GcatWiki - User contributions [en]

Post-transcriptional Regulation Technologies - Erin Zwack

2007-12-06T20:39:35Z

Erzwack: /* References */

==Post-transcriptional Regulation Technologies==

==Overview:==

Regulation of translation provides an excellent tool for research on metabolic and other pathways in organisms, and for the production of different sensors by controlling the translation of specific genes depending on cellular conditions. Further development of these types of technology could provide a “knock-down” equivalent to RNAi, which exist in some eukaryotes. A gene of interest could be expressed normally at all times when the regulator is not active; thus, no ill effects will result before a particular pathway activates and produces a specific ligand if the gene has another purpose as well. Other synthetic biologists could use these technologies to engineer fast-responding, RNA-based biological sensors for environmental chemicals, or novel pathways.

Using RNA regulatory molecules instead of regulatory proteins to control gene expression provides several benefits to synthetic biologists. Regulatory proteins bind to specific sites such as sites on the promoter or sites upstream of the promoter called operators ([http://en.wikipedia.org/wiki/Gene_regulation#Regulatory_protein wikipedia]). Control by these proteins can rely heavily on [[CellularMemory:Mathematical Models#Cooperativity_and_Bistability| cooperativity]] (Gardner et al. 2000), that is multiple proteins binding to one site, in order to see an effect. Regulatory RNA molecules on the other hand need a one to one ratio of regulatory molecule to target. As long as the molecule is expressed in an equal or greater amount than the target, the regulatory RNAs will normally be able to bind to their targets and control transcription.

While synthetic biologists could use the regulatory proteins and their binding sites that are found in nature, rational design of more new regulatory proteins is difficult. With the oligo and gene sythesis technology in existence today, RNA can be engineered that complements and thus targets any other RNA sequence. Regulatory proteins are also controlled mainly by promoter when determining whether they are active or not. As only so many inducible promoters exist, there is a small number of stimuli that can be used to determine under what conditions the gene under a regulatory protein's control will be expressed or repressed. An aptamer, RNA sequence that binds to a small molecule such as theophylline (Bayer and Smolke 2005), can be used not only to regulate gene expression but can also regulate under what conditions an RNA regulatory molecule is active. New aptamers are easily developed through rational design, and the number in existence is continually increasing and providing new molecules that can act as ligands.

Finally, regulatory proteins stop gene expression before transcription. When the stimulus changes and the gene is expressed (either because a regulatory protein has now bound to or has released its site), the time it takes for the phenotype to be expressed is longer because both transcription and translation must occur instead of just translation. With RNA, the gene expression is halted after transcription. Once the stimulus is removed, the RNA already produced by the gene simply needs to be translated.

==Development of Systems==

In most cases, post-transcriptional regulatory mechanisms that were developed and worked in eukaryotes cannot be directly transferred to prokaryotes. Modifications are necessary because eukaryotic and prokaryotic transcription and translation do not follow the exact same path. In eukaryotes, mRNA must have introns spliced out before translation begins; thus, any mechanism that regulates translation has time to bind or manipulate the mRNA (figure 1). In prokaryotes, translation begins as soon as the ribosomal binding site (RBS) is transcribed and accessible to a ribosome (figure 2).

[[Image:Eukaryotic.jpg]]

'''Figure 1:''' Inside of the nucleus of the eukaryote, the gene is transcribed into pre-mRNA, which contain both introns (orange) and exons (red). The pre-mRNA is then modified so that the introns are spliced out and the exons are put together. Finally the mRNA are translated by the ribosome in the cytoplasm.

[[Image:Prokaryote.jpg]]

'''Figure 2:''' In a prokaryotic cell the DNA is transcribed by RNA polymerase. As soon as the polymerase transcribes the ribosomal binding site, a ribosome binds and begins translating the sequence into protein. There is no modification step between transcription and translation.

==Eukaryotes:==

[[Antiswitches]]

==Prokaryotes:==

[[Riboregulators]]

[[Riboswitches]]

==Use of Post-transciptional Regulatory Technologies==

Regulation of translation provides an excellent tool for research on metabolic and other pathways in organisms, and for the production of different sensors by controlling the translation of specific genes depending on cellular conditions. Researchers can turn-off translation of certain genes in response to different pathways being activated, such as metabolic pathways. If a researcher wanted to know if a particular gene were necessary to proper function of a pathway, the aptamer of the antiswitch or riboswitch could be designed to have a molecule produced upstream in the pathway be its ligand. The gene would be expressed normally at all times when the pathway is not active; thus, no ill effects will result before the pathway activates if the gene has another purpose as well. This would provide a “knock-down” equivalent to RNAi, which exists in some eukaryotes.

Synthetic biologists could engineer fast-responding, RNA-based biological sensors for environmental chemicals. The sensors could be used to detect harmful chemical like arsenic or chemical indicative of explosives by using post-transcriptional regulatory technologies that would activate or be produced in the presence of the chemical and turn-on a reporter gene like GFP. The technology could also be used to engineer novel pathways that only activate when the environmental conditions are favorable. By regulating when a newly engineered pathway is on, biologists could possibly achieve optimal efficiency in generating a desired product by not taxing the cells when they do not have enough resources to thrive and produce a product that is not natural to the organism.

==The Labs==

[http://www.che.caltech.edu/groups/cds/index.htm Smolke]

[http://www.bu.edu/abl/ Collins]

[http://www.bu.edu/abl/files/naturebiotech_isaacs.pdf Dr. Isaacs's Review of RNA Synthetic Biology]

[http://gallivan1.chem.emory.edu/Gallivan%20Lab/Home.html Gallivan]

==References==

[http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=Retrieve&db=pubmed&dopt=AbstractPlus&list_uids=15723047 Bayer TS and Smolke CD. Programmable ligand-controlled riboregulators of eukaryotic gene expression. ''Nat Biotechnol''. (2005) 3:337-43.]

[http://pubs.acs.org/cgi-bin/article.cgi/jacsat/2004/126/i41/pdf/ja048634j.pdf Desai SK and Gallivan JP. Genetic Screens and Selections for Small Molecules Based on a Synthetic Riboswitch That Activates Protein Translation. ''J. Am. Chem. Soc.''(2004) 126:13247-54.]

[http://www.bio.davidson.edu/courses/synthetic/papers/Collins_ToggleSwitch.pdf Gardner, T.S., Cantor, C.R., and Collins, J.J. Construction of a genetic toggle switch in Eschreichia coli. ''Nature'' (2000) 403: 339-342.]

Isaacs FJ, et al. Engineered riboregulators enable post-transcriptional control of gene expression. ''Nat Biotechnol.'' (2004) 22:841-47.[http://www.bio.davidson.edu/Courses/Synthetic/papers/RNA_Regulation.pdf]

Regulatory Proteins. Wikipedia. December 2007. http://en.wikipedia.org/wiki/Gene_regulation#Regulatory_protein

[[A Review of Synthetic Biology| Return to Main Page]]

Post-transcriptional Regulation Technologies - Erin Zwack

2007-12-06T20:39:12Z

Erzwack: /* References */

==Post-transcriptional Regulation Technologies==

==Overview:==

Regulation of translation provides an excellent tool for research on metabolic and other pathways in organisms, and for the production of different sensors by controlling the translation of specific genes depending on cellular conditions. Further development of these types of technology could provide a “knock-down” equivalent to RNAi, which exist in some eukaryotes. A gene of interest could be expressed normally at all times when the regulator is not active; thus, no ill effects will result before a particular pathway activates and produces a specific ligand if the gene has another purpose as well. Other synthetic biologists could use these technologies to engineer fast-responding, RNA-based biological sensors for environmental chemicals, or novel pathways.

Using RNA regulatory molecules instead of regulatory proteins to control gene expression provides several benefits to synthetic biologists. Regulatory proteins bind to specific sites such as sites on the promoter or sites upstream of the promoter called operators ([http://en.wikipedia.org/wiki/Gene_regulation#Regulatory_protein wikipedia]). Control by these proteins can rely heavily on [[CellularMemory:Mathematical Models#Cooperativity_and_Bistability| cooperativity]] (Gardner et al. 2000), that is multiple proteins binding to one site, in order to see an effect. Regulatory RNA molecules on the other hand need a one to one ratio of regulatory molecule to target. As long as the molecule is expressed in an equal or greater amount than the target, the regulatory RNAs will normally be able to bind to their targets and control transcription.

While synthetic biologists could use the regulatory proteins and their binding sites that are found in nature, rational design of more new regulatory proteins is difficult. With the oligo and gene sythesis technology in existence today, RNA can be engineered that complements and thus targets any other RNA sequence. Regulatory proteins are also controlled mainly by promoter when determining whether they are active or not. As only so many inducible promoters exist, there is a small number of stimuli that can be used to determine under what conditions the gene under a regulatory protein's control will be expressed or repressed. An aptamer, RNA sequence that binds to a small molecule such as theophylline (Bayer and Smolke 2005), can be used not only to regulate gene expression but can also regulate under what conditions an RNA regulatory molecule is active. New aptamers are easily developed through rational design, and the number in existence is continually increasing and providing new molecules that can act as ligands.

Finally, regulatory proteins stop gene expression before transcription. When the stimulus changes and the gene is expressed (either because a regulatory protein has now bound to or has released its site), the time it takes for the phenotype to be expressed is longer because both transcription and translation must occur instead of just translation. With RNA, the gene expression is halted after transcription. Once the stimulus is removed, the RNA already produced by the gene simply needs to be translated.

==Development of Systems==

In most cases, post-transcriptional regulatory mechanisms that were developed and worked in eukaryotes cannot be directly transferred to prokaryotes. Modifications are necessary because eukaryotic and prokaryotic transcription and translation do not follow the exact same path. In eukaryotes, mRNA must have introns spliced out before translation begins; thus, any mechanism that regulates translation has time to bind or manipulate the mRNA (figure 1). In prokaryotes, translation begins as soon as the ribosomal binding site (RBS) is transcribed and accessible to a ribosome (figure 2).

[[Image:Eukaryotic.jpg]]

'''Figure 1:''' Inside of the nucleus of the eukaryote, the gene is transcribed into pre-mRNA, which contain both introns (orange) and exons (red). The pre-mRNA is then modified so that the introns are spliced out and the exons are put together. Finally the mRNA are translated by the ribosome in the cytoplasm.

[[Image:Prokaryote.jpg]]

'''Figure 2:''' In a prokaryotic cell the DNA is transcribed by RNA polymerase. As soon as the polymerase transcribes the ribosomal binding site, a ribosome binds and begins translating the sequence into protein. There is no modification step between transcription and translation.

==Eukaryotes:==

[[Antiswitches]]

==Prokaryotes:==

[[Riboregulators]]

[[Riboswitches]]

==Use of Post-transciptional Regulatory Technologies==

Regulation of translation provides an excellent tool for research on metabolic and other pathways in organisms, and for the production of different sensors by controlling the translation of specific genes depending on cellular conditions. Researchers can turn-off translation of certain genes in response to different pathways being activated, such as metabolic pathways. If a researcher wanted to know if a particular gene were necessary to proper function of a pathway, the aptamer of the antiswitch or riboswitch could be designed to have a molecule produced upstream in the pathway be its ligand. The gene would be expressed normally at all times when the pathway is not active; thus, no ill effects will result before the pathway activates if the gene has another purpose as well. This would provide a “knock-down” equivalent to RNAi, which exists in some eukaryotes.

Synthetic biologists could engineer fast-responding, RNA-based biological sensors for environmental chemicals. The sensors could be used to detect harmful chemical like arsenic or chemical indicative of explosives by using post-transcriptional regulatory technologies that would activate or be produced in the presence of the chemical and turn-on a reporter gene like GFP. The technology could also be used to engineer novel pathways that only activate when the environmental conditions are favorable. By regulating when a newly engineered pathway is on, biologists could possibly achieve optimal efficiency in generating a desired product by not taxing the cells when they do not have enough resources to thrive and produce a product that is not natural to the organism.

==The Labs==

[http://www.che.caltech.edu/groups/cds/index.htm Smolke]

[http://www.bu.edu/abl/ Collins]

[http://www.bu.edu/abl/files/naturebiotech_isaacs.pdf Dr. Isaacs's Review of RNA Synthetic Biology]

[http://gallivan1.chem.emory.edu/Gallivan%20Lab/Home.html Gallivan]

==References==

[http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=Retrieve&db=pubmed&dopt=AbstractPlus&list_uids=15723047 Bayer TS and Smolke CD. Programmable ligand-controlled riboregulators of eukaryotic gene expression. ''Nat Biotechnol''. (2005) 3:337-43.]

[http://pubs.acs.org/cgi-bin/article.cgi/jacsat/2004/126/i41/pdf/ja048634j.pdf Desai SK and Gallivan JP. Genetic Screens and Selections for Small Molecules Based on a Synthetic Riboswitch That Activates Protein Translation. ''J. Am. Chem. Soc.''(2004) 126:13247-54.]

[http://www.bio.davidson.edu/courses/synthetic/papers/Collins_ToggleSwitch.pdf Gardner, T.S., Cantor, C.R., and Collins, J.J. Construction of a genetic toggle switch in Eschreichia coli. ''Nature'' (2000) 403: 339-342.]

Isaacs FJ, et al. Engineered riboregulators enable post-transcriptional control of gene expression. ''Nat Biotechnol.'' (2004) 22:841-47.[http://www.bio.davidson.edu/Courses/Synthetic/papers/RNA_Regulation.pdf}

Regulatory Proteins. Wikipedia. December 2007. http://en.wikipedia.org/wiki/Gene_regulation#Regulatory_protein

[[A Review of Synthetic Biology| Return to Main Page]]

Post-transcriptional Regulation Technologies - Erin Zwack

2007-12-06T20:33:11Z

Erzwack: /* References */

==Post-transcriptional Regulation Technologies==

==Overview:==

Regulation of translation provides an excellent tool for research on metabolic and other pathways in organisms, and for the production of different sensors by controlling the translation of specific genes depending on cellular conditions. Further development of these types of technology could provide a “knock-down” equivalent to RNAi, which exist in some eukaryotes. A gene of interest could be expressed normally at all times when the regulator is not active; thus, no ill effects will result before a particular pathway activates and produces a specific ligand if the gene has another purpose as well. Other synthetic biologists could use these technologies to engineer fast-responding, RNA-based biological sensors for environmental chemicals, or novel pathways.

Using RNA regulatory molecules instead of regulatory proteins to control gene expression provides several benefits to synthetic biologists. Regulatory proteins bind to specific sites such as sites on the promoter or sites upstream of the promoter called operators ([http://en.wikipedia.org/wiki/Gene_regulation#Regulatory_protein wikipedia]). Control by these proteins can rely heavily on [[CellularMemory:Mathematical Models#Cooperativity_and_Bistability| cooperativity]] (Gardner et al. 2000), that is multiple proteins binding to one site, in order to see an effect. Regulatory RNA molecules on the other hand need a one to one ratio of regulatory molecule to target. As long as the molecule is expressed in an equal or greater amount than the target, the regulatory RNAs will normally be able to bind to their targets and control transcription.

While synthetic biologists could use the regulatory proteins and their binding sites that are found in nature, rational design of more new regulatory proteins is difficult. With the oligo and gene sythesis technology in existence today, RNA can be engineered that complements and thus targets any other RNA sequence. Regulatory proteins are also controlled mainly by promoter when determining whether they are active or not. As only so many inducible promoters exist, there is a small number of stimuli that can be used to determine under what conditions the gene under a regulatory protein's control will be expressed or repressed. An aptamer, RNA sequence that binds to a small molecule such as theophylline (Bayer and Smolke 2005), can be used not only to regulate gene expression but can also regulate under what conditions an RNA regulatory molecule is active. New aptamers are easily developed through rational design, and the number in existence is continually increasing and providing new molecules that can act as ligands.

Finally, regulatory proteins stop gene expression before transcription. When the stimulus changes and the gene is expressed (either because a regulatory protein has now bound to or has released its site), the time it takes for the phenotype to be expressed is longer because both transcription and translation must occur instead of just translation. With RNA, the gene expression is halted after transcription. Once the stimulus is removed, the RNA already produced by the gene simply needs to be translated.

==Development of Systems==

In most cases, post-transcriptional regulatory mechanisms that were developed and worked in eukaryotes cannot be directly transferred to prokaryotes. Modifications are necessary because eukaryotic and prokaryotic transcription and translation do not follow the exact same path. In eukaryotes, mRNA must have introns spliced out before translation begins; thus, any mechanism that regulates translation has time to bind or manipulate the mRNA (figure 1). In prokaryotes, translation begins as soon as the ribosomal binding site (RBS) is transcribed and accessible to a ribosome (figure 2).

[[Image:Eukaryotic.jpg]]

'''Figure 1:''' Inside of the nucleus of the eukaryote, the gene is transcribed into pre-mRNA, which contain both introns (orange) and exons (red). The pre-mRNA is then modified so that the introns are spliced out and the exons are put together. Finally the mRNA are translated by the ribosome in the cytoplasm.

[[Image:Prokaryote.jpg]]

'''Figure 2:''' In a prokaryotic cell the DNA is transcribed by RNA polymerase. As soon as the polymerase transcribes the ribosomal binding site, a ribosome binds and begins translating the sequence into protein. There is no modification step between transcription and translation.

==Eukaryotes:==

[[Antiswitches]]

==Prokaryotes:==

[[Riboregulators]]

[[Riboswitches]]

==Use of Post-transciptional Regulatory Technologies==

Regulation of translation provides an excellent tool for research on metabolic and other pathways in organisms, and for the production of different sensors by controlling the translation of specific genes depending on cellular conditions. Researchers can turn-off translation of certain genes in response to different pathways being activated, such as metabolic pathways. If a researcher wanted to know if a particular gene were necessary to proper function of a pathway, the aptamer of the antiswitch or riboswitch could be designed to have a molecule produced upstream in the pathway be its ligand. The gene would be expressed normally at all times when the pathway is not active; thus, no ill effects will result before the pathway activates if the gene has another purpose as well. This would provide a “knock-down” equivalent to RNAi, which exists in some eukaryotes.

Synthetic biologists could engineer fast-responding, RNA-based biological sensors for environmental chemicals. The sensors could be used to detect harmful chemical like arsenic or chemical indicative of explosives by using post-transcriptional regulatory technologies that would activate or be produced in the presence of the chemical and turn-on a reporter gene like GFP. The technology could also be used to engineer novel pathways that only activate when the environmental conditions are favorable. By regulating when a newly engineered pathway is on, biologists could possibly achieve optimal efficiency in generating a desired product by not taxing the cells when they do not have enough resources to thrive and produce a product that is not natural to the organism.

==The Labs==

[http://www.che.caltech.edu/groups/cds/index.htm Smolke]

[http://www.bu.edu/abl/ Collins]

[http://www.bu.edu/abl/files/naturebiotech_isaacs.pdf Dr. Isaacs's Review of RNA Synthetic Biology]

[http://gallivan1.chem.emory.edu/Gallivan%20Lab/Home.html Gallivan]

==References==

[http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=Retrieve&db=pubmed&dopt=AbstractPlus&list_uids=15723047 Bayer TS and Smolke CD. Programmable ligand-controlled riboregulators of eukaryotic gene expression. ''Nat Biotechnol''. (2005) 3:337-43.]

[http://pubs.acs.org/cgi-bin/article.cgi/jacsat/2004/126/i41/pdf/ja048634j.pdf Desai SK and Gallivan JP. Genetic Screens and Selections for Small Molecules Based on a Synthetic Riboswitch That Activates Protein Translation. ''J. Am. Chem. Soc.''(2004) 126:13247-54.]

[http://www.bio.davidson.edu/courses/synthetic/papers/Collins_ToggleSwitch.pdf Gardner, T.S., Cantor, C.R., and Collins, J.J. Construction of a genetic toggle switch in Eschreichia coli. ''Nature'' (2000) 403: 339-342.]

