vectors. Lancet Oncology 4:548-56.

Ro D, et al. (2006). Production of the antimalarial drug precursor artemisinic acid in engineered yeast. Nature 440:940-43.