[http://www.bio.davidson.edu/Courses/Synthetic/papers/RNA_Regulation.pdf Isaacs FJ, et al. Engineered riboregulators enable post-transcriptional control of gene expression. ''Nat Biotechnol.'' (2004) 22:841-47.]

Regulatory Proteins. Wikipedia. December 2007. http://en.wikipedia.org/wiki/Gene_regulation#Regulatory_protein

[[A Review of Synthetic Biology| Return to Main Page]]

Post-transcriptional Regulation Technologies - Erin Zwack

2007-12-06T20:32:52Z

Erzwack: /* References */

==Post-transcriptional Regulation Technologies==

==Overview:==

Regulation of translation provides an excellent tool for research on metabolic and other pathways in organisms, and for the production of different sensors by controlling the translation of specific genes depending on cellular conditions. Further development of these types of technology could provide a “knock-down” equivalent to RNAi, which exist in some eukaryotes. A gene of interest could be expressed normally at all times when the regulator is not active; thus, no ill effects will result before a particular pathway activates and produces a specific ligand if the gene has another purpose as well. Other synthetic biologists could use these technologies to engineer fast-responding, RNA-based biological sensors for environmental chemicals, or novel pathways.

Using RNA regulatory molecules instead of regulatory proteins to control gene expression provides several benefits to synthetic biologists. Regulatory proteins bind to specific sites such as sites on the promoter or sites upstream of the promoter called operators ([http://en.wikipedia.org/wiki/Gene_regulation#Regulatory_protein wikipedia]). Control by these proteins can rely heavily on [[CellularMemory:Mathematical Models#Cooperativity_and_Bistability| cooperativity]] (Gardner et al. 2000), that is multiple proteins binding to one site, in order to see an effect. Regulatory RNA molecules on the other hand need a one to one ratio of regulatory molecule to target. As long as the molecule is expressed in an equal or greater amount than the target, the regulatory RNAs will normally be able to bind to their targets and control transcription.

While synthetic biologists could use the regulatory proteins and their binding sites that are found in nature, rational design of more new regulatory proteins is difficult. With the oligo and gene sythesis technology in existence today, RNA can be engineered that complements and thus targets any other RNA sequence. Regulatory proteins are also controlled mainly by promoter when determining whether they are active or not. As only so many inducible promoters exist, there is a small number of stimuli that can be used to determine under what conditions the gene under a regulatory protein's control will be expressed or repressed. An aptamer, RNA sequence that binds to a small molecule such as theophylline (Bayer and Smolke 2005), can be used not only to regulate gene expression but can also regulate under what conditions an RNA regulatory molecule is active. New aptamers are easily developed through rational design, and the number in existence is continually increasing and providing new molecules that can act as ligands.

Finally, regulatory proteins stop gene expression before transcription. When the stimulus changes and the gene is expressed (either because a regulatory protein has now bound to or has released its site), the time it takes for the phenotype to be expressed is longer because both transcription and translation must occur instead of just translation. With RNA, the gene expression is halted after transcription. Once the stimulus is removed, the RNA already produced by the gene simply needs to be translated.

==Development of Systems==

In most cases, post-transcriptional regulatory mechanisms that were developed and worked in eukaryotes cannot be directly transferred to prokaryotes. Modifications are necessary because eukaryotic and prokaryotic transcription and translation do not follow the exact same path. In eukaryotes, mRNA must have introns spliced out before translation begins; thus, any mechanism that regulates translation has time to bind or manipulate the mRNA (figure 1). In prokaryotes, translation begins as soon as the ribosomal binding site (RBS) is transcribed and accessible to a ribosome (figure 2).

[[Image:Eukaryotic.jpg]]

'''Figure 1:''' Inside of the nucleus of the eukaryote, the gene is transcribed into pre-mRNA, which contain both introns (orange) and exons (red). The pre-mRNA is then modified so that the introns are spliced out and the exons are put together. Finally the mRNA are translated by the ribosome in the cytoplasm.

[[Image:Prokaryote.jpg]]

'''Figure 2:''' In a prokaryotic cell the DNA is transcribed by RNA polymerase. As soon as the polymerase transcribes the ribosomal binding site, a ribosome binds and begins translating the sequence into protein. There is no modification step between transcription and translation.

==Eukaryotes:==

[[Antiswitches]]

==Prokaryotes:==

[[Riboregulators]]

[[Riboswitches]]

==Use of Post-transciptional Regulatory Technologies==

Regulation of translation provides an excellent tool for research on metabolic and other pathways in organisms, and for the production of different sensors by controlling the translation of specific genes depending on cellular conditions. Researchers can turn-off translation of certain genes in response to different pathways being activated, such as metabolic pathways. If a researcher wanted to know if a particular gene were necessary to proper function of a pathway, the aptamer of the antiswitch or riboswitch could be designed to have a molecule produced upstream in the pathway be its ligand. The gene would be expressed normally at all times when the pathway is not active; thus, no ill effects will result before the pathway activates if the gene has another purpose as well. This would provide a “knock-down” equivalent to RNAi, which exists in some eukaryotes.

Synthetic biologists could engineer fast-responding, RNA-based biological sensors for environmental chemicals. The sensors could be used to detect harmful chemical like arsenic or chemical indicative of explosives by using post-transcriptional regulatory technologies that would activate or be produced in the presence of the chemical and turn-on a reporter gene like GFP. The technology could also be used to engineer novel pathways that only activate when the environmental conditions are favorable. By regulating when a newly engineered pathway is on, biologists could possibly achieve optimal efficiency in generating a desired product by not taxing the cells when they do not have enough resources to thrive and produce a product that is not natural to the organism.

==The Labs==

[http://www.che.caltech.edu/groups/cds/index.htm Smolke]

[http://www.bu.edu/abl/ Collins]

[http://www.bu.edu/abl/files/naturebiotech_isaacs.pdf Dr. Isaacs's Review of RNA Synthetic Biology]

[http://gallivan1.chem.emory.edu/Gallivan%20Lab/Home.html Gallivan]

==References==

[http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=Retrieve&db=pubmed&dopt=AbstractPlus&list_uids=15723047 Bayer TS and Smolke CD. Programmable ligand-controlled riboregulators of eukaryotic gene expression. ''Nat Biotechnol''. (2005) 3:337-43.]

[http://pubs.acs.org/cgi-bin/article.cgi/jacsat/2004/126/i41/pdf/ja048634j.pdf Desai SK and Gallivan JP. Genetic Screens and Selections for Small Molecules Based on a Synthetic Riboswitch That Activates Protein Translation. ''J. Am. Chem. Soc.''(2004) 126:13247-54.]

[http://www.bio.davidson.edu/courses/synthetic/papers/Collins_ToggleSwitch.pdf Gardner, T.S., Cantor, C.R., and Collins, J.J. Construction of a genetic toggle switch in Eschreichia coli. ''Nature'' (2000) 403: 339-342.]

[http://www.bio.davidson.edu/Courses/Synthetic/papers/RNA_Regulation.pdf Isaacs FJ, et al. Engineered riboregulators enable post-transcriptional control of gene expression. ''Nat Biotechnol.'' (2004) 22:841-47.]

Regulatory Proteins. Wikipedia. December 2007. [http://en.wikipedia.org/wiki/Gene_regulation#Regulatory_protein]

[[A Review of Synthetic Biology| Return to Main Page]]

Post-transcriptional Regulation Technologies - Erin Zwack

2007-12-06T20:31:23Z

Erzwack: /* Use of Post-transciptional Regulatory Technologies */

==Post-transcriptional Regulation Technologies==

==Overview:==

Regulation of translation provides an excellent tool for research on metabolic and other pathways in organisms, and for the production of different sensors by controlling the translation of specific genes depending on cellular conditions. Further development of these types of technology could provide a “knock-down” equivalent to RNAi, which exist in some eukaryotes. A gene of interest could be expressed normally at all times when the regulator is not active; thus, no ill effects will result before a particular pathway activates and produces a specific ligand if the gene has another purpose as well. Other synthetic biologists could use these technologies to engineer fast-responding, RNA-based biological sensors for environmental chemicals, or novel pathways.

Using RNA regulatory molecules instead of regulatory proteins to control gene expression provides several benefits to synthetic biologists. Regulatory proteins bind to specific sites such as sites on the promoter or sites upstream of the promoter called operators ([http://en.wikipedia.org/wiki/Gene_regulation#Regulatory_protein wikipedia]). Control by these proteins can rely heavily on [[CellularMemory:Mathematical Models#Cooperativity_and_Bistability| cooperativity]] (Gardner et al. 2000), that is multiple proteins binding to one site, in order to see an effect. Regulatory RNA molecules on the other hand need a one to one ratio of regulatory molecule to target. As long as the molecule is expressed in an equal or greater amount than the target, the regulatory RNAs will normally be able to bind to their targets and control transcription.

While synthetic biologists could use the regulatory proteins and their binding sites that are found in nature, rational design of more new regulatory proteins is difficult. With the oligo and gene sythesis technology in existence today, RNA can be engineered that complements and thus targets any other RNA sequence. Regulatory proteins are also controlled mainly by promoter when determining whether they are active or not. As only so many inducible promoters exist, there is a small number of stimuli that can be used to determine under what conditions the gene under a regulatory protein's control will be expressed or repressed. An aptamer, RNA sequence that binds to a small molecule such as theophylline (Bayer and Smolke 2005), can be used not only to regulate gene expression but can also regulate under what conditions an RNA regulatory molecule is active. New aptamers are easily developed through rational design, and the number in existence is continually increasing and providing new molecules that can act as ligands.

Finally, regulatory proteins stop gene expression before transcription. When the stimulus changes and the gene is expressed (either because a regulatory protein has now bound to or has released its site), the time it takes for the phenotype to be expressed is longer because both transcription and translation must occur instead of just translation. With RNA, the gene expression is halted after transcription. Once the stimulus is removed, the RNA already produced by the gene simply needs to be translated.

==Development of Systems==

In most cases, post-transcriptional regulatory mechanisms that were developed and worked in eukaryotes cannot be directly transferred to prokaryotes. Modifications are necessary because eukaryotic and prokaryotic transcription and translation do not follow the exact same path. In eukaryotes, mRNA must have introns spliced out before translation begins; thus, any mechanism that regulates translation has time to bind or manipulate the mRNA (figure 1). In prokaryotes, translation begins as soon as the ribosomal binding site (RBS) is transcribed and accessible to a ribosome (figure 2).

[[Image:Eukaryotic.jpg]]

'''Figure 1:''' Inside of the nucleus of the eukaryote, the gene is transcribed into pre-mRNA, which contain both introns (orange) and exons (red). The pre-mRNA is then modified so that the introns are spliced out and the exons are put together. Finally the mRNA are translated by the ribosome in the cytoplasm.

[[Image:Prokaryote.jpg]]

'''Figure 2:''' In a prokaryotic cell the DNA is transcribed by RNA polymerase. As soon as the polymerase transcribes the ribosomal binding site, a ribosome binds and begins translating the sequence into protein. There is no modification step between transcription and translation.

==Eukaryotes:==

[[Antiswitches]]

==Prokaryotes:==

[[Riboregulators]]

[[Riboswitches]]

==Use of Post-transciptional Regulatory Technologies==

Regulation of translation provides an excellent tool for research on metabolic and other pathways in organisms, and for the production of different sensors by controlling the translation of specific genes depending on cellular conditions. Researchers can turn-off translation of certain genes in response to different pathways being activated, such as metabolic pathways. If a researcher wanted to know if a particular gene were necessary to proper function of a pathway, the aptamer of the antiswitch or riboswitch could be designed to have a molecule produced upstream in the pathway be its ligand. The gene would be expressed normally at all times when the pathway is not active; thus, no ill effects will result before the pathway activates if the gene has another purpose as well. This would provide a “knock-down” equivalent to RNAi, which exists in some eukaryotes.

Synthetic biologists could engineer fast-responding, RNA-based biological sensors for environmental chemicals. The sensors could be used to detect harmful chemical like arsenic or chemical indicative of explosives by using post-transcriptional regulatory technologies that would activate or be produced in the presence of the chemical and turn-on a reporter gene like GFP. The technology could also be used to engineer novel pathways that only activate when the environmental conditions are favorable. By regulating when a newly engineered pathway is on, biologists could possibly achieve optimal efficiency in generating a desired product by not taxing the cells when they do not have enough resources to thrive and produce a product that is not natural to the organism.

==The Labs==

[http://www.che.caltech.edu/groups/cds/index.htm Smolke]

[http://www.bu.edu/abl/ Collins]

[http://www.bu.edu/abl/files/naturebiotech_isaacs.pdf Dr. Isaacs's Review of RNA Synthetic Biology]

[http://gallivan1.chem.emory.edu/Gallivan%20Lab/Home.html Gallivan]

==References==

[http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=Retrieve&db=pubmed&dopt=AbstractPlus&list_uids=15723047 Bayer TS and Smolke CD. Programmable ligand-controlled riboregulators of eukaryotic gene expression. ''Nat Biotechnol''. (2005) 3:337-43.]

[http://pubs.acs.org/cgi-bin/article.cgi/jacsat/2004/126/i41/pdf/ja048634j.pdf Desai SK and Gallivan JP. Genetic Screens and Selections for Small Molecules Based on a Synthetic Riboswitch That Activates Protein Translation. ''J. Am. Chem. Soc.''(2004) 126:13247-54.]

[http://www.bio.davidson.edu/courses/synthetic/papers/Collins_ToggleSwitch.pdf Gardner, T.S., Cantor, C.R., and Collins, J.J. Construction of a genetic toggle switch in Eschreichia coli. ''Nature'' (2000) 403: 339-342.]

[http://www.bio.davidson.edu/Courses/Synthetic/papers/RNA_Regulation.pdf Isaacs FJ, et al. Engineered riboregulators enable post-transcriptional control of gene expression. ''Nat Biotechnol.'' (2004) 22:841-47.]

[[A Review of Synthetic Biology| Return to Main Page]]

Riboswitches

2007-12-06T20:22:41Z

Erzwack:

All information on Riboswitches came from Desai and Gallivan (2004).

Riboswitches are small sequences in mRNA that bind small molecules to regulate translation and occasionally transcription. Riboswitches occur naturally in both eukaryotes and prokaryotes. Desai and Gallivan hoped to find new synthetic riboswitches (riboswitches with new ligand specificities) by creating libraries of mutant riboswitches and using genetic selection to pick the functional ones of interest. Desai and Gallivan also employed riboswitches to screen for the presence of specific small molecules. In theory riboswitches are perfect because the number of aptamers already in existence and our capbaility to engineer new aptamers through rational design provide great versatility in shoosing stimuli and conditions.

==Design==

Reviews of previous research showed that the theophylline aptamer worked in riboswitches in wheat germ, a eukaryote, and ''Bacillus subtilis'', a gram positive bacterium. Desai and Gallivan decided to translate the technology to gram negative bacteria. To do this they cloned the theophylline aptamer five base pairs upstream of the RBS for ''lacZ'', a gene that produces the enzyme beta-galactosidase. The gene is controlled by a weak promoter and a weak RBS allowing for sensitivity to changes in translation because of the presence of theophylline. The construct was then transformed into ''E. coli''. When theophylline is added, translation should be turned on again.

==From Concept to Wet Lab==

The aptamer for theophylline was inserted in front of the gene for beta-galactosidase gene to create an mRNA with a riboswitch. Beta-dalactosidase activity is dependent on the amount of the enzyme beta-galactosidase; therefore, measurements of beta-galactosidase activity indicate whether translation of the mRNA is taking place. Testing and comparing beta-galactosidase activity for cells with the riboswitch in the presence of theophylline, caffeine, and no ligand as well as testing and comparing beta-galactosidase activity for cells without the riboswitch under the same conditions demonstrated that the riboswitch does control gene expression in response to theophylline (figure 14). Significant increases in beta-galactosidase activity in cells with the riboswitch were only found when theophylline was added. Because beta-galactosidase activity in the presence of theophylline, caffeine, or no ligand for cells without a riboswitch did not significantly vary, the increase in beta-galactosidase activity in the presence of theophylline for cells with a riboswitch most likely resulted from the theophylline binding to the riboswitch and allowing translation.

[[Image:Plate.JPG]][[Image:Beta.JPG]][[Image:Beta_bar.JPG]]

(Desai and Gallivan 2004)

'''Figure 14:''' '''a)''' On the plate are cells with the riboswitch that were grown in three different conditions: theophylline present, caffeine present, and no ligand present. Cells that were grown in the presence of theophylline have a distinct blue/green color, which indicates beta-galactosidase activity, compared to the cells grown with caffeine or without ligand. The lack of color in the cells grown with caffeine or without ligand indicates that the riboswitch most likely prevents translation of the mRNA encoding beta-galactosidase. The blue color of the cells grown with theophylline indicates that theophylline probably is binding to the riboswitch and allowing for translation.''' b)''' The green line represents cells that have had theophylline added to them while the red line represents cells that had caffeine added. Beta-galactosidase activity's increasing, as measured by Miller Units, significantly only in the presence of high enough concentrations of theophylline but not caffeine suggests that the riboswitch is highly specific for its ligand. '''c)''' When no riboswitch existed in the beta-galactosidase transcript, beta-galactosidase activity was not significantly different for cells grown in the presence of theophylline, caffeine, or no ligand. When a riboswitch existed in the beta-galactosidase transcript, cells grown in the presence of theophylline exhibited significantly greater beta-galactosidase activity than cells grown in the presence of caffeine or no ligand. Beta-galactosidase activity for cells grown in the presence of caffeine did not vary significantly from activity for cells grown without any ligand present.

To further support that the theophylline was interacting with the aptamer and not just increasing protein translation through another route, a single point mutation that ''in vitro'' decreases affinity for theophylline and increases affinity for 3-methylxanthine was introduced to the aptamer. When using this mutant riboswitch, the beta-galactosidase activity, in the presence of theophylline was almost the same as beta-galactosidase activity without any small molecule while beta-galactosidase in the presence of 3-methylxanthine was significantly higher than base-line. These results suggest that the change in translation as indicated by the increase of beta-galactosidase is controlled by the riboswitch and not some other mechanism (figure 15).

[[Image:Mutation.JPG]]

(Desai and Gallivan 2004)

'''Figure 15:''' Beta-galactosidase activity was low and basically the same for cells with the mutant riboswitch that had no molecule (black), caffeine (red), or theophylline (green) added. Cells with the mutant riboswitch showed significant increase in beta-galactosidase activity when 3-methylxanthine (blue) was added.

In synthetic biology, parts are often tweaked until the efficiency, detectability, and other properties are enhanced to work as a part in a device. Gallivan's lab worked on optimization of the riboswitch by changing the position of the aptamer in relation to the RBS. The riboswitch showed a greater increase in beta-galactosidase activity when the aptamer was 8 base pairs upstream of the RBS than when the aptamer was either 2 or 5 base pairs upstream (figure 16). This result could be because of the surrounding bases being purines in this particular transcript or the actual distance.

[[Image:Optimization.JPG]]

(Desai and Gallivan 2004)

'''Figure 16:''' Cells exposed to theophylline (green) showed greater beta-galactosidase activity than cells exposed to caffeine (red) or cells exposed to nothing (black) no matter the distance of the riboswitch from the RBS, but the greatest increase was experienced by cells with the riboswitch 8 base pairs (the farthest tested distance) from the RBS when exposed to theophylline.

==Modularity==

An advantage of riboswitches is that they are modular. The aptamer that is in front of the RBS does not have the restriction that it only works in front of specific sequences. A riboswitch can technically be integrated into any mRNA without redesigning the whole riboswitch component unlike with antiswitches. Because riboswitches do not have a complement to any specific RBS, they can function in front of RBS's that vary drastically from each other while Isaac's riboregulators may need to be redesigned to accomodate the different RBS's.

==References==

Desai SK and Gallivan JP. Genetic Screens and Selections for Small Molecules Based on a Synthetic Riboswitch That Activates Protein Translation. J. Am. Chem. Soc.(2004) 126:13247-54.

[[Post-transcriptional Regulation Technologies - Erin Zwack|Return to Post-transcriptional Regulation Technologies]]

Antiswitches

2007-12-06T20:13:36Z

Erzwack: /* From Concept to Wet Lab */

All information about antiswitches came from Bayer and Smolke (2005).

Antiswitches, trans-RNA molecules that regulate translation of mRNA based on the presence or absence of specific ligands, were first developed by Smolke and Bayer (2005) in ''Saccharomyces cerevisiae''. Two types of antiswitches were engineered: on-switches and off-switches. On-switches turn on gene expression in the presence of the ligand while off-switches turn off gene expression in the presence of the ligand. Control by the ligand allows researchers to regulate pathways’ protein production with less leakiness, low level transcription even when “off”, than using a specific promoter.

==Design==

Antiswitches are made of an aptamer and two stems: the aptamer stem and the antisense stem (figure 3).

[[Image:Switch.JPG]]

(Bayer and Smolke 2005 Permission Pending)

'''Figure 3:''' The antisense stem can bind to itself (duplex). The antisense stem also contains the complement to the RNA that a researcher desires to regulate. The aptamer stem swings either toward the antisense stem and disrupts the duplex or swings away from the antisense stem and allows the antisense stem to duplex with itself depending on whether the aptamer is bound to its ligand.

The aptamer is the sequence that binds the ligand and causes a conformational (shape) change of the antiswitch molecule. Aptamers can be highly specific for their particular molecules. The theophylline aptamer used by Smolke and Bayer can distinguish between caffeine and theophylline, which differ by a single methyl (figure 4).

[[Image:Caffeine.JPG]]

'''Figure 4.''' Caffeine (a) and Theophylline (b) differ by one methyl group (circled in red).

While Smolke and Bayer mainly used the aptamer for the ligand theophylline, aptamers for many other molecules are now being generated using rational design (Win and Smolke 2007). The large number of possible aptamers provides versatility in what will control gene expression.

The antisense stem contains a sequence that complements a targeted RNA transcript and a second sequence that sequesters this complementary sequence to keep it from binding the transcript. When the antisense stem is not duplexed with itself, it prevents translation by binding to the complementary mRNA (figure 5).

The aptamer stem is a short sequence that complements a portion of the sequestering sequence of the antisense stem. In an off-switch, the aptamer stem swings towards the antisense stem and displaces the portion that complements the targeted transcript when the liand binds to the aptamer (figure 5). In an on-switch, the aptamer swings away from the antisense stem when the ligand binds to the aptamer; thus, the antisense stem can duplex and is no longer free to bind to the transcript.

[[Image:ANTI.jpg]]

(Bayer and Smolke 2005 Permission Pending)

'''Figure 5:''' a) An inactive off-switch. The antisense stem is duplexed with itself. b) The same switch after being activated by theophylline (blue ellipse). The antisense stem is duplexed with the mRNA; thus, translation is stopped.

==From Concept to Wet Lab==

Smolke and Bayer synthesized genes for antiswitches with antisense stems that contained complements to either GFP or YFP transcripts and the aptamer to either theophylline or tetracycline. These genes were then transformed into ''Saccharomyces cerevisiae''. Several experiments, including dose-response curves to the appropriate ligand and fluorescence measurements of cells when exposed to both the appropriate ligand and wrong ligand, demonstrated that these antiswitches effectively regulate translation of their specific target in response to only the correct ligand.

In the dose-response experiments for off-switches when enough ligand (~ 1 mM theophylline or tetracycline depending on the switch) was present, the inactive off-switches became active and relative GFP expression dropped to almost zero. These experimental results support the antiswitch technology being functional. As off-switches with either theophylline or tetracycline aptamers worked, the versatility of antiswitches is supported (figure 6).

[[Image:Offswitch_experiment.JPG]]

(Bayer and Smolke 2005 Permission Pending)

'''Figure 6:''' The red line represents the tetracycline controlled off-switch while blue line represents the theophylline controlled off-switch. When the concentration of ligand is high enough, enough off-switches are active and binding to mRNA to stop translation of the gene.

In the dose-response experiments for the on-switches when ~ 1 mM of theophylline was added to cells containing the inactive on-switch, the cells relative expression of GFP jumped from near zero to approximately 0.9. This data show that the on-switch is also functional (figure 7).

[[Image:Onswitch_experiment.JPG]]

(Bayer and Smolke 2005 Permission Pending)

'''Figure 7:''' The red line represents the on-switch while the blue Line represents the off-switch. When a high enough concentration of ligand is present, enough on-switches are activated and have thus released their target mRNA for detectable near normal translation to occur as measured by fluorescence. The off-switch follows the opposite path as shown in figure 6.

Finally, antiswitches in eukaryotes can be combined to regulate multiple genes for different conditions like the taRNA/crRNA system in prokaryotes. By simply switching the aptamer and antisense stem targeting sequence, the switch now controls a different gene by a different stimulus. Off-switch experiments using a theophylline aptamer with a GFP targeting antisense stem and a tetracycline aptamer with a YFP targeting antisense stem showed that the switches regulate the genes independently and can be used in combination to create complex regulatory mechanisms (figure 8).

[[Image:Combinatorial.JPG]]

(Bayer and Smolke 2005 Permission Pending)

'''Figure 8:''' a) On the left, theophylline binds to the antiswitch's aptamer and activates the off-switch, which then binds to the GFP transcript and prevents translation. On the right, tetracycline binds to the antiswitch's aptamer and activates the off-switch, which then binds to the YFP transcript and prevents translation. If only one ligand is present (either theophylline or tetracycline), only one gene's transcripts (either GFP or YFP) are not translated. b) When no theophylline or tetracycline is present, both GFP and YFP are expressed at near normal levels. When only tetracycline is present, GFP is expressed at near normal levels while YFP expression is close to zero. When only theophylline is present, YFP is expressed at near normal levels while GFP expression is close to zero. When both tetracycline and theophylline are present, GFP and YFP expression are both close to zero.

==Modularity==

While antiswitches have many advantages, they do lack modularity, the ability to be integrated with any gene without redesigning a new sequence specific part (Isaacs et al. 2004). If you want to regulate a new gene, you have to synthesize a whole new antiswitch because the antisense stem is specific to a particular gene and the aptamer stem is specific to a particular antisense stem.

==References==

Bayer TS and Smolke CD. Programmable ligand-controlled riboregulators of eukaryotic gene expression. Nat Biotechnol. (2005) 3:337-43.

Isaacs FJ, et al. Engineered riboregulators enable post-transcriptional control of gene expression. Nat Biotechnol. (2004) 22:841-47.

Win MN, Smolke CD (2007). A modular and extensible RNA-based gene-regulatory platform for engineering cellular function. PNAS 104(36):14283-8. Epub 2007 Aug 20. [http://www.pnas.org/cgi/content/abstract/104/36/14283 Abstract].

[[Post-transcriptional Regulation Technologies - Erin Zwack|Return to Post-transcriptional Regulation Technologies]]

Riboregulators

2007-12-06T20:11:37Z

Erzwack: /* Modularity */

All information on riboregulators came from Isaacs et al. 2004.

After observing the many and varied naturally occuring post-transcriptional, regulatory RNA systems, Isaacs et al. designed and engineered a modular synthetic system where RNA turns on and off gene expression by controlling translation. The modularity of their system allows any gene to be regulated instead of only a specific gene to which the riboregulator is targeted.

==Design==

The design itself has two components: a short cis-repressed RNA sequence (crRNA) that is inserted upstream of the gene and a transactivating RNA sequence (taRNA) that targets the crRNA and is free floating. The crRNA sequence contains two fundamental components: the complement of the ribosomal binding site (RBS) and a pyrimidine-uracil-nucleotide-purine (YUNR) sequence (figure 9). When not interacting with the taRNA, the complement of the RBS binds to the RBS, causing the crRNA to loop and block the ribosome's access to the RBS. When the RBS is blocked, translation does not occur; the gene expression is off. The YUNR sequence has a complement on the taRNA. When the taRNA finds a crRNA, the interaction with the YUNR sequence begins pulling the crRNA off the RBS (figure 11).

[[Image:CrRNA.JPG ]]

(Isaacs et al. 2004)

'''Figure 9:''' Cis repressing RNA, crRNA, sequence is inserted upstream of the RBS. Part of the crRNA (red) complements the RBS and a few bases on either side. This section of the crRNA is not an exact complement; thus, the crRNA can be peeled off the RBS by the trans activating RNA, taRNA. The complementary sequence to YUNR is found in the ta-RNA and begins peeling off the crRNA by binding to it (figure 11).

The taRNA is free floating and regulated by an inducible promoter. The inducible promoter allows the researcher to determine under what conditions translation will be allowed. Besides the complement to the YUNR sequence, the taRNA contains a section of high complementarity to the section of the crRNA that folds over to cover the RBS. This complementary sequence allows the taRNA to keep the crRNA sequestered from the RBS (figure 10).

[[Image:TaRNA.JPG]]

(Isaacs et al. 2004)

'''Figure 10:''' taRNA when not in contact with crRNA sequesters the complement to the crRNA as it is extremely close to the RBS sequence. If the complement to the crRNA was not sequestered until the taRNA comes in contact with the YUNR sequence, ribosomes could potentially bind to the taRNA and decrease the efficiency of translation in the cell. When the YUNR complement binds to the YUNR sequence in the cr-RNA, the binding of the crRNA to the rest of its complement in the taRNA begins, and the crRNA is pulled off the RBS (figure 11).

To recap, gene expression is off when there is crRNA upstream of the gene but no taRNA is in the system. In the presence of taRNA, gene expression is turned back on.

[[Image:Riboregulator system.JPG ]]

(Isaacs et al. 2004)

'''Figure 11:''' a. the crRNA is bound to the RBS and blocks ribosomes from translating the mRNA into protein. b. the taRNA comes in contact with the YUNR sequence on the crRNA and begins to bind to the rest of the crRNA. c. the taRNA fully bound to the crRNA and peeled the crRNA off the RBS. The RBS is now free for the ribosome to bind to and begin translation.

==From Concept to Wet Lab==

In wet lab, the crRNA sequence was placed in front of the GFP gene and introduced to cells. Using flow cytometry, Isaacs et al. measured fluorescence of cells that had just the GFP gene, the GFP gene with crRNA upstream, the GFP gene with crRNA upstream and taRNA, and no GFP at all. The crRNA decreased fluorescence to near basal levels. When taRNA was present in the system, fluorescence increased by approximately a power of ten. While the fluorescence did not equal fluorescence of cells with just GFP, the repression of gene expression with crRNA and return of expression with taRNA are strong enough to suggest the riboregulator system works (figure 12).

[[Image:experimental_taRNA.JPG]]

(Isaacs et al. 2004)

'''Figure 12:''' The black curve represents fluorescence in cells that do not have a GFP gene (ie. the cells natural autofluorescence). The red curve represents fluorescence in cells that have GFP with crRNA upstream. The green curve represents fluorescence in cells that have GFP with crRNA upstream and produce the taRNA. The blue curve represents the fluorescence of cells that have normal GFP minus the cells' autofluorescence.

Finally, riboregulators can control different genes in response to different stimuli by using different crRNA/taRNA pairs as the pairs are specific to each other (figure 13).

[[Image: Specific.JPG]]

(Isaacs et al. 2004)

'''Figure 13:''' The graph shows both GFP fluorescence (black and white bars) when the taRNA promoter, pBad, is off (- arabinose) and on (+ arabinose). All data is normalized to + arabinose GFP and RNA levels. The low level GFP fluorescence when no arabinose is present shows that the efficiency of crRNA is not 100% but is still high. As high GFP fluorescence is seen only when high amounts of the matching taRNA is present and not simply when any taRNA variant is present, this data shows that riboregulator pairs are specific and multiple pairs can be used to regulate multiple genes without fear that any taRNA produced would activate all the RNAs and not just its target RNA.

==Modularity==

While all of these experiments were done with the GFP gene, Isaacs et al. designed their riboregulator to be modular, capable of being used with any gene (Isaacs et al. 2004). As the taRNA simply targets the crRNA and the crRNA can be placed in front of any gene, it can be considered modular. The above experiments were done with pBad and pLac controlling the production of taRNA. As the system worked when the taRNA was under control of either promoter and the crRNA can be inserted upstream of any gene, this system is basically considered modular.

The only caveat is that the crRNA construct added to the gene will need to contain the RBS unless the gene's RBS is close enough to the complement to bind to it. Even small changes to a ribosomal binding site can can change the transcription rate of genes (Gardner et al. 2000). If the original RBS is not close enough to the complement in the crRNA and you desire to keep the original transcriptional rate and level, you would have to redesign the crRNA and the taRNA as the complement to the RBS in the crRNA and the complement to the crRNA in the taRNA would need to be different.

==Further Work==

As mentioned in the overview, using regulatory proteins or inducible promoters limits the number of stimuli (molecules) that can be used to determine when expression should occur. Generating taRNA that is ligand controlled like an antiswitch or riboswitch would provide a greater versatility for riboregulator use. Ligand controlled riboregulators may also be more effective as control by a ligand may decrease leaky activity, which occurs with promoters as they always allow for some basal level of transcription. Isaacs has continued working to engineer a ligand controlled version of the riboregulator.

==References==

Gardner, T.S., Cantor, C.R., and Collins, J.J. Construction of a genetic toggle switch in Eschreichia coli. Nature (2000) 403: 339-342.

Isaacs FJ, et al. Engineered riboregulators enable post-transcriptional control of gene expression. Nat Biotechnol. (2004) 22:841-47.

[[Post-transcriptional Regulation Technologies - Erin Zwack|Return to Post-transcriptional Regulation Technologies]]

Riboregulators

2007-12-06T20:10:28Z

Erzwack: /* From Concept to Wet Lab */

All information on riboregulators came from Isaacs et al. 2004.

After observing the many and varied naturally occuring post-transcriptional, regulatory RNA systems, Isaacs et al. designed and engineered a modular synthetic system where RNA turns on and off gene expression by controlling translation. The modularity of their system allows any gene to be regulated instead of only a specific gene to which the riboregulator is targeted.

==Design==

The design itself has two components: a short cis-repressed RNA sequence (crRNA) that is inserted upstream of the gene and a transactivating RNA sequence (taRNA) that targets the crRNA and is free floating. The crRNA sequence contains two fundamental components: the complement of the ribosomal binding site (RBS) and a pyrimidine-uracil-nucleotide-purine (YUNR) sequence (figure 9). When not interacting with the taRNA, the complement of the RBS binds to the RBS, causing the crRNA to loop and block the ribosome's access to the RBS. When the RBS is blocked, translation does not occur; the gene expression is off. The YUNR sequence has a complement on the taRNA. When the taRNA finds a crRNA, the interaction with the YUNR sequence begins pulling the crRNA off the RBS (figure 11).

[[Image:CrRNA.JPG ]]

(Isaacs et al. 2004)

'''Figure 9:''' Cis repressing RNA, crRNA, sequence is inserted upstream of the RBS. Part of the crRNA (red) complements the RBS and a few bases on either side. This section of the crRNA is not an exact complement; thus, the crRNA can be peeled off the RBS by the trans activating RNA, taRNA. The complementary sequence to YUNR is found in the ta-RNA and begins peeling off the crRNA by binding to it (figure 11).

The taRNA is free floating and regulated by an inducible promoter. The inducible promoter allows the researcher to determine under what conditions translation will be allowed. Besides the complement to the YUNR sequence, the taRNA contains a section of high complementarity to the section of the crRNA that folds over to cover the RBS. This complementary sequence allows the taRNA to keep the crRNA sequestered from the RBS (figure 10).

[[Image:TaRNA.JPG]]

(Isaacs et al. 2004)

'''Figure 10:''' taRNA when not in contact with crRNA sequesters the complement to the crRNA as it is extremely close to the RBS sequence. If the complement to the crRNA was not sequestered until the taRNA comes in contact with the YUNR sequence, ribosomes could potentially bind to the taRNA and decrease the efficiency of translation in the cell. When the YUNR complement binds to the YUNR sequence in the cr-RNA, the binding of the crRNA to the rest of its complement in the taRNA begins, and the crRNA is pulled off the RBS (figure 11).

To recap, gene expression is off when there is crRNA upstream of the gene but no taRNA is in the system. In the presence of taRNA, gene expression is turned back on.

[[Image:Riboregulator system.JPG ]]

(Isaacs et al. 2004)

'''Figure 11:''' a. the crRNA is bound to the RBS and blocks ribosomes from translating the mRNA into protein. b. the taRNA comes in contact with the YUNR sequence on the crRNA and begins to bind to the rest of the crRNA. c. the taRNA fully bound to the crRNA and peeled the crRNA off the RBS. The RBS is now free for the ribosome to bind to and begin translation.

==From Concept to Wet Lab==

In wet lab, the crRNA sequence was placed in front of the GFP gene and introduced to cells. Using flow cytometry, Isaacs et al. measured fluorescence of cells that had just the GFP gene, the GFP gene with crRNA upstream, the GFP gene with crRNA upstream and taRNA, and no GFP at all. The crRNA decreased fluorescence to near basal levels. When taRNA was present in the system, fluorescence increased by approximately a power of ten. While the fluorescence did not equal fluorescence of cells with just GFP, the repression of gene expression with crRNA and return of expression with taRNA are strong enough to suggest the riboregulator system works (figure 12).

[[Image:experimental_taRNA.JPG]]

(Isaacs et al. 2004)

'''Figure 12:''' The black curve represents fluorescence in cells that do not have a GFP gene (ie. the cells natural autofluorescence). The red curve represents fluorescence in cells that have GFP with crRNA upstream. The green curve represents fluorescence in cells that have GFP with crRNA upstream and produce the taRNA. The blue curve represents the fluorescence of cells that have normal GFP minus the cells' autofluorescence.

Finally, riboregulators can control different genes in response to different stimuli by using different crRNA/taRNA pairs as the pairs are specific to each other (figure 13).

[[Image: Specific.JPG]]

(Isaacs et al. 2004)

'''Figure 13:''' The graph shows both GFP fluorescence (black and white bars) when the taRNA promoter, pBad, is off (- arabinose) and on (+ arabinose). All data is normalized to + arabinose GFP and RNA levels. The low level GFP fluorescence when no arabinose is present shows that the efficiency of crRNA is not 100% but is still high. As high GFP fluorescence is seen only when high amounts of the matching taRNA is present and not simply when any taRNA variant is present, this data shows that riboregulator pairs are specific and multiple pairs can be used to regulate multiple genes without fear that any taRNA produced would activate all the RNAs and not just its target RNA.

==Modularity==

While all of these experiments were done with the GFP gene, Isaacs et al. designed their riboregulator to be modular, capable of being used with any gene (Isaacs et al. 2004). As the taRNA simply targets the crRNA and the crRNA can be placed in front of any gene, it can be considered modular. The only caveat is that the crRNA construct added to the gene will need to contain the RBS unless the gene's RBS is close enough to the complement to bind to it. Even small changes to a ribosomal binding site can can change the transcription rate of genes (Gardner et al. 2000). If the original RBS is not close enough to the complement in the crRNA and you desire to keep the original transcriptional rate and level, you would have to redesign the crRNA and the taRNA as the complement to the RBS in the crRNA and the complement to the crRNA in the taRNA would need to be different.

==Further Work==

As mentioned in the overview, using regulatory proteins or inducible promoters limits the number of stimuli (molecules) that can be used to determine when expression should occur. Generating taRNA that is ligand controlled like an antiswitch or riboswitch would provide a greater versatility for riboregulator use. Ligand controlled riboregulators may also be more effective as control by a ligand may decrease leaky activity, which occurs with promoters as they always allow for some basal level of transcription. Isaacs has continued working to engineer a ligand controlled version of the riboregulator.

==References==

Gardner, T.S., Cantor, C.R., and Collins, J.J. Construction of a genetic toggle switch in Eschreichia coli. Nature (2000) 403: 339-342.

Isaacs FJ, et al. Engineered riboregulators enable post-transcriptional control of gene expression. Nat Biotechnol. (2004) 22:841-47.

[[Post-transcriptional Regulation Technologies - Erin Zwack|Return to Post-transcriptional Regulation Technologies]]

Riboregulators

2007-12-06T20:09:55Z

Erzwack: /* From Concept to Wet Lab */

All information on riboregulators came from Isaacs et al. 2004.

After observing the many and varied naturally occuring post-transcriptional, regulatory RNA systems, Isaacs et al. designed and engineered a modular synthetic system where RNA turns on and off gene expression by controlling translation. The modularity of their system allows any gene to be regulated instead of only a specific gene to which the riboregulator is targeted.

==Design==

The design itself has two components: a short cis-repressed RNA sequence (crRNA) that is inserted upstream of the gene and a transactivating RNA sequence (taRNA) that targets the crRNA and is free floating. The crRNA sequence contains two fundamental components: the complement of the ribosomal binding site (RBS) and a pyrimidine-uracil-nucleotide-purine (YUNR) sequence (figure 9). When not interacting with the taRNA, the complement of the RBS binds to the RBS, causing the crRNA to loop and block the ribosome's access to the RBS. When the RBS is blocked, translation does not occur; the gene expression is off. The YUNR sequence has a complement on the taRNA. When the taRNA finds a crRNA, the interaction with the YUNR sequence begins pulling the crRNA off the RBS (figure 11).

[[Image:CrRNA.JPG ]]

(Isaacs et al. 2004)

'''Figure 9:''' Cis repressing RNA, crRNA, sequence is inserted upstream of the RBS. Part of the crRNA (red) complements the RBS and a few bases on either side. This section of the crRNA is not an exact complement; thus, the crRNA can be peeled off the RBS by the trans activating RNA, taRNA. The complementary sequence to YUNR is found in the ta-RNA and begins peeling off the crRNA by binding to it (figure 11).

The taRNA is free floating and regulated by an inducible promoter. The inducible promoter allows the researcher to determine under what conditions translation will be allowed. Besides the complement to the YUNR sequence, the taRNA contains a section of high complementarity to the section of the crRNA that folds over to cover the RBS. This complementary sequence allows the taRNA to keep the crRNA sequestered from the RBS (figure 10).

[[Image:TaRNA.JPG]]

(Isaacs et al. 2004)

'''Figure 10:''' taRNA when not in contact with crRNA sequesters the complement to the crRNA as it is extremely close to the RBS sequence. If the complement to the crRNA was not sequestered until the taRNA comes in contact with the YUNR sequence, ribosomes could potentially bind to the taRNA and decrease the efficiency of translation in the cell. When the YUNR complement binds to the YUNR sequence in the cr-RNA, the binding of the crRNA to the rest of its complement in the taRNA begins, and the crRNA is pulled off the RBS (figure 11).

To recap, gene expression is off when there is crRNA upstream of the gene but no taRNA is in the system. In the presence of taRNA, gene expression is turned back on.

[[Image:Riboregulator system.JPG ]]

(Isaacs et al. 2004)

'''Figure 11:''' a. the crRNA is bound to the RBS and blocks ribosomes from translating the mRNA into protein. b. the taRNA comes in contact with the YUNR sequence on the crRNA and begins to bind to the rest of the crRNA. c. the taRNA fully bound to the crRNA and peeled the crRNA off the RBS. The RBS is now free for the ribosome to bind to and begin translation.

==From Concept to Wet Lab==

In wet lab, the crRNA sequence was placed in front of the GFP gene and introduced to cells. Using flow cytometry, Isaacs et al. measured fluorescence of cells that had just the GFP gene, the GFP gene with crRNA upstream, the GFP gene with crRNA upstream and taRNA, and no GFP at all. The crRNA decreased fluorescence to near basal levels. When taRNA was present in the system, fluorescence increased by approximately a power of ten. While the fluorescence did not equal fluorescence of cells with just GFP, the repression of gene expression with crRNA and return of expression with taRNA are strong enough to suggest the riboregulator system works (figure 12).

[[Image:experimental_taRNA.JPG]]

(Isaacs et al. 2004)

'''Figure 12:''' The black curve represents fluorescence in cells that do not have a GFP gene (ie. the cells natural autofluorescence). The red curve represents fluorescence in cells that have GFP with crRNA upstream. The green curve represents fluorescence in cells that have GFP with crRNA upstream and produce the taRNA. The blue curve represents the fluorescence of cells that have normal GFP minus the cells' autofluorescence.

These experiments were done with pBad and pLac controlling the production of taRNA. As the system worked when the taRNA was under control of either promoter and the crRNA can be inserted upstream of any gene, this system is considered modular.

Finally, riboregulators can control different genes in response to different stimuli by using different crRNA/taRNA pairs as the pairs are specific to each other (figure 13).

[[Image: Specific.JPG]]

(Isaacs et al. 2004)

'''Figure 13:''' The graph shows both GFP fluorescence (black and white bars) when the taRNA promoter, pBad, is off (- arabinose) and on (+ arabinose). All data is normalized to + arabinose GFP and RNA levels. The low level GFP fluorescence when no arabinose is present shows that the efficiency of crRNA is not 100% but is still high. As high GFP fluorescence is seen only when high amounts of the matching taRNA is present and not simply when any taRNA variant is present, this data shows that riboregulator pairs are specific and multiple pairs can be used to regulate multiple genes without fear that any taRNA produced would activate all the RNAs and not just its target RNA.

==Modularity==

While all of these experiments were done with the GFP gene, Isaacs et al. designed their riboregulator to be modular, capable of being used with any gene (Isaacs et al. 2004). As the taRNA simply targets the crRNA and the crRNA can be placed in front of any gene, it can be considered modular. The only caveat is that the crRNA construct added to the gene will need to contain the RBS unless the gene's RBS is close enough to the complement to bind to it. Even small changes to a ribosomal binding site can can change the transcription rate of genes (Gardner et al. 2000). If the original RBS is not close enough to the complement in the crRNA and you desire to keep the original transcriptional rate and level, you would have to redesign the crRNA and the taRNA as the complement to the RBS in the crRNA and the complement to the crRNA in the taRNA would need to be different.

==Further Work==

As mentioned in the overview, using regulatory proteins or inducible promoters limits the number of stimuli (molecules) that can be used to determine when expression should occur. Generating taRNA that is ligand controlled like an antiswitch or riboswitch would provide a greater versatility for riboregulator use. Ligand controlled riboregulators may also be more effective as control by a ligand may decrease leaky activity, which occurs with promoters as they always allow for some basal level of transcription. Isaacs has continued working to engineer a ligand controlled version of the riboregulator.

==References==

Gardner, T.S., Cantor, C.R., and Collins, J.J. Construction of a genetic toggle switch in Eschreichia coli. Nature (2000) 403: 339-342.

Isaacs FJ, et al. Engineered riboregulators enable post-transcriptional control of gene expression. Nat Biotechnol. (2004) 22:841-47.

[[Post-transcriptional Regulation Technologies - Erin Zwack|Return to Post-transcriptional Regulation Technologies]]

Riboregulators

2007-12-06T20:05:55Z

Erzwack: /* Design */

All information on riboregulators came from Isaacs et al. 2004.

After observing the many and varied naturally occuring post-transcriptional, regulatory RNA systems, Isaacs et al. designed and engineered a modular synthetic system where RNA turns on and off gene expression by controlling translation. The modularity of their system allows any gene to be regulated instead of only a specific gene to which the riboregulator is targeted.

==Design==

The design itself has two components: a short cis-repressed RNA sequence (crRNA) that is inserted upstream of the gene and a transactivating RNA sequence (taRNA) that targets the crRNA and is free floating. The crRNA sequence contains two fundamental components: the complement of the ribosomal binding site (RBS) and a pyrimidine-uracil-nucleotide-purine (YUNR) sequence (figure 9). When not interacting with the taRNA, the complement of the RBS binds to the RBS, causing the crRNA to loop and block the ribosome's access to the RBS. When the RBS is blocked, translation does not occur; the gene expression is off. The YUNR sequence has a complement on the taRNA. When the taRNA finds a crRNA, the interaction with the YUNR sequence begins pulling the crRNA off the RBS (figure 11).

[[Image:CrRNA.JPG ]]

(Isaacs et al. 2004)

'''Figure 9:''' Cis repressing RNA, crRNA, sequence is inserted upstream of the RBS. Part of the crRNA (red) complements the RBS and a few bases on either side. This section of the crRNA is not an exact complement; thus, the crRNA can be peeled off the RBS by the trans activating RNA, taRNA. The complementary sequence to YUNR is found in the ta-RNA and begins peeling off the crRNA by binding to it (figure 11).

The taRNA is free floating and regulated by an inducible promoter. The inducible promoter allows the researcher to determine under what conditions translation will be allowed. Besides the complement to the YUNR sequence, the taRNA contains a section of high complementarity to the section of the crRNA that folds over to cover the RBS. This complementary sequence allows the taRNA to keep the crRNA sequestered from the RBS (figure 10).

[[Image:TaRNA.JPG]]

(Isaacs et al. 2004)

'''Figure 10:''' taRNA when not in contact with crRNA sequesters the complement to the crRNA as it is extremely close to the RBS sequence. If the complement to the crRNA was not sequestered until the taRNA comes in contact with the YUNR sequence, ribosomes could potentially bind to the taRNA and decrease the efficiency of translation in the cell. When the YUNR complement binds to the YUNR sequence in the cr-RNA, the binding of the crRNA to the rest of its complement in the taRNA begins, and the crRNA is pulled off the RBS (figure 11).

To recap, gene expression is off when there is crRNA upstream of the gene but no taRNA is in the system. In the presence of taRNA, gene expression is turned back on.

[[Image:Riboregulator system.JPG ]]

(Isaacs et al. 2004)

'''Figure 11:''' a. the crRNA is bound to the RBS and blocks ribosomes from translating the mRNA into protein. b. the taRNA comes in contact with the YUNR sequence on the crRNA and begins to bind to the rest of the crRNA. c. the taRNA fully bound to the crRNA and peeled the crRNA off the RBS. The RBS is now free for the ribosome to bind to and begin translation.

==From Concept to Wet Lab==

In wet lab, the crRNA sequence was placed in front of the GFP gene and introduced to cells. Using flow cytometry, Isaacs et al. measured fluorescence of cells that had just the GFP gene, the GFP gene with crRNA upstream, the GFP gene with crRNA upstream and taRNA, and no GFP at all. The crRNA decreased fluorescence to near basal levels. When taRNA was present in the system, fluorescence increased by approximately a power of ten. While the fluorescence did not equal fluorescence of cells with just GFP, the repression of gene expression with crRNA and return of expression with taRNA are strong enough to suggest the riboregulator system works.

[[Image:experimental_taRNA.JPG]]

(Isaacs et al. 2004)

'''Figure 12:''' The black curve represents fluoresnce in cells that do not have a GFP gene (ie. the cells natural autofluoresence). The red curve represents fluoresence in cells that have GFP with crRNA upstream. The green curve represents fluoresence in cells that have GFP with crRNA upstream and produce the taRNA. The blue curve represents the fluorescence of cells that have normal GFP minus the cells' autofluoresence.

These experiments were done with pBad and pLac controlling the production of taRNA. As the system worked when the taRNA was under control of either promoter and the crRNA can be inserted upstream of any gene, this system is considered modular.

Finally, riboregulators can control different genes in response to different stinuli by using different crRNA/taRNA pairs as the pairs are specific to each other.

[[Image: Specific.JPG]]

(Isaacs et al. 2004)

'''Figure 13:''' The graph shows both GFP fluorescence (black and white bars) when the ta-RNA promoter, pBad, is off (- arabinose) and on (+ arabinose). All data is normalized to + arabinose GFP an RNA levels. The low level GFP fluorescence when no arabinose is present shows that the efficiency of cr-RNA is not 100% but is still high. As high GFP fluorescence is seen only when high amounts of the matching ta-RNA is present and not simply when any ta-RNA variant is present, this data shows that riboregulator pairs are specific and multiple pairs can be used to regulate multiple genes without fear that any ta-RNA produced would activated all the RNAs and not just its target RNA.

==Modularity==

While all of these experiments were done with the GFP gene, Isaacs et al. designed their riboregulator to be modular, capable of being used with any gene (Isaacs et al. 2004). As the taRNA simply targets the crRNA and the crRNA can be placed in front of any gene, it can be considered modular. The only caveat is that the crRNA construct added to the gene will need to contain the RBS unless the gene's RBS is close enough to the complement to bind to it. Even small changes to a ribosomal binding site can can change the transcription rate of genes (Gardner et al. 2000). If the original RBS is not close enough to the complement in the crRNA and you desire to keep the original transcriptional rate and level, you would have to redesign the crRNA and the taRNA as the complement to the RBS in the crRNA and the complement to the crRNA in the taRNA would need to be different.

==Further Work==

As mentioned in the overview, using regulatory proteins or inducible promoters limits the number of stimuli (molecules) that can be used to determine when expression should occur. Generating taRNA that is ligand controlled like an antiswitch or riboswitch would provide a greater versatility for riboregulator use. Ligand controlled riboregulators may also be more effective as control by a ligand may decrease leaky activity, which occurs with promoters as they always allow for some basal level of transcription. Isaacs has continued working to engineer a ligand controlled version of the riboregulator.

==References==

Gardner, T.S., Cantor, C.R., and Collins, J.J. Construction of a genetic toggle switch in Eschreichia coli. Nature (2000) 403: 339-342.

Isaacs FJ, et al. Engineered riboregulators enable post-transcriptional control of gene expression. Nat Biotechnol. (2004) 22:841-47.

[[Post-transcriptional Regulation Technologies - Erin Zwack|Return to Post-transcriptional Regulation Technologies]]

Riboregulators

2007-12-06T19:59:35Z

Erzwack: /* Design */

All information on riboregulators came from Isaacs et al. 2004.

After observing the many and varied naturally occuring post-transcriptional, regulatory RNA systems, Isaacs et al. designed and engineered a modular synthetic system where RNA turns on and off gene expression by controlling translation. The modularity of their system allows any gene to be regulated instead of only a specific gene to which the riboregulator is targeted.

==Design==

The design itself has two components: a short cis-repressed RNA sequence (crRNA) that is inserted upstream of the gene and a transactivating RNA sequence (taRNA) that targets the crRNA and is free floating. The crRNA sequence contains two fundamental components: the complement of the ribosomal binding site (RBS) and a pyrimidine-uracil-nucleotide-purine (YUNR) sequence (figure 9). When not interacting with the taRNA, the complement of the RBS binds to the RBS, causing the crRNA to loop and block the ribosome's access to the RBS. When the RBS is blocked, translation does not occur; the gene expression is off. The YUNR sequence has a complement on the taRNA. When the taRNA finds a crRNA, the interaction with the YUNR sequence begins pulling the crRNA off the RBS (figure 11).

[[Image:CrRNA.JPG ]]

(Isaacs et al. 2004)

'''Figure 9:''' Cis repressing RNA, crRNA, sequence is inserted upstream of the RBS. Part of the crRNA (red) complements the RBS and a few bases on either side. This section of the crRNA is not an exact complement; thus, the crRNA can be peeled off the RBS by the trans activating RNA, taRNA. The complementary sequence to YUNR is found in the ta-RNA and begins peeling off the crRNA by binding to it (figure 11).

The taRNA is free floating and regulated by an inducible promoter. The inducible promoter allows the researcher to determine under what conditions translation will be allowed. Besides the complement to the YUNR sequence, the taRNA contains a section of high complementarity to section of the crRNA that folds over to cover the RBS, which allows the taRNA to keep the crRNA sequestered from the RBS (figure 10).

[[Image:TaRNA.JPG]]

(Isaacs et al. 2004)

'''Figure 10:''' taRNA when not in contact with crRNA sequesters the complement to the crRNA as it is extremely close to the RBS sequence. When the YUNR complement binds to the YUNR sequence in the cr-RNA, the binding of the crRNA to the rest of its complement in the taRNA begins, and the crRNA is pulled off the RBS (figure 11).

To recap, gene expression is off when there is crRNA upstream of the gene but no taRNA is in the system. In the presence of taRNA, gene expression is turned back on.

[[Image:Riboregulator system.JPG ]]

(Isaacs et al. 2004)

'''Figure 11:''' In a. the crRNA is bound to the RBS and blocks ribosomes from translating the mRNA into protein. In b. the ta-RNA comes in contact with the YUNR sequence on the crRNA and begins to bind to the rest of the cr-RNA. In c. the taRNA fully bound to the crRNA and peeled the crRNA off the RBS. The RBS is now free for the ribosome to bind to and begin translation.

==From Concept to Wet Lab==

In wet lab, the crRNA sequence was placed in front of the GFP gene and introduced to cells. Using flow cytometry, Isaacs et al. measured fluorescence of cells that had just the GFP gene, the GFP gene with crRNA upstream, the GFP gene with crRNA upstream and taRNA, and no GFP at all. The crRNA decreased fluorescence to near basal levels. When taRNA was present in the system, fluorescence increased by approximately a power of ten. While the fluorescence did not equal fluorescence of cells with just GFP, the repression of gene expression with crRNA and return of expression with taRNA are strong enough to suggest the riboregulator system works.

[[Image:experimental_taRNA.JPG]]

(Isaacs et al. 2004)

'''Figure 12:''' The black curve represents fluoresnce in cells that do not have a GFP gene (ie. the cells natural autofluoresence). The red curve represents fluoresence in cells that have GFP with crRNA upstream. The green curve represents fluoresence in cells that have GFP with crRNA upstream and produce the taRNA. The blue curve represents the fluorescence of cells that have normal GFP minus the cells' autofluoresence.

These experiments were done with pBad and pLac controlling the production of taRNA. As the system worked when the taRNA was under control of either promoter and the crRNA can be inserted upstream of any gene, this system is considered modular.

Finally, riboregulators can control different genes in response to different stinuli by using different crRNA/taRNA pairs as the pairs are specific to each other.

[[Image: Specific.JPG]]

(Isaacs et al. 2004)

'''Figure 13:''' The graph shows both GFP fluorescence (black and white bars) when the ta-RNA promoter, pBad, is off (- arabinose) and on (+ arabinose). All data is normalized to + arabinose GFP an RNA levels. The low level GFP fluorescence when no arabinose is present shows that the efficiency of cr-RNA is not 100% but is still high. As high GFP fluorescence is seen only when high amounts of the matching ta-RNA is present and not simply when any ta-RNA variant is present, this data shows that riboregulator pairs are specific and multiple pairs can be used to regulate multiple genes without fear that any ta-RNA produced would activated all the RNAs and not just its target RNA.

==Modularity==

While all of these experiments were done with the GFP gene, Isaacs et al. designed their riboregulator to be modular, capable of being used with any gene (Isaacs et al. 2004). As the taRNA simply targets the crRNA and the crRNA can be placed in front of any gene, it can be considered modular. The only caveat is that the crRNA construct added to the gene will need to contain the RBS unless the gene's RBS is close enough to the complement to bind to it. Even small changes to a ribosomal binding site can can change the transcription rate of genes (Gardner et al. 2000). If the original RBS is not close enough to the complement in the crRNA and you desire to keep the original transcriptional rate and level, you would have to redesign the crRNA and the taRNA as the complement to the RBS in the crRNA and the complement to the crRNA in the taRNA would need to be different.

==Further Work==

As mentioned in the overview, using regulatory proteins or inducible promoters limits the number of stimuli (molecules) that can be used to determine when expression should occur. Generating taRNA that is ligand controlled like an antiswitch or riboswitch would provide a greater versatility for riboregulator use. Ligand controlled riboregulators may also be more effective as control by a ligand may decrease leaky activity, which occurs with promoters as they always allow for some basal level of transcription. Isaacs has continued working to engineer a ligand controlled version of the riboregulator.

==References==

Gardner, T.S., Cantor, C.R., and Collins, J.J. Construction of a genetic toggle switch in Eschreichia coli. Nature (2000) 403: 339-342.

Isaacs FJ, et al. Engineered riboregulators enable post-transcriptional control of gene expression. Nat Biotechnol. (2004) 22:841-47.

[[Post-transcriptional Regulation Technologies - Erin Zwack|Return to Post-transcriptional Regulation Technologies]]

Antiswitches

2007-12-06T19:54:50Z

Erzwack: /* From Concept to Wet Lab */

All information about antiswitches came from Bayer and Smolke (2005).

Antiswitches, trans-RNA molecules that regulate translation of mRNA based on the presence or absence of specific ligands, were first developed by Smolke and Bayer (2005) in ''Saccharomyces cerevisiae''. Two types of antiswitches were engineered: on-switches and off-switches. On-switches turn on gene expression in the presence of the ligand while off-switches turn off gene expression in the presence of the ligand. Control by the ligand allows researchers to regulate pathways’ protein production with less leakiness, low level transcription even when “off”, than using a specific promoter.

==Design==

Antiswitches are made of an aptamer and two stems: the aptamer stem and the antisense stem (figure 3).

[[Image:Switch.JPG]]

(Bayer and Smolke 2005 Permission Pending)

'''Figure 3:''' The antisense stem can bind to itself (duplex). The antisense stem also contains the complement to the RNA that a researcher desires to regulate. The aptamer stem swings either toward the antisense stem and disrupts the duplex or swings away from the antisense stem and allows the antisense stem to duplex with itself depending on whether the aptamer is bound to its ligand.

The aptamer is the sequence that binds the ligand and causes a conformational (shape) change of the antiswitch molecule. Aptamers can be highly specific for their particular molecules. The theophylline aptamer used by Smolke and Bayer can distinguish between caffeine and theophylline, which differ by a single methyl (figure 4).

[[Image:Caffeine.JPG]]

'''Figure 4.''' Caffeine (a) and Theophylline (b) differ by one methyl group (circled in red).

While Smolke and Bayer mainly used the aptamer for the ligand theophylline, aptamers for many other molecules are now being generated using rational design (Win and Smolke 2007). The large number of possible aptamers provides versatility in what will control gene expression.

The antisense stem contains a sequence that complements a targeted RNA transcript and a second sequence that sequesters this complementary sequence to keep it from binding the transcript. When the antisense stem is not duplexed with itself, it prevents translation by binding to the complementary mRNA (figure 5).

The aptamer stem is a short sequence that complements a portion of the sequestering sequence of the antisense stem. In an off-switch, the aptamer stem swings towards the antisense stem and displaces the portion that complements the targeted transcript when the liand binds to the aptamer (figure 5). In an on-switch, the aptamer swings away from the antisense stem when the ligand binds to the aptamer; thus, the antisense stem can duplex and is no longer free to bind to the transcript.

[[Image:ANTI.jpg]]

(Bayer and Smolke 2005 Permission Pending)

'''Figure 5:''' a) An inactive off-switch. The antisense stem is duplexed with itself. b) The same switch after being activated by theophylline (blue ellipse). The antisense stem is duplexed with the mRNA; thus, translation is stopped.

==From Concept to Wet Lab==

Smolke and Bayer synthesized genes for antiswitches with antisense stems that contained complements to either GFP or YFP transcripts and the aptamer to either theophylline or tetracycline. These genes were then transformed into ''Saccharomyces cerevisiae''. Several experiments, including dose-response curves to the appropriate ligand and fluoresence measurements of cells when exposed to both the appropriate ligand and wrong ligand, demonstrated that these antiswitches effectively regulate translation of their specific target in response to only the correct ligand.

In the dose-response experiments for off-switches when enough ligand (~ 1 mM theophylline or tetracycline depending on the switch) was present, the inactive off-switches became active and relative GFP expression dropped to almost zero. These experimental results support the antiswitch technology being functional. As off-switches with either theophylline or tetracycline aptamers worked, the versatility of antiswitches is supported (figure 6).

[[Image:Offswitch_experiment.JPG]]

(Bayer and Smolke 2005 Permission Pending)

'''Figure 6:''' The red line represents the tetracycline controlled off-switch while blue line represents the theophylline controlled off-switch. When the concentration of ligand is high enough, enough off-switches are active and binding to mRNA to stop translation of the gene.

In the dose-response experiments for the on-switches when ~ 1 mM of theophylline was added to cells containing the inactive on-switch, the cells relative expression of GFP jumped from near zero to approximately 0.9. This data show that the on-switch is also functional (figure 7).

[[Image:Onswitch_experiment.JPG]]

(Bayer and Smolke 2005 Permission Pending)

'''Figure 7:''' The red line represents the on-switch while the blue Line represents the off-switch. When a high enough concentration of ligand is present, enough on-switches are activated and have thus released their target mRNA for detectable near normal translation to occur as measured by fluoresence. The off-switch follows the opposite path as shown in figure 6.

Finally, antiswitches in eukaryotes can be combined to regulate multiple genes for different conditions like the taRNA/crRNA system in prokaryotes. By simply switching the aptamer and antisense stem targeting sequence, the switch now controls a different gene by a different stimulus. Off-switch experiments using a theophylline aptamer with a GFP targeting antisense stem and a tetracycline aptamer with a YFP targeting antisense stem showed that the switches regulate the genes independently and can be used in combination to create complex regulatory mechanisms (figure 8).

[[Image:Combinatorial.JPG]]

(Bayer and Smolke 2005 Permission Pending)

'''Figure 8:''' a) On the left, theophylline binds to the antiswitch's aptamer and activates the off-switch, which then binds to the GFP transcript and prevents translation. On the right, tetracycline binds to the antiswitch's aptamer and activates the off-switch, which then binds to the YFP transcript and prevents translation. If only one ligand is present (either theophylline or tetracycline), only one gene's transcripts (either GFP or YFP) are not translated. b) When no theophylline or tetracycline is present, both GFP and YFP are expressed at near normal levels. When only tetracycline is present, GFP is expressed at near normal levels while YFP expression is close to zero. When only theophylline is present, YFP is expressed at near normal levels while GFP expression is close to zero. When both tetracycline and theophylline are present, GFP and YFP expression are both close to zero.

==Modularity==

While antiswitches have many advantages, they do lack modularity, the ability to be integrated with any gene without redesigning a new sequence specific part (Isaacs et al. 2004). If you want to regulate a new gene, you have to synthesize a whole new antiswitch because the antisense stem is specific to a particular gene and the aptamer stem is specific to a particular antisense stem.

==References==

Bayer TS and Smolke CD. Programmable ligand-controlled riboregulators of eukaryotic gene expression. Nat Biotechnol. (2005) 3:337-43.

Isaacs FJ, et al. Engineered riboregulators enable post-transcriptional control of gene expression. Nat Biotechnol. (2004) 22:841-47.

Win MN, Smolke CD (2007). A modular and extensible RNA-based gene-regulatory platform for engineering cellular function. PNAS 104(36):14283-8. Epub 2007 Aug 20. [http://www.pnas.org/cgi/content/abstract/104/36/14283 Abstract].

[[Post-transcriptional Regulation Technologies - Erin Zwack|Return to Post-transcriptional Regulation Technologies]]

Antiswitches

2007-12-06T19:47:04Z

Erzwack: /* From Concept to Wet Lab */

All information about antiswitches came from Bayer and Smolke (2005).

Antiswitches, trans-RNA molecules that regulate translation of mRNA based on the presence or absence of specific ligands, were first developed by Smolke and Bayer (2005) in ''Saccharomyces cerevisiae''. Two types of antiswitches were engineered: on-switches and off-switches. On-switches turn on gene expression in the presence of the ligand while off-switches turn off gene expression in the presence of the ligand. Control by the ligand allows researchers to regulate pathways’ protein production with less leakiness, low level transcription even when “off”, than using a specific promoter.

==Design==

Antiswitches are made of an aptamer and two stems: the aptamer stem and the antisense stem (figure 3).

[[Image:Switch.JPG]]

(Bayer and Smolke 2005 Permission Pending)

'''Figure 3:''' The antisense stem can bind to itself (duplex). The antisense stem also contains the complement to the RNA that a researcher desires to regulate. The aptamer stem swings either toward the antisense stem and disrupts the duplex or swings away from the antisense stem and allows the antisense stem to duplex with itself depending on whether the aptamer is bound to its ligand.

The aptamer is the sequence that binds the ligand and causes a conformational (shape) change of the antiswitch molecule. Aptamers can be highly specific for their particular molecules. The theophylline aptamer used by Smolke and Bayer can distinguish between caffeine and theophylline, which differ by a single methyl (figure 4).

[[Image:Caffeine.JPG]]

'''Figure 4.''' Caffeine (a) and Theophylline (b) differ by one methyl group (circled in red).

While Smolke and Bayer mainly used the aptamer for the ligand theophylline, aptamers for many other molecules are now being generated using rational design (Win and Smolke 2007). The large number of possible aptamers provides versatility in what will control gene expression.

The antisense stem contains a sequence that complements a targeted RNA transcript and a second sequence that sequesters this complementary sequence to keep it from binding the transcript. When the antisense stem is not duplexed with itself, it prevents translation by binding to the complementary mRNA (figure 5).

The aptamer stem is a short sequence that complements a portion of the sequestering sequence of the antisense stem. In an off-switch, the aptamer stem swings towards the antisense stem and displaces the portion that complements the targeted transcript when the liand binds to the aptamer (figure 5). In an on-switch, the aptamer swings away from the antisense stem when the ligand binds to the aptamer; thus, the antisense stem can duplex and is no longer free to bind to the transcript.

[[Image:ANTI.jpg]]

(Bayer and Smolke 2005 Permission Pending)

'''Figure 5:''' a) An inactive off-switch. The antisense stem is duplexed with itself. b) The same switch after being activated by theophylline (blue ellipse). The antisense stem is duplexed with the mRNA; thus, translation is stopped.

==From Concept to Wet Lab==

Smolke and Bayer synthesized genes for antiswitches with antisense stems that contained complements to either GFP or YFP transcripts and the aptamer to either theophylline or tetracycline. These genes were then transformed into ''Saccharomyces cerevisiae''. Several experiments, including dose-response curves to the appropriate ligand and fluoresence measurements of cells when exposed to both the appropriate ligand and wrong ligand, demonstrated that these antiswitches effectively regulate translation of their specific target in response to only the correct ligand.

In the dose-response experiments for off-switches when enough ligand (~ 1 mM theophylline or tetracycline depending on the switch) was present, the inactive off-switches became active and relative GFP expression dropped to almost zero. These experimental results support the antiswitch technology being functional. As off-switches with either theophylline or tetracycline aptamers worked, the versatility of antiswitches is supported (figure 6).

[[Image:Offswitch_experiment.JPG]]

(Bayer and Smolke 2005 Permission Pending)

'''Figure 6:''' The red line represents the tetracycline controlled switch while blue line represents the theophylline controlled switch. When the concentration of ligand is high enough, enough switches are active and binding to mRNA to stop translation of the gene.

In the dose-response experiments for the on-switches when ~ 1 mM of theophylline was added to cells containing the inactive on-switch, the cells relative expression of GFP jumped from near zero to approximately 0.9. This data show that the on-switch is also functional (figure 7).

[[Image:Onswitch_experiment.JPG]]

(Bayer and Smolke 2005 Permission Pending)

'''Figure 7:''' The red line represents the onswitch while the blue Line represents the offswitch. When a high enough concentration of ligand is present, enough on-switches are activated and have thus released their target mRNA for detectable near normal translation to occur. The off-switch follows the opposite path as in figure 6.

Finally, antiswitches in eukaryotes can be combined to regulate multiple genes for different conditions like the taRNA/crRNA system in prokaryotes. By simply switching the aptamer and antisense stem targeting sequence, the switch now controls a different gene by a different stimulus. Off-switch experiments using a theophylline aptamer with a GFP targeting antisense stem and a tetracycline aptamer with a YFP targeting antisense stem showed that the switches regulate the genes independently and can be used in combination to create complex regulatory mechanisms (figure 8).

[[Image:Combinatorial.JPG]]

(Bayer and Smolke 2005 Permission Pending)

'''Figure 8:''' a) On the left, theophylline activates the antiswitch's aptamer, which then binds to the GFP transcript and prevents translation. On the right, tetracycline binds to the antiswitch's aptamer, which then binds to the YFP transcript and prevents translation. If only one ligand is present (either theophylline or tetracycline), only one gene's transcripts (either GFP or YFP)is not translated. b) When no theophylline or tetracycline is present, both GFP and YFP are expressed at near normal levels. When only tetracycline is present, GFP is expressed at near normal levels while YFP expression is close to zero. When only theophylline is present, YFP is expressed at near normal levels while GFP expression is close to zero.
When both tetracycline and theophylline are present, GFP and YFP expression are both close to zero.

==Modularity==

While antiswitches have many advantages, they do lack modularity, the ability to be integrated with any gene without redesigning a new sequence specific part (Isaacs et al. 2004). If you want to regulate a new gene, you have to synthesize a whole new antiswitch because the antisense stem is specific to a particular gene and the aptamer stem is specific to a particular antisense stem.

==References==

Bayer TS and Smolke CD. Programmable ligand-controlled riboregulators of eukaryotic gene expression. Nat Biotechnol. (2005) 3:337-43.

Isaacs FJ, et al. Engineered riboregulators enable post-transcriptional control of gene expression. Nat Biotechnol. (2004) 22:841-47.

Win MN, Smolke CD (2007). A modular and extensible RNA-based gene-regulatory platform for engineering cellular function. PNAS 104(36):14283-8. Epub 2007 Aug 20. [http://www.pnas.org/cgi/content/abstract/104/36/14283 Abstract].

[[Post-transcriptional Regulation Technologies - Erin Zwack|Return to Post-transcriptional Regulation Technologies]]

Antiswitches

2007-12-06T19:45:06Z

Erzwack: /* From Concept to Wet Lab */

All information about antiswitches came from Bayer and Smolke (2005).

Antiswitches, trans-RNA molecules that regulate translation of mRNA based on the presence or absence of specific ligands, were first developed by Smolke and Bayer (2005) in ''Saccharomyces cerevisiae''. Two types of antiswitches were engineered: on-switches and off-switches. On-switches turn on gene expression in the presence of the ligand while off-switches turn off gene expression in the presence of the ligand. Control by the ligand allows researchers to regulate pathways’ protein production with less leakiness, low level transcription even when “off”, than using a specific promoter.

==Design==

Antiswitches are made of an aptamer and two stems: the aptamer stem and the antisense stem (figure 3).

[[Image:Switch.JPG]]

(Bayer and Smolke 2005 Permission Pending)

'''Figure 3:''' The antisense stem can bind to itself (duplex). The antisense stem also contains the complement to the RNA that a researcher desires to regulate. The aptamer stem swings either toward the antisense stem and disrupts the duplex or swings away from the antisense stem and allows the antisense stem to duplex with itself depending on whether the aptamer is bound to its ligand.

The aptamer is the sequence that binds the ligand and causes a conformational (shape) change of the antiswitch molecule. Aptamers can be highly specific for their particular molecules. The theophylline aptamer used by Smolke and Bayer can distinguish between caffeine and theophylline, which differ by a single methyl (figure 4).

[[Image:Caffeine.JPG]]

'''Figure 4.''' Caffeine (a) and Theophylline (b) differ by one methyl group (circled in red).

While Smolke and Bayer mainly used the aptamer for the ligand theophylline, aptamers for many other molecules are now being generated using rational design (Win and Smolke 2007). The large number of possible aptamers provides versatility in what will control gene expression.

The antisense stem contains a sequence that complements a targeted RNA transcript and a second sequence that sequesters this complementary sequence to keep it from binding the transcript. When the antisense stem is not duplexed with itself, it prevents translation by binding to the complementary mRNA (figure 5).

The aptamer stem is a short sequence that complements a portion of the sequestering sequence of the antisense stem. In an off-switch, the aptamer stem swings towards the antisense stem and displaces the portion that complements the targeted transcript when the liand binds to the aptamer (figure 5). In an on-switch, the aptamer swings away from the antisense stem when the ligand binds to the aptamer; thus, the antisense stem can duplex and is no longer free to bind to the transcript.

[[Image:ANTI.jpg]]

(Bayer and Smolke 2005 Permission Pending)

'''Figure 5:''' a) An inactive off-switch. The antisense stem is duplexed with itself. b) The same switch after being activated by theophylline (blue ellipse). The antisense stem is duplexed with the mRNA; thus, translation is stopped.

==From Concept to Wet Lab==

Smolke and Bayer synthesized genes for antiswitches with antisense stems that contained complements to either GFP or YFP transcripts and the aptamer to either theophylline or tetracycline. These genes were then transformed into ''Saccharomyces cerevisiae''. Several experiments, including dose-response curves to the appropriate ligand and fluoresence measurements of cells when exposed to both the appropriate ligand and wrong ligand, demonstrated that these antiswitches effectively regulate translation of their specific target in response to only the correct ligand.

In the dose-response experiments for off-switches when enough ligand (~ 1 mM theophylline or tetracycline depending on the switch) was present, the inactive off-switches became active and relative GFP expression dropped to almost zero. These experimental results support the antiswitch technology being functional. As off-switches with theophylline and tetracycline binding aptamers worked, the versatility of antiswitches is supported (figure 6).

[[Image:Offswitch_experiment.JPG]]

(Bayer and Smolke 2005 Permission Pending)

'''Figure 6:''' The red line represents the tetracycline controlled switch while blue line represents the theophylline controlled switch. When the concentration of ligand is high enough, enough switches are active and binding to mRNA to stop translation of the gene.

In the dose-response experiments for the on-switches when ~ 1 mM of theophylline was added to cells containing the inactive on-switch, the cells relative expression of GFP jumped from near zero to approximately 0.9. This data show that the on-switch is also functional (figure 7).

[[Image:Onswitch_experiment.JPG]]

(Bayer and Smolke 2005 Permission Pending)

'''Figure 7:''' The red line represents the onswitch while the blue Line represents the offswitch. When a high enough concentration of ligand is present, enough on-switches are activated and have thus released their target mRNA for detectable near normal translation to occur. The off-switch follows the opposite path as in figure 6.

Finally, antiswitches in eukaryotes can be combined to regulate multiple genes for different conditions like the taRNA/crRNA system in prokaryotes. By simply switching the aptamer and antisense stem targeting sequence, the switch now controls a different gene by a different stimulus. Off-switch experiments using a theophylline aptamer with a GFP targeting antisense stem and a tetracycline aptamer with a YFP targeting antisense stem showed that the switches regulate the genes independently and can be used in combination to create complex regulatory mechanisms (figure 8).

[[Image:Combinatorial.JPG]]

(Bayer and Smolke 2005 Permission Pending)

'''Figure 8:''' a) On the left, theophylline activates the antiswitch's aptamer, which then binds to the GFP transcript and prevents translation. On the right, tetracycline binds to the antiswitch's aptamer, which then binds to the YFP transcript and prevents translation. If only one ligand is present (either theophylline or tetracycline), only one gene's transcripts (either GFP or YFP)is not translated. b) When no theophylline or tetracycline is present, both GFP and YFP are expressed at near normal levels. When only tetracycline is present, GFP is expressed at near normal levels while YFP expression is close to zero. When only theophylline is present, YFP is expressed at near normal levels while GFP expression is close to zero.
When both tetracycline and theophylline are present, GFP and YFP expression are both close to zero.

==Modularity==

While antiswitches have many advantages, they do lack modularity, the ability to be integrated with any gene without redesigning a new sequence specific part (Isaacs et al. 2004). If you want to regulate a new gene, you have to synthesize a whole new antiswitch because the antisense stem is specific to a particular gene and the aptamer stem is specific to a particular antisense stem.

==References==

Bayer TS and Smolke CD. Programmable ligand-controlled riboregulators of eukaryotic gene expression. Nat Biotechnol. (2005) 3:337-43.

Isaacs FJ, et al. Engineered riboregulators enable post-transcriptional control of gene expression. Nat Biotechnol. (2004) 22:841-47.

Win MN, Smolke CD (2007). A modular and extensible RNA-based gene-regulatory platform for engineering cellular function. PNAS 104(36):14283-8. Epub 2007 Aug 20. [http://www.pnas.org/cgi/content/abstract/104/36/14283 Abstract].

[[Post-transcriptional Regulation Technologies - Erin Zwack|Return to Post-transcriptional Regulation Technologies]]

Antiswitches

2007-12-06T19:44:15Z

Erzwack: /* Design */

All information about antiswitches came from Bayer and Smolke (2005).

Antiswitches, trans-RNA molecules that regulate translation of mRNA based on the presence or absence of specific ligands, were first developed by Smolke and Bayer (2005) in ''Saccharomyces cerevisiae''. Two types of antiswitches were engineered: on-switches and off-switches. On-switches turn on gene expression in the presence of the ligand while off-switches turn off gene expression in the presence of the ligand. Control by the ligand allows researchers to regulate pathways’ protein production with less leakiness, low level transcription even when “off”, than using a specific promoter.

==Design==

Antiswitches are made of an aptamer and two stems: the aptamer stem and the antisense stem (figure 3).

[[Image:Switch.JPG]]

(Bayer and Smolke 2005 Permission Pending)

'''Figure 3:''' The antisense stem can bind to itself (duplex). The antisense stem also contains the complement to the RNA that a researcher desires to regulate. The aptamer stem swings either toward the antisense stem and disrupts the duplex or swings away from the antisense stem and allows the antisense stem to duplex with itself depending on whether the aptamer is bound to its ligand.

The aptamer is the sequence that binds the ligand and causes a conformational (shape) change of the antiswitch molecule. Aptamers can be highly specific for their particular molecules. The theophylline aptamer used by Smolke and Bayer can distinguish between caffeine and theophylline, which differ by a single methyl (figure 4).

[[Image:Caffeine.JPG]]

'''Figure 4.''' Caffeine (a) and Theophylline (b) differ by one methyl group (circled in red).

While Smolke and Bayer mainly used the aptamer for the ligand theophylline, aptamers for many other molecules are now being generated using rational design (Win and Smolke 2007). The large number of possible aptamers provides versatility in what will control gene expression.

The antisense stem contains a sequence that complements a targeted RNA transcript and a second sequence that sequesters this complementary sequence to keep it from binding the transcript. When the antisense stem is not duplexed with itself, it prevents translation by binding to the complementary mRNA (figure 5).

The aptamer stem is a short sequence that complements a portion of the sequestering sequence of the antisense stem. In an off-switch, the aptamer stem swings towards the antisense stem and displaces the portion that complements the targeted transcript when the liand binds to the aptamer (figure 5). In an on-switch, the aptamer swings away from the antisense stem when the ligand binds to the aptamer; thus, the antisense stem can duplex and is no longer free to bind to the transcript.

[[Image:ANTI.jpg]]

(Bayer and Smolke 2005 Permission Pending)

'''Figure 5:''' a) An inactive off-switch. The antisense stem is duplexed with itself. b) The same switch after being activated by theophylline (blue ellipse). The antisense stem is duplexed with the mRNA; thus, translation is stopped.

==From Concept to Wet Lab==

Smolke and Bayer synthesized genes for antiswitches with antisense stems that contained complements to either GFP or YFP transcripts and the aptamer to either theophylline or tetracycline. These genes were then transformed into '''Sacchromyces cerevisea'''. Several experiments, including dose-response curves to the appropriate ligand and fluoresence measurements of cells when exposed to both the appropriate ligand and wrong ligand, demonstrated that these antiswitches effectively regulate translation of their specific target in response to only the correct ligand.

In the dose-response experiments for off-switches when enough ligand (~ 1 mM theophylline or tetracycline depending on the switch) was present, the inactive off-switches became active and relative GFP expression dropped to almost zero. These experimental results support the antiswitch technology being functional. As off-switches with theophylline and tetracycline binding aptamers worked, the versatility of antiswitches is supported (figure 6).

[[Image:Offswitch_experiment.JPG]]

(Bayer and Smolke 2005 Permission Pending)

'''Figure 6:''' The red line represents the tetracycline controlled switch while blue line represents the theophylline controlled switch. When the concentration of ligand is high enough, enough switches are active and binding to mRNA to stop translation of the gene.

In the dose-response experiments for the on-switches when ~ 1 mM of theophylline was added to cells containing the inactive on-switch, the cells relative expression of GFP jumped from near zero to approximately 0.9. This data show that the on-switch is also functional (figure 7).

[[Image:Onswitch_experiment.JPG]]

(Bayer and Smolke 2005 Permission Pending)

'''Figure 7:''' The red line represents the onswitch while the blue Line represents the offswitch. When a high enough concentration of ligand is present, enough on-switches are activated and have thus released their target mRNA for detectable near normal translation to occur. The off-switch follows the opposite path as in figure 6.

Finally, antiswitches in eukaryotes can be combined to regulate multiple genes for different conditions like the taRNA/crRNA system in prokaryotes. By simply switching the aptamer and antisense stem targeting sequence, the switch now controls a different gene by a different stimulus. Off-switch experiments using a theophylline aptamer with a GFP targeting antisense stem and a tetracycline aptamer with a YFP targeting antisense stem showed that the switches regulate the genes independently and can be used in combination to create complex regulatory mechanisms (figure 8).

[[Image:Combinatorial.JPG]]

(Bayer and Smolke 2005 Permission Pending)

'''Figure 8:''' a) On the left, theophylline activates the antiswitch's aptamer, which then binds to the GFP transcript and prevents translation. On the right, tetracycline binds to the antiswitch's aptamer, which then binds to the YFP transcript and prevents translation. If only one ligand is present (either theophylline or tetracycline), only one gene's transcripts (either GFP or YFP)is not translated. b) When no theophylline or tetracycline is present, both GFP and YFP are expressed at near normal levels. When only tetracycline is present, GFP is expressed at near normal levels while YFP expression is close to zero. When only theophylline is present, YFP is expressed at near normal levels while GFP expression is close to zero.
When both tetracycline and theophylline are present, GFP and YFP expression are both close to zero.

==Modularity==

While antiswitches have many advantages, they do lack modularity, the ability to be integrated with any gene without redesigning a new sequence specific part (Isaacs et al. 2004). If you want to regulate a new gene, you have to synthesize a whole new antiswitch because the antisense stem is specific to a particular gene and the aptamer stem is specific to a particular antisense stem.

==References==

Bayer TS and Smolke CD. Programmable ligand-controlled riboregulators of eukaryotic gene expression. Nat Biotechnol. (2005) 3:337-43.

Isaacs FJ, et al. Engineered riboregulators enable post-transcriptional control of gene expression. Nat Biotechnol. (2004) 22:841-47.

Win MN, Smolke CD (2007). A modular and extensible RNA-based gene-regulatory platform for engineering cellular function. PNAS 104(36):14283-8. Epub 2007 Aug 20. [http://www.pnas.org/cgi/content/abstract/104/36/14283 Abstract].

[[Post-transcriptional Regulation Technologies - Erin Zwack|Return to Post-transcriptional Regulation Technologies]]

Antiswitches

2007-12-06T19:43:01Z

Erzwack: /* Design */

All information about antiswitches came from Bayer and Smolke (2005).

Antiswitches, trans-RNA molecules that regulate translation of mRNA based on the presence or absence of specific ligands, were first developed by Smolke and Bayer (2005) in ''Saccharomyces cerevisiae''. Two types of antiswitches were engineered: on-switches and off-switches. On-switches turn on gene expression in the presence of the ligand while off-switches turn off gene expression in the presence of the ligand. Control by the ligand allows researchers to regulate pathways’ protein production with less leakiness, low level transcription even when “off”, than using a specific promoter.

==Design==

Antiswitches are made of an aptamer and two stems: the aptamer stem and the antisense stem (figure 3).

[[Image:Switch.JPG]]

(Bayer and Smolke 2005 Permission Pending)

'''Figure 3:''' The antisense stem can bind to itself (duplex). The antisense stem also contains the complement to the RNA that a researcher desires to regulate. The aptamer stem swings either toward the antisense stem and disrupts the duplex or swings away from the antisense stem and allows the antisense stem to duplex with itself depending on whether the aptamer is bound to its ligand.

The aptamer is the sequence that binds the ligand and causes a conformational (shape) change of the antiswitch molecule. Aptamers can be highly specific for their particular molecules. The theophylline aptamer used by Smolke and Bayer can distinguish between caffeine and theophylline, which differ by a single methyl (figure 4).

[[Image:Caffeine.JPG]]

'''Figure 4.''' Caffeine (a) and Theophylline (b) differ by one methyl group (circled in red).

While Smolke and Bayer mainly used the aptamer for the ligand theophylline, aptamers for many other molecules are now being generated using rational design (Win and Smolke 2007). The large number of possible aptamers provides versatility in what will control gene expression.

The antisense stem contains a sequence that complements a targeted RNA transcript and a second sequence that sequesters this complementary sequence to keep it from binding the transcript. When the antisense stem is not duplexed with itself, it prevents translation by binding to the complementary mRNA (figure 5).

The aptamer stem is a short sequence that complements a portion of the sequestering sequence of the antisense stem. In an off-switch, the aptamer stem swings towards the antisense stem and displaces the portion that complements the targeted transcript when the liand binds to the aptamer (figure 5). In an on-switch, the aptamer swings away from the antisense stem when the ligand bids to the aptamer; thus, the antisense stem can duplex and is no longer free to bind to the transcript.

[[Image:ANTI.jpg]]

(Bayer and Smolke 2005 Permission Pending)

'''Figure 5:''' a) An inactive off-switch. The antisense stem is duplexed with itself. b) The same switch after being activated by theophylline (blue ellipse). The antisense stem is duplexed with the mRNA; thus, translation is stopped.

==From Concept to Wet Lab==

Smolke and Bayer synthesized genes for antiswitches with antisense stems that contained complements to either GFP or YFP transcripts and the aptamer to either theophylline or tetracycline. These genes were then transformed into '''Sacchromyces cerevisea'''. Several experiments, including dose-response curves to the appropriate ligand and fluoresence measurements of cells when exposed to both the appropriate ligand and wrong ligand, demonstrated that these antiswitches effectively regulate translation of their specific target in response to only the correct ligand.

In the dose-response experiments for off-switches when enough ligand (~ 1 mM theophylline or tetracycline depending on the switch) was present, the inactive off-switches became active and relative GFP expression dropped to almost zero. These experimental results support the antiswitch technology being functional. As off-switches with theophylline and tetracycline binding aptamers worked, the versatility of antiswitches is supported (figure 6).

[[Image:Offswitch_experiment.JPG]]

(Bayer and Smolke 2005 Permission Pending)

'''Figure 6:''' The red line represents the tetracycline controlled switch while blue line represents the theophylline controlled switch. When the concentration of ligand is high enough, enough switches are active and binding to mRNA to stop translation of the gene.

In the dose-response experiments for the on-switches when ~ 1 mM of theophylline was added to cells containing the inactive on-switch, the cells relative expression of GFP jumped from near zero to approximately 0.9. This data show that the on-switch is also functional (figure 7).

[[Image:Onswitch_experiment.JPG]]

(Bayer and Smolke 2005 Permission Pending)

'''Figure 7:''' The red line represents the onswitch while the blue Line represents the offswitch. When a high enough concentration of ligand is present, enough on-switches are activated and have thus released their target mRNA for detectable near normal translation to occur. The off-switch follows the opposite path as in figure 6.

Finally, antiswitches in eukaryotes can be combined to regulate multiple genes for different conditions like the taRNA/crRNA system in prokaryotes. By simply switching the aptamer and antisense stem targeting sequence, the switch now controls a different gene by a different stimulus. Off-switch experiments using a theophylline aptamer with a GFP targeting antisense stem and a tetracycline aptamer with a YFP targeting antisense stem showed that the switches regulate the genes independently and can be used in combination to create complex regulatory mechanisms (figure 8).

[[Image:Combinatorial.JPG]]

(Bayer and Smolke 2005 Permission Pending)

'''Figure 8:''' a) On the left, theophylline activates the antiswitch's aptamer, which then binds to the GFP transcript and prevents translation. On the right, tetracycline binds to the antiswitch's aptamer, which then binds to the YFP transcript and prevents translation. If only one ligand is present (either theophylline or tetracycline), only one gene's transcripts (either GFP or YFP)is not translated. b) When no theophylline or tetracycline is present, both GFP and YFP are expressed at near normal levels. When only tetracycline is present, GFP is expressed at near normal levels while YFP expression is close to zero. When only theophylline is present, YFP is expressed at near normal levels while GFP expression is close to zero.
When both tetracycline and theophylline are present, GFP and YFP expression are both close to zero.

==Modularity==

While antiswitches have many advantages, they do lack modularity, the ability to be integrated with any gene without redesigning a new sequence specific part (Isaacs et al. 2004). If you want to regulate a new gene, you have to synthesize a whole new antiswitch because the antisense stem is specific to a particular gene and the aptamer stem is specific to a particular antisense stem.

==References==

Bayer TS and Smolke CD. Programmable ligand-controlled riboregulators of eukaryotic gene expression. Nat Biotechnol. (2005) 3:337-43.

Isaacs FJ, et al. Engineered riboregulators enable post-transcriptional control of gene expression. Nat Biotechnol. (2004) 22:841-47.

Win MN, Smolke CD (2007). A modular and extensible RNA-based gene-regulatory platform for engineering cellular function. PNAS 104(36):14283-8. Epub 2007 Aug 20. [http://www.pnas.org/cgi/content/abstract/104/36/14283 Abstract].

[[Post-transcriptional Regulation Technologies - Erin Zwack|Return to Post-transcriptional Regulation Technologies]]

Antiswitches

2007-12-06T19:41:21Z

Erzwack: /* References */

All information about antiswitches came from Bayer and Smolke (2005).

Antiswitches, trans-RNA molecules that regulate translation of mRNA based on the presence or absence of specific ligands, were first developed by Smolke and Bayer (2005) in ''Saccharomyces cerevisiae''. Two types of antiswitches were engineered: on-switches and off-switches. On-switches turn on gene expression in the presence of the ligand while off-switches turn off gene expression in the presence of the ligand. Control by the ligand allows researchers to regulate pathways’ protein production with less leakiness, low level transcription even when “off”, than using a specific promoter.

==Design==

Antiswitches are made of an aptamer and two stems: the aptamer stem and the antisense stem (figure 3).

[[Image:Switch.JPG]]

(Bayer and Smolke 2005 Permission Pending)

'''Figure 3:''' The antisense stem can bind to itself (duplex). The antisense stem also contains the complement to the RNA that a researcher desires to regulate. The aptamer stem swings either toward the antisense stem and disrupts the duplex or swings away from the antisense stem and allows the antisense stem to duplex with itself depending on whether the aptamer is bound to its ligand.

The aptamer is the sequence that binds the ligand and causes a conformational (shape) change of the antiswitch molecule. Aptamers can be highly specific for their particular molecules. The theophylline aptamer used by Smolke and Bayer can distinguish between caffeine and theophylline, which differ by a single methyl.

[[Image:Caffeine.JPG]]

'''Figure 4.''' Caffeine (a) and Theophylline (b) differ by one methyl group (circled in red).

While Smolke and Bayer mainly used the aptamer for the ligand theophylline, aptamers for many other molecules are now being generated using rational design (Win and Smolke 2007). The large number of possible aptamers provides versatility in what will control gene expression.

The antisense stem contains a sequence that complements a targeted RNA transcript and a second sequence that sequesters this complementary sequence to keep it from binding the transcript. When the antisense stem is not duplexed with itself, it prevents translation by binding to the complementary mRNA.

The aptamer stem is a short sequence that complements a portion of the sequestering sequence of the antisense stem. In an off-switch, the aptamer stem swings towards the antisense stem and displaces the portion that complements the targeted transcript when the liand binds to the aptamer. In an on-switch, the aptamer swings away from the antisense stem when the ligand bids to the aptamer; thus, the antisense stem can duplex and is no longer free to bind to the transcript.

[[Image:ANTI.jpg]]

(Bayer and Smolke 2005 Permission Pending)

'''Figure 5:''' a) An inactive off-switch. The antisense stem is duplexed with itself. b) The same switch after being activated by theophylline (blue ellipse). The antisense stem is duplexed with the mRNA; thus, translation is stopped.

==From Concept to Wet Lab==

Smolke and Bayer synthesized genes for antiswitches with antisense stems that contained complements to either GFP or YFP transcripts and the aptamer to either theophylline or tetracycline. These genes were then transformed into '''Sacchromyces cerevisea'''. Several experiments, including dose-response curves to the appropriate ligand and fluoresence measurements of cells when exposed to both the appropriate ligand and wrong ligand, demonstrated that these antiswitches effectively regulate translation of their specific target in response to only the correct ligand.

In the dose-response experiments for off-switches when enough ligand (~ 1 mM theophylline or tetracycline depending on the switch) was present, the inactive off-switches became active and relative GFP expression dropped to almost zero. These experimental results support the antiswitch technology being functional. As off-switches with theophylline and tetracycline binding aptamers worked, the versatility of antiswitches is supported (figure 6).

[[Image:Offswitch_experiment.JPG]]

(Bayer and Smolke 2005 Permission Pending)

'''Figure 6:''' The red line represents the tetracycline controlled switch while blue line represents the theophylline controlled switch. When the concentration of ligand is high enough, enough switches are active and binding to mRNA to stop translation of the gene.

In the dose-response experiments for the on-switches when ~ 1 mM of theophylline was added to cells containing the inactive on-switch, the cells relative expression of GFP jumped from near zero to approximately 0.9. This data show that the on-switch is also functional (figure 7).

[[Image:Onswitch_experiment.JPG]]

(Bayer and Smolke 2005 Permission Pending)

'''Figure 7:''' The red line represents the onswitch while the blue Line represents the offswitch. When a high enough concentration of ligand is present, enough on-switches are activated and have thus released their target mRNA for detectable near normal translation to occur. The off-switch follows the opposite path as in figure 6.

Finally, antiswitches in eukaryotes can be combined to regulate multiple genes for different conditions like the taRNA/crRNA system in prokaryotes. By simply switching the aptamer and antisense stem targeting sequence, the switch now controls a different gene by a different stimulus. Off-switch experiments using a theophylline aptamer with a GFP targeting antisense stem and a tetracycline aptamer with a YFP targeting antisense stem showed that the switches regulate the genes independently and can be used in combination to create complex regulatory mechanisms (figure 8).

[[Image:Combinatorial.JPG]]

(Bayer and Smolke 2005 Permission Pending)

'''Figure 8:''' a) On the left, theophylline activates the antiswitch's aptamer, which then binds to the GFP transcript and prevents translation. On the right, tetracycline binds to the antiswitch's aptamer, which then binds to the YFP transcript and prevents translation. If only one ligand is present (either theophylline or tetracycline), only one gene's transcripts (either GFP or YFP)is not translated. b) When no theophylline or tetracycline is present, both GFP and YFP are expressed at near normal levels. When only tetracycline is present, GFP is expressed at near normal levels while YFP expression is close to zero. When only theophylline is present, YFP is expressed at near normal levels while GFP expression is close to zero.
When both tetracycline and theophylline are present, GFP and YFP expression are both close to zero.

==Modularity==

While antiswitches have many advantages, they do lack modularity, the ability to be integrated with any gene without redesigning a new sequence specific part (Isaacs et al. 2004). If you want to regulate a new gene, you have to synthesize a whole new antiswitch because the antisense stem is specific to a particular gene and the aptamer stem is specific to a particular antisense stem.

==References==

Bayer TS and Smolke CD. Programmable ligand-controlled riboregulators of eukaryotic gene expression. Nat Biotechnol. (2005) 3:337-43.

Isaacs FJ, et al. Engineered riboregulators enable post-transcriptional control of gene expression. Nat Biotechnol. (2004) 22:841-47.

Win MN, Smolke CD (2007). A modular and extensible RNA-based gene-regulatory platform for engineering cellular function. PNAS 104(36):14283-8. Epub 2007 Aug 20. [http://www.pnas.org/cgi/content/abstract/104/36/14283 Abstract].

[[Post-transcriptional Regulation Technologies - Erin Zwack|Return to Post-transcriptional Regulation Technologies]]

Antiswitches

2007-12-06T19:41:07Z

Erzwack:

All information about antiswitches came from Bayer and Smolke (2005).

Antiswitches, trans-RNA molecules that regulate translation of mRNA based on the presence or absence of specific ligands, were first developed by Smolke and Bayer (2005) in ''Saccharomyces cerevisiae''. Two types of antiswitches were engineered: on-switches and off-switches. On-switches turn on gene expression in the presence of the ligand while off-switches turn off gene expression in the presence of the ligand. Control by the ligand allows researchers to regulate pathways’ protein production with less leakiness, low level transcription even when “off”, than using a specific promoter.

==Design==

Antiswitches are made of an aptamer and two stems: the aptamer stem and the antisense stem (figure 3).

[[Image:Switch.JPG]]

(Bayer and Smolke 2005 Permission Pending)

'''Figure 3:''' The antisense stem can bind to itself (duplex). The antisense stem also contains the complement to the RNA that a researcher desires to regulate. The aptamer stem swings either toward the antisense stem and disrupts the duplex or swings away from the antisense stem and allows the antisense stem to duplex with itself depending on whether the aptamer is bound to its ligand.

The aptamer is the sequence that binds the ligand and causes a conformational (shape) change of the antiswitch molecule. Aptamers can be highly specific for their particular molecules. The theophylline aptamer used by Smolke and Bayer can distinguish between caffeine and theophylline, which differ by a single methyl.

[[Image:Caffeine.JPG]]

'''Figure 4.''' Caffeine (a) and Theophylline (b) differ by one methyl group (circled in red).

While Smolke and Bayer mainly used the aptamer for the ligand theophylline, aptamers for many other molecules are now being generated using rational design (Win and Smolke 2007). The large number of possible aptamers provides versatility in what will control gene expression.

The antisense stem contains a sequence that complements a targeted RNA transcript and a second sequence that sequesters this complementary sequence to keep it from binding the transcript. When the antisense stem is not duplexed with itself, it prevents translation by binding to the complementary mRNA.

The aptamer stem is a short sequence that complements a portion of the sequestering sequence of the antisense stem. In an off-switch, the aptamer stem swings towards the antisense stem and displaces the portion that complements the targeted transcript when the liand binds to the aptamer. In an on-switch, the aptamer swings away from the antisense stem when the ligand bids to the aptamer; thus, the antisense stem can duplex and is no longer free to bind to the transcript.

[[Image:ANTI.jpg]]

(Bayer and Smolke 2005 Permission Pending)

'''Figure 5:''' a) An inactive off-switch. The antisense stem is duplexed with itself. b) The same switch after being activated by theophylline (blue ellipse). The antisense stem is duplexed with the mRNA; thus, translation is stopped.

==From Concept to Wet Lab==

Smolke and Bayer synthesized genes for antiswitches with antisense stems that contained complements to either GFP or YFP transcripts and the aptamer to either theophylline or tetracycline. These genes were then transformed into '''Sacchromyces cerevisea'''. Several experiments, including dose-response curves to the appropriate ligand and fluoresence measurements of cells when exposed to both the appropriate ligand and wrong ligand, demonstrated that these antiswitches effectively regulate translation of their specific target in response to only the correct ligand.

In the dose-response experiments for off-switches when enough ligand (~ 1 mM theophylline or tetracycline depending on the switch) was present, the inactive off-switches became active and relative GFP expression dropped to almost zero. These experimental results support the antiswitch technology being functional. As off-switches with theophylline and tetracycline binding aptamers worked, the versatility of antiswitches is supported (figure 6).

[[Image:Offswitch_experiment.JPG]]

(Bayer and Smolke 2005 Permission Pending)

'''Figure 6:''' The red line represents the tetracycline controlled switch while blue line represents the theophylline controlled switch. When the concentration of ligand is high enough, enough switches are active and binding to mRNA to stop translation of the gene.

In the dose-response experiments for the on-switches when ~ 1 mM of theophylline was added to cells containing the inactive on-switch, the cells relative expression of GFP jumped from near zero to approximately 0.9. This data show that the on-switch is also functional (figure 7).

[[Image:Onswitch_experiment.JPG]]

(Bayer and Smolke 2005 Permission Pending)

'''Figure 7:''' The red line represents the onswitch while the blue Line represents the offswitch. When a high enough concentration of ligand is present, enough on-switches are activated and have thus released their target mRNA for detectable near normal translation to occur. The off-switch follows the opposite path as in figure 6.

Finally, antiswitches in eukaryotes can be combined to regulate multiple genes for different conditions like the taRNA/crRNA system in prokaryotes. By simply switching the aptamer and antisense stem targeting sequence, the switch now controls a different gene by a different stimulus. Off-switch experiments using a theophylline aptamer with a GFP targeting antisense stem and a tetracycline aptamer with a YFP targeting antisense stem showed that the switches regulate the genes independently and can be used in combination to create complex regulatory mechanisms (figure 8).

[[Image:Combinatorial.JPG]]

(Bayer and Smolke 2005 Permission Pending)

'''Figure 8:''' a) On the left, theophylline activates the antiswitch's aptamer, which then binds to the GFP transcript and prevents translation. On the right, tetracycline binds to the antiswitch's aptamer, which then binds to the YFP transcript and prevents translation. If only one ligand is present (either theophylline or tetracycline), only one gene's transcripts (either GFP or YFP)is not translated. b) When no theophylline or tetracycline is present, both GFP and YFP are expressed at near normal levels. When only tetracycline is present, GFP is expressed at near normal levels while YFP expression is close to zero. When only theophylline is present, YFP is expressed at near normal levels while GFP expression is close to zero.
When both tetracycline and theophylline are present, GFP and YFP expression are both close to zero.

==Modularity==

While antiswitches have many advantages, they do lack modularity, the ability to be integrated with any gene without redesigning a new sequence specific part (Isaacs et al. 2004). If you want to regulate a new gene, you have to synthesize a whole new antiswitch because the antisense stem is specific to a particular gene and the aptamer stem is specific to a particular antisense stem.

==References==

Bayer TS and Smolke CD. Programmable ligand-controlled riboregulators of eukaryotic gene expression. Nat Biotechnol. (2005) 3:337-43.

Isaacs FJ, et al. Engineered riboregulators enable post-transcriptional control of gene expression. Nat Biotechnol. (2004) 22:841-47.

[[Post-transcriptional Regulation Technologies - Erin Zwack|Return to Post-transcriptional Regulation Technologies]]

Post-transcriptional Regulation Technologies - Erin Zwack

2007-12-06T19:33:51Z

Erzwack: /* Overview: */

==Post-transcriptional Regulation Technologies==

==Overview:==

Regulation of translation provides an excellent tool for research on metabolic and other pathways in organisms, and for the production of different sensors by controlling the translation of specific genes depending on cellular conditions. Further development of these types of technology could provide a “knock-down” equivalent to RNAi, which exist in some eukaryotes. A gene of interest could be expressed normally at all times when the regulator is not active; thus, no ill effects will result before a particular pathway activates and produces a specific ligand if the gene has another purpose as well. Other synthetic biologists could use these technologies to engineer fast-responding, RNA-based biological sensors for environmental chemicals, or novel pathways.

Using RNA regulatory molecules instead of regulatory proteins to control gene expression provides several benefits to synthetic biologists. Regulatory proteins bind to specific sites such as sites on the promoter or sites upstream of the promoter called operators ([http://en.wikipedia.org/wiki/Gene_regulation#Regulatory_protein wikipedia]). Control by these proteins can rely heavily on [[CellularMemory:Mathematical Models#Cooperativity_and_Bistability| cooperativity]] (Gardner et al. 2000), that is multiple proteins binding to one site, in order to see an effect. Regulatory RNA molecules on the other hand need a one to one ratio of regulatory molecule to target. As long as the molecule is expressed in an equal or greater amount than the target, the regulatory RNAs will normally be able to bind to their targets and control transcription.

While synthetic biologists could use the regulatory proteins and their binding sites that are found in nature, rational design of more new regulatory proteins is difficult. With the oligo and gene sythesis technology in existence today, RNA can be engineered that complements and thus targets any other RNA sequence. Regulatory proteins are also controlled mainly by promoter when determining whether they are active or not. As only so many inducible promoters exist, there is a small number of stimuli that can be used to determine under what conditions the gene under a regulatory protein's control will be expressed or repressed. An aptamer, RNA sequence that binds to a small molecule such as theophylline (Bayer and Smolke 2005), can be used not only to regulate gene expression but can also regulate under what conditions an RNA regulatory molecule is active. New aptamers are easily developed through rational design, and the number in existence is continually increasing and providing new molecules that can act as ligands.

Finally, regulatory proteins stop gene expression before transcription. When the stimulus changes and the gene is expressed (either because a regulatory protein has now bound to or has released its site), the time it takes for the phenotype to be expressed is longer because both transcription and translation must occur instead of just translation. With RNA, the gene expression is halted after transcription. Once the stimulus is removed, the RNA already produced by the gene simply needs to be translated.

==Development of Systems==

In most cases, post-transcriptional regulatory mechanisms that were developed and worked in eukaryotes cannot be directly transferred to prokaryotes. Modifications are necessary because eukaryotic and prokaryotic transcription and translation do not follow the exact same path. In eukaryotes, mRNA must have introns spliced out before translation begins; thus, any mechanism that regulates translation has time to bind or manipulate the mRNA (figure 1). In prokaryotes, translation begins as soon as the ribosomal binding site (RBS) is transcribed and accessible to a ribosome (figure 2).

[[Image:Eukaryotic.jpg]]

'''Figure 1:''' Inside of the nucleus of the eukaryote, the gene is transcribed into pre-mRNA, which contain both introns (orange) and exons (red). The pre-mRNA is then modified so that the introns are spliced out and the exons are put together. Finally the mRNA are translated by the ribosome in the cytoplasm.

[[Image:Prokaryote.jpg]]

'''Figure 2:''' In a prokaryotic cell the DNA is transcribed by RNA polymerase. As soon as the polymerase transcribes the ribosomal binding site, a ribosome binds and begins translating the sequence into protein. There is no modification step between transcription and translation.

==Eukaryotes:==

[[Antiswitches]]

==Prokaryotes:==

[[Riboregulators]]

[[Riboswitches]]

==Use of Post-transciptional Regulatory Technologies==

Regulation of translation provides an excellent tool for research on metabolic and other pathways in organisms, and for the production of different sensors by controlling the translation of specific genes depending on cellular conditions. Researchers can turn-off translation of certain genes in response to different pathways being activated, such as metabolic pathways. If a researcher wanted to know if a particular gene was necessary to proper function of a pathway, the aptamer of the antiswitch or riboswitch could be designed to have a molecule produced in the pathway to be its ligand. The gene would be expressed normally at all times when the pathway is not active; thus, no ill effects will result before the pathway activates if the gene has another purpose as well. This would provide a “knock-down” equivalent to RNAi available in some eukaryotes.

Synthetic biologists could engineer fast-responding, RNA-based biological sensors for environmental chemicals, or novel pathways that only activate when the environmental conditions are favorable.

==The Labs==

[http://www.che.caltech.edu/groups/cds/index.htm Smolke]

[http://www.bu.edu/abl/ Collins]

[http://www.bu.edu/abl/files/naturebiotech_isaacs.pdf Dr. Isaacs's Review of RNA Synthetic Biology]

[http://gallivan1.chem.emory.edu/Gallivan%20Lab/Home.html Gallivan]

==References==

[http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=Retrieve&db=pubmed&dopt=AbstractPlus&list_uids=15723047 Bayer TS and Smolke CD. Programmable ligand-controlled riboregulators of eukaryotic gene expression. ''Nat Biotechnol''. (2005) 3:337-43.]

[http://pubs.acs.org/cgi-bin/article.cgi/jacsat/2004/126/i41/pdf/ja048634j.pdf Desai SK and Gallivan JP. Genetic Screens and Selections for Small Molecules Based on a Synthetic Riboswitch That Activates Protein Translation. ''J. Am. Chem. Soc.''(2004) 126:13247-54.]

[http://www.bio.davidson.edu/courses/synthetic/papers/Collins_ToggleSwitch.pdf Gardner, T.S., Cantor, C.R., and Collins, J.J. Construction of a genetic toggle switch in Eschreichia coli. ''Nature'' (2000) 403: 339-342.]

[http://www.bio.davidson.edu/Courses/Synthetic/papers/RNA_Regulation.pdf Isaacs FJ, et al. Engineered riboregulators enable post-transcriptional control of gene expression. ''Nat Biotechnol.'' (2004) 22:841-47.]

[[A Review of Synthetic Biology| Return to Main Page]]

Post-transcriptional Regulation Technologies - Erin Zwack

2007-12-06T19:30:35Z

Erzwack: /* Overview: */

==Post-transcriptional Regulation Technologies==

==Overview:==

Regulation of translation provides an excellent tool for research on metabolic and other pathways in organisms, and for the production of different sensors by controlling the translation of specific genes depending on cellular conditions. Further development of these types of technology could provide a “knock-down” equivalent to RNAi, which exist in some eukaryotes. A gene of interest could be expressed normally at all times when the regulator is not active; thus, no ill effects will result before a particular pathway activates and produces a specific ligand if the gene has another purpose as well. Other synthetic biologists could use these technologies to engineer fast-responding, RNA-based biological sensors for environmental chemicals, or novel pathways.

Using RNA regulatory molecules instead of regulatory proteins to control gene expression provides several benefits to synthetic biologists. Regulatory proteins bind to specific sites such as sites on the promoter or sites upstream of the promoter called operators ([http://en.wikipedia.org/wiki/Gene_regulation#Regulatory_protein wikipedia]). Control by these proteins can rely heavily on [[CellularMemory:Mathematical Models#Cooperativity_and_Bistability| cooperativity]] (Gardner et al. 2000), that is multiple proteins binding to one site, in order to see an effect. Regulatory RNA molecules on the other hand need a one to one ratio of regulatory molecule to target. As long as the molecule is expressed in an equal or greater amount than the target, the regulatory RNAs will normally be able to bind to their targets and control transcription.

While synthetic biologists could use the regulatory proteins and their binding sites that are found in nature, rational design of more new regulatory proteins is difficult. With the oligo and gene sythesis technology in existence today, RNA can be engineered that complements and thus targets any other RNA sequence. Regulatory proteins are also controlled mainly by promoter when determining whether they are active or not. As only so many inducible promoters exist, their is a small number of stimuli that can be used to determine under what conditions the gene under a regulatory protein's control will be expressed or repressed. An aptamer, RNA sequence that binds to a small molecule such as theophylline, can be used not only to regulate gene expression but can also regulate under what conditions an RNA regulatory molecule is active. New aptamers are easily developed through rational design, and the number in existence is continually increasing and providing new molecules that can act as ligands.

Finally, regulatory proteins stop gene expression before transcription. When the stimulus changes and the gene is expressed (either because a regulatory protein has now bound to or has released its site), the time it takes for the phenotype to be expressed is longer because both transcription and translation must occur instead of just translation. With RNA, the gene expression is halted after transcription. Once the stimulus is removed, the RNA already produced by the gene simply need to be translated.

==Development of Systems==

In most cases, post-transcriptional regulatory mechanisms that were developed and worked in eukaryotes cannot be directly transferred to prokaryotes. Modifications are necessary because eukaryotic and prokaryotic transcription and translation do not follow the exact same path. In eukaryotes, mRNA must have introns spliced out before translation begins; thus, any mechanism that regulates translation has time to bind or manipulate the mRNA (figure 1). In prokaryotes, translation begins as soon as the ribosomal binding site (RBS) is transcribed and accessible to a ribosome (figure 2).

[[Image:Eukaryotic.jpg]]

'''Figure 1:''' Inside of the nucleus of the eukaryote, the gene is transcribed into pre-mRNA, which contain both introns (orange) and exons (red). The pre-mRNA is then modified so that the introns are spliced out and the exons are put together. Finally the mRNA are translated by the ribosome in the cytoplasm.

[[Image:Prokaryote.jpg]]

'''Figure 2:''' In a prokaryotic cell the DNA is transcribed by RNA polymerase. As soon as the polymerase transcribes the ribosomal binding site, a ribosome binds and begins translating the sequence into protein. There is no modification step between transcription and translation.

==Eukaryotes:==

[[Antiswitches]]

==Prokaryotes:==

[[Riboregulators]]

[[Riboswitches]]

==Use of Post-transciptional Regulatory Technologies==

Regulation of translation provides an excellent tool for research on metabolic and other pathways in organisms, and for the production of different sensors by controlling the translation of specific genes depending on cellular conditions. Researchers can turn-off translation of certain genes in response to different pathways being activated, such as metabolic pathways. If a researcher wanted to know if a particular gene was necessary to proper function of a pathway, the aptamer of the antiswitch or riboswitch could be designed to have a molecule produced in the pathway to be its ligand. The gene would be expressed normally at all times when the pathway is not active; thus, no ill effects will result before the pathway activates if the gene has another purpose as well. This would provide a “knock-down” equivalent to RNAi available in some eukaryotes.

Synthetic biologists could engineer fast-responding, RNA-based biological sensors for environmental chemicals, or novel pathways that only activate when the environmental conditions are favorable.

==The Labs==

[http://www.che.caltech.edu/groups/cds/index.htm Smolke]

[http://www.bu.edu/abl/ Collins]

[http://www.bu.edu/abl/files/naturebiotech_isaacs.pdf Dr. Isaacs's Review of RNA Synthetic Biology]

[http://gallivan1.chem.emory.edu/Gallivan%20Lab/Home.html Gallivan]

==References==

[http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=Retrieve&db=pubmed&dopt=AbstractPlus&list_uids=15723047 Bayer TS and Smolke CD. Programmable ligand-controlled riboregulators of eukaryotic gene expression. ''Nat Biotechnol''. (2005) 3:337-43.]

[http://pubs.acs.org/cgi-bin/article.cgi/jacsat/2004/126/i41/pdf/ja048634j.pdf Desai SK and Gallivan JP. Genetic Screens and Selections for Small Molecules Based on a Synthetic Riboswitch That Activates Protein Translation. ''J. Am. Chem. Soc.''(2004) 126:13247-54.]

[http://www.bio.davidson.edu/courses/synthetic/papers/Collins_ToggleSwitch.pdf Gardner, T.S., Cantor, C.R., and Collins, J.J. Construction of a genetic toggle switch in Eschreichia coli. ''Nature'' (2000) 403: 339-342.]

[http://www.bio.davidson.edu/Courses/Synthetic/papers/RNA_Regulation.pdf Isaacs FJ, et al. Engineered riboregulators enable post-transcriptional control of gene expression. ''Nat Biotechnol.'' (2004) 22:841-47.]

[[A Review of Synthetic Biology| Return to Main Page]]

Post-transcriptional Regulation Technologies - Erin Zwack

2007-12-06T19:26:46Z

Erzwack: /* Overview: */

==Post-transcriptional Regulation Technologies==

==Overview:==

Regulation of translation provides an excellent tool for research on metabolic and other pathways in organisms, and for the production of different sensors by controlling the translation of specific genes depending on cellular conditions. Further development of these types of technology could provide a “knock-down” equivalent to RNAi, which exist in some eukaryotes. A gene of interest could be expressed normally at all times when the regulator is not active; thus, no ill effects will result before a particular pathway activates and produces a specific ligand if the gene has another purpose as well. Other sythetic biologists could use these technologies to engineer fast-responding, RNA-based biological sensors for environmental chemicals, or novel pathways.

Using RNA regulatory molecules instead of regulatory proteins to control gene expression provides several benefits to synthetic biologists. Regulatory proteins bind to specific sites such as sites on the promoter or sites upstream of the promoter called operators. Control by these proteins can rely heavily on [[CellularMemory:Mathematical Models#Cooperativity_and_Bistability| cooperativity]] (Gardner et al. 2000), that is multiple proteins binding to one site, in order to see an effect. Regulatory RNA molecules on the other hand need a one to one ratio of regulatory molecule to target. As long as the molecule is expressed in an equal or greater amount than the target, the regulatory RNAs will normally be able to bind to their targets and control transcription.

While synthetic biologists could use the regulatory proteins and their binding sites that are found in nature, rational design of more new regulatory proteins is difficult. With the oligo and gene sythesis technology in existence today, RNA can be engineered that complements and thus targets any other RNA sequence. Regulatory proteins are also controlled mainly by promoter when determining whether they are active or not. As only so many inducible promoters exist, their is a small number of stimuli that can be used to determine under what conditions the gene under a regulatory protein's control will be expressed or repressed. An aptamer, RNA sequence that binds to a small molecule such as theophylline, can be used not only to regulate gene expression but can also regulate under what conditions an RNA regulatory molecule is active. New aptamers are easily developed through rational design, and the number in existence is continually increasing and providing new molecules that can act as ligands.

Finally, regulatory proteins stop gene expression before transcription. When the stimulus changes and the gene is expressed (either because a regulatory protein has now bound to or has released its site), the time it takes for the phenotype to be expressed is longer because both transcription and translation must occur instead of just translation. With RNA, the gene expression is halted after transcription. Once the stimulus is removed, the RNA already produced by the gene simply need to be translated.

==Development of Systems==

In most cases, post-transcriptional regulatory mechanisms that were developed and worked in eukaryotes cannot be directly transferred to prokaryotes. Modifications are necessary because eukaryotic and prokaryotic transcription and translation do not follow the exact same path. In eukaryotes, mRNA must have introns spliced out before translation begins; thus, any mechanism that regulates translation has time to bind or manipulate the mRNA (figure 1). In prokaryotes, translation begins as soon as the ribosomal binding site (RBS) is transcribed and accessible to a ribosome (figure 2).

[[Image:Eukaryotic.jpg]]

'''Figure 1:''' Inside of the nucleus of the eukaryote, the gene is transcribed into pre-mRNA, which contain both introns (orange) and exons (red). The pre-mRNA is then modified so that the introns are spliced out and the exons are put together. Finally the mRNA are translated by the ribosome in the cytoplasm.

[[Image:Prokaryote.jpg]]

'''Figure 2:''' In a prokaryotic cell the DNA is transcribed by RNA polymerase. As soon as the polymerase transcribes the ribosomal binding site, a ribosome binds and begins translating the sequence into protein. There is no modification step between transcription and translation.

==Eukaryotes:==

[[Antiswitches]]

==Prokaryotes:==

[[Riboregulators]]

[[Riboswitches]]

==Use of Post-transciptional Regulatory Technologies==

Regulation of translation provides an excellent tool for research on metabolic and other pathways in organisms, and for the production of different sensors by controlling the translation of specific genes depending on cellular conditions. Researchers can turn-off translation of certain genes in response to different pathways being activated, such as metabolic pathways. If a researcher wanted to know if a particular gene was necessary to proper function of a pathway, the aptamer of the antiswitch or riboswitch could be designed to have a molecule produced in the pathway to be its ligand. The gene would be expressed normally at all times when the pathway is not active; thus, no ill effects will result before the pathway activates if the gene has another purpose as well. This would provide a “knock-down” equivalent to RNAi available in some eukaryotes.

Synthetic biologists could engineer fast-responding, RNA-based biological sensors for environmental chemicals, or novel pathways that only activate when the environmental conditions are favorable.

==The Labs==

[http://www.che.caltech.edu/groups/cds/index.htm Smolke]

[http://www.bu.edu/abl/ Collins]

[http://www.bu.edu/abl/files/naturebiotech_isaacs.pdf Dr. Isaacs's Review of RNA Synthetic Biology]

[http://gallivan1.chem.emory.edu/Gallivan%20Lab/Home.html Gallivan]

==References==

[http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=Retrieve&db=pubmed&dopt=AbstractPlus&list_uids=15723047 Bayer TS and Smolke CD. Programmable ligand-controlled riboregulators of eukaryotic gene expression. ''Nat Biotechnol''. (2005) 3:337-43.]

[http://pubs.acs.org/cgi-bin/article.cgi/jacsat/2004/126/i41/pdf/ja048634j.pdf Desai SK and Gallivan JP. Genetic Screens and Selections for Small Molecules Based on a Synthetic Riboswitch That Activates Protein Translation. ''J. Am. Chem. Soc.''(2004) 126:13247-54.]

[http://www.bio.davidson.edu/courses/synthetic/papers/Collins_ToggleSwitch.pdf Gardner, T.S., Cantor, C.R., and Collins, J.J. Construction of a genetic toggle switch in Eschreichia coli. ''Nature'' (2000) 403: 339-342.]

[http://www.bio.davidson.edu/Courses/Synthetic/papers/RNA_Regulation.pdf Isaacs FJ, et al. Engineered riboregulators enable post-transcriptional control of gene expression. ''Nat Biotechnol.'' (2004) 22:841-47.]

[[A Review of Synthetic Biology| Return to Main Page]]

Antiswitches

2007-12-06T18:29:29Z

Erzwack: /* From Concept to Wet Lab */

All information about antiswitches came from Bayer and Smolke (2005).

Antiswitches, trans-RNA molecules that regulate translation of mRNA based on ligands, were first developed by Smolke and Bayer (2005) in Saccharomyces cerevisiae. Two types of antiswitches were engineered: on-switches and off-switches. On-switches turn on gene expression in the presence of the ligand while off-switches turn off gene expression in the presence of ligand. Control by the ligand allows researchers to regulate pathways’ protein production with less leakiness,low level transcription even when “off”, than using a specific promoter.

==Design==

Antiswitches are made of an aptamer and two stems: the aptamer stem and the antisense stem (figure 3).

[[Image:Switch.JPG]]

(Bayer and Smolke 2005 Permission Pending)

'''Figure 3:''' The antisense stem can bind to itself (duplex). The antisense stem also contains the complement to the RNA that a researcher desires to regulate. The aptamer stem swings either toward the antisense stem and disrupts the duplex or swings away from the antisense stem and allows the antisense stem to duplex depending on whether the aptamer is bound to its ligand.

The aptamer is the sequence that binds the ligand and causes a conformational (shape) change of the antiswitch molecule. Aptamers can be highly specific for their particular molecules. The theophylline aptamer used by Smolke and Bayer can distinguish between caffeine and theophylline, which differ by a single methyl.

[[Image:Caffeine.JPG]]

'''Figure 4.''' Caffeine (a) and Theophylline (b) differ by one methyl group (circled in red).

While Smolke and Bayer mainly used the aptamer for the ligand theophylline, aptamers for many other molecules are now being generated using rational design. The large number of possible aptamers provides versatility in what will control gene expression.

The antisense stem contains a sequence that complements a targeted RNA transcript and a second sequence that sequesters this complementary sequence to keep it from binding the transcript. When the antisense stem is not duplexed with itself, it prevents translation by binding to the complementary mRNA.

The aptamer stem is a short sequence that complements a portion of the sequestering sequence of the antisense stem. In an off-switch, the aptamer stem swings towards the antisense stem and displaces the portion that complements the targeted transcript when the liand binds to the aptamer. In an on-switch, the aptamer swings away from the antisense stem when the ligand bids to the aptamer; thus, the antisense stem can duplex and is no longer free to bind to the transcript.

[[Image:ANTI.jpg]]

(Bayer and Smolke 2005 Permission Pending)

'''Figure 5:''' a) An inactive off-switch. The antisense stem is duplexed with itself. b) The same switch after being activated by theophylline (blue ellipse). The antisense stem is duplexed with the mRNA; thus, translation is stopped.

==From Concept to Wet Lab==

Smolke and Bayer synthesized genes for antiswitches with antisense stems that contained complements to either GFP or YFP transcripts and the aptamer to either theophylline or tetracycline. These genes were then transformed into '''Sacchromyces cerevisea'''. Several experiments, including dose-response curves to the appropriate ligand and fluoresence measurements of cells when exposed to both the appropriate ligand and wrong ligand, demonstrated that these antiswitches effectively regulate translation of their specific target in response to only the correct ligand.

In the dose-response experiments for off-switches when enough ligand (~ 1 mM theophylline or tetracycline depending on the switch) was present, the inactive off-switches became active and relative GFP expression dropped to almost zero. These experimental results support the antiswitch technology being functional. As off-switches with theophylline and tetracycline binding aptamers worked, the versatility of antiswitches is supported (figure 6).

[[Image:Offswitch_experiment.JPG]]

(Bayer and Smolke 2005 Permission Pending)

'''Figure 6:''' The red line represents the tetracycline controlled switch while blue line represents the theophylline controlled switch. When the concentration of ligand is high enough, enough switches are active and binding to mRNA to stop translation of the gene.

In the dose-response experiments for the on-switches when ~ 1 mM of theophylline was added to cells containing the inactive on-switch, the cells relative expression of GFP jumped from near zero to approximately 0.9. This data show that the on-switch is also functional (figure 7).

[[Image:Onswitch_experiment.JPG]]

(Bayer and Smolke 2005 Permission Pending)

'''Figure 7:''' The red line represents the onswitch while the blue Line represents the offswitch. When a high enough concentration of ligand is present, enough on-switches are activated and have thus released their target mRNA for detectable near normal translation to occur. The off-switch follows the opposite path as in figure 6.

Finally, antiswitches in eukaryotes can be combined to regulate multiple genes for different conditions like the taRNA/crRNA system in prokaryotes. By simply switching the aptamer and antisense stem targeting sequence, the switch now controls a different gene by a different stimulus. Off-switch experiments using a theophylline aptamer with a GFP targeting antisense stem and a tetracycline aptamer with a YFP targeting antisense stem showed that the switches regulate the genes independently and can be used in combination to create complex regulatory mechanisms (figure 8).

[[Image:Combinatorial.JPG]]

(Bayer and Smolke 2005 Permission Pending)

'''Figure 8:''' a) On the left, theophylline activates the antiswitch's aptamer, which then binds to the GFP transcript and prevents translation. On the right, tetracycline binds to the antiswitch's aptamer, which then binds to the YFP transcript and prevents translation. If only one ligand is present (either theophylline or tetracycline), only one gene's transcripts (either GFP or YFP)is not translated. b) When no theophylline or tetracycline is present, both GFP and YFP are expressed at near normal levels. When only tetracycline is present, GFP is expressed at near normal levels while YFP expression is close to zero. When only theophylline is present, YFP is expressed at near normal levels while GFP expression is close to zero.
When both tetracycline and theophylline are present, GFP and YFP expression are both close to zero.

==Modularity==

While antiswitches have many advantages, they do lack modularity, the ability to be integrated with any gene without redesigning a new sequence specific part (Isaacs et al. 2004). If you want to regulate a new gene, you have to synthesize a whole new antiswitch because the antisense stem is specific to a particular gene and the aptamer stem is specific to a particular antisense stem.

==References==

Bayer TS and Smolke CD. Programmable ligand-controlled riboregulators of eukaryotic gene expression. Nat Biotechnol. (2005) 3:337-43.

Isaacs FJ, et al. Engineered riboregulators enable post-transcriptional control of gene expression. Nat Biotechnol. (2004) 22:841-47.

[[Post-transcriptional Regulation Technologies - Erin Zwack|Return to Post-transcriptional Regulation Technologies]]

Riboregulators

2007-12-06T18:28:57Z

Erzwack: /* From Concept to Wet Lab */

All information on riboregulators came from Isaacs et al. 2004.

After observing the many and varied naturally occuring post-transcriptional, regulatory RNA systems, Isaacs et al. designed and engineered a modular synthetic system where RNA turns on and off gene expression by controlling translation. The modularity of their system allows any gene to be regulated instead of only a specific gene to which the riboregulator is targeted.

==Design==

The design itself has two components: a short cis-repressed RNA sequence (crRNA) that is inserted upstream of the gene and a transactivating RNA sequence (taRNA) that targets the crRNA and is not attached to the target. The crRNA sequence contains two fundamental components: the complement of the ribosomal binding site (RBS) and a pyrimidine-uracil-nucleotide-purine (YUNR) sequence (figure 9). When not interacting with the taRNA, the complement of the RBS binds to the RBS, causing the crRNA to loop and block the ribosome's access to the RBS. When the RBS is blocked, translation does not occur; the gene expression is off. The YUNR sequence has a complement on the taRNA. When the taRNA finds a crRNA, the interaction with the YUNR sequence begins pulling the crRNA off the RBS.

[[Image:CrRNA.JPG ]]

(Isaacs et al. 2004)

'''Figure 9:''' Cis repressing RNA, cr-RNA, sequence is inserted upstream of the RBS. Part of the cr-RNA (red) complements the RBS and a few bases on either side. This section of the cr-RNA is not an exact complement; thus, the cr-RNA can be peeled off the RBS by the trans activating RNA, ta-RNA. The complementary sequence to YUNR is found in the ta-RNA and begins peeling off the cr-RNA by binding to it (figure 11).

The taRNA is free floating and regulated by an inducible promoter. The inducible promoter allows the researcher to determine under what conditions translation will be allowed. Besides the complement to the YUNR sequence, the taRNA contains a section of high complementarity to section of the crRNA that folds over to cover the RBS, which allows the taRNA to keep the crRNA sequestered from the RBS (figure 10).

[[Image:TaRNA.JPG]]

(Isaacs et al. 2004)

'''Figure 10:''' ta-RNA when not in contact with cr-RNA sequesters the complement to the cr-RNA as it is extremely close to the RBS sequence. When the YUNR complement binds to the YUNR sequence in the cr-RNA, the binding of the cr-RNA to the rest of its complement in the ta-RNA begins, and the cr-RNA is pulled off the RBS (figure 11).

To recap, gene expression is off when there is crRNA upstream of the gene but no taRNA is in the system. In the presence of taRNA, gene expression is turned back on.

[[Image:Riboregulator system.JPG ]]

(Isaacs et al. 2004)

'''Figure 11:''' In a. the cr-RNA is bound to the RBS and blocks ribosomes from translating the mRNA into protein. In b. the ta-RNA comes in contact with the YUNR sequence on the cr-RNA and begins to bind to the rest of the cr-RNA. In c. the ta-RNA fully bound to the cr-RNA and peeled the cr-RNA off the RBS. The RBS is now free for the ribosome to bind to and begin translation.

==From Concept to Wet Lab==

In wet lab, the crRNA sequence was placed in front of the GFP gene and introduced to cells. Using flow cytometry, Isaacs et al. measured fluorescence of cells that had just the GFP gene, the GFP gene with crRNA upstream, the GFP gene with crRNA upstream and taRNA, and no GFP at all. The crRNA decreased fluorescence to near basal levels. When taRNA was present in the system, fluorescence increased by approximately a power of ten. While the fluorescence did not equal fluorescence of cells with just GFP, the repression of gene expression with crRNA and return of expression with taRNA are strong enough to suggest the riboregulator system works.

[[Image:experimental_taRNA.JPG]]

(Isaacs et al. 2004)

'''Figure 12:''' The black curve represents fluoresnce in cells that do not have a GFP gene (ie. the cells natural autofluoresence). The red curve represents fluoresence in cells that have GFP with crRNA upstream. The green curve represents fluoresence in cells that have GFP with crRNA upstream and produce the taRNA. The blue curve represents the fluorescence of cells that have normal GFP minus the cells' autofluoresence.

These experiments were done with pBad and pLac controlling the production of taRNA. As the system worked when the taRNA was under control of either promoter and the crRNA can be inserted upstream of any gene, this system is considered modular.

Finally, riboregulators can control different genes in response to different stinuli by using different crRNA/taRNA pairs as the pairs are specific to each other.

[[Image: Specific.JPG]]

(Isaacs et al. 2004)

'''Figure 13:''' The graph shows both GFP fluorescence (black and white bars) when the ta-RNA promoter, pBad, is off (- arabinose) and on (+ arabinose). All data is normalized to + arabinose GFP an RNA levels. The low level GFP fluorescence when no arabinose is present shows that the efficiency of cr-RNA is not 100% but is still high. As high GFP fluorescence is seen only when high amounts of the matching ta-RNA is present and not simply when any ta-RNA variant is present, this data shows that riboregulator pairs are specific and multiple pairs can be used to regulate multiple genes without fear that any ta-RNA produced would activated all the RNAs and not just its target RNA.

==Modularity==

While all of these experiments were done with the GFP gene, Isaacs et al. designed their riboregulator to be modular, capable of being used with any gene (Isaacs et al. 2004). As the taRNA simply targets the crRNA and the crRNA can be placed in front of any gene, it can be considered modular. The only caveat is that the crRNA construct added to the gene will need to contain the RBS unless the gene's RBS is close enough to the complement to bind to it. Even small changes to a ribosomal binding site can can change the transcription rate of genes (Gardner et al. 2000). If the original RBS is not close enough to the complement in the crRNA and you desire to keep the original transcriptional rate and level, you would have to redesign the crRNA and the taRNA as the complement to the RBS in the crRNA and the complement to the crRNA in the taRNA would need to be different.

==Further Work==

As mentioned in the overview, using regulatory proteins or inducible promoters limits the number of stimuli (molecules) that can be used to determine when expression should occur. Generating taRNA that is ligand controlled like an antiswitch or riboswitch would provide a greater versatility for riboregulator use. Ligand controlled riboregulators may also be more effective as control by a ligand may decrease leaky activity, which occurs with promoters as they always allow for some basal level of transcription. Isaacs has continued working to engineer a ligand controlled version of the riboregulator.

==References==

Gardner, T.S., Cantor, C.R., and Collins, J.J. Construction of a genetic toggle switch in Eschreichia coli. Nature (2000) 403: 339-342.

Isaacs FJ, et al. Engineered riboregulators enable post-transcriptional control of gene expression. Nat Biotechnol. (2004) 22:841-47.

[[Post-transcriptional Regulation Technologies - Erin Zwack|Return to Post-transcriptional Regulation Technologies]]

Riboswitches

2007-12-06T18:28:23Z

Erzwack: /* From Concept to Wet Lab */

All information on Riboswitches came from Desai and Gallivan (2004).

Riboswitches are small sequences in mRNA that bind small molecules to regulate translation and occasionally transcription. Riboswitches occur naturally in both eukaryotes and prokaryotes. Desai and Gallivan hoped to find new synthetic riboswitches (riboswitches with new ligand specificities) by creating libraries of mutant riboswitches and using genetic selection to pick the functional ones of interest. Desai and Gallivan also employed riboswitches to screen for the presence of specific small molecules. In theory riboswitches are perfect because the number of aptamers already in existence and our capbaility to engineer new aptamers through rational design.

==Design==

Reviews of previous research showed that the theophylline aptamer worked in riboswitches in wheat germ, a eukaryote, and ''Bacillus subtilis'', a gram positive bacterium. Desai and Gallivan decided to translate the technology to gram negative bacteria. To do this they cloned the theophylline aptamer five base pairs upstream of the RBS for ''lacZ''. The gene is controlled by a weak promoter and a weak RBS allowing for sensitivity to changes in translation because of the presence of theophylline. The construct was then transformed into ''E. coli''. When theophylline is added, translation should be turned on again.

==From Concept to Wet Lab==

The aptamer for theophylline was inserted in front of the gene for beta-galactosidase gene to create an mRNA with a riboswitch. Beta-dalactosidase activity is dependent on the amount of the enzyme beta-galactosidase; therefore, measurements of beta-galactosidase indicate whether translation of the mRNA is taking place. Testing and comparing beta-galactosidase activity for cells with the riboswitch in the presence of theophylline, caffeine, and no ligand as well as testing and comparing beta-galactosidase activity for cells without the riboswitch under the same conditions demonstrated that the riboswitch does control gene expression in response to theophylline (figure 14). Significant increases in beta-galactosidase activity in cells with the riboswitch were only found when theophylline was added. Because beta-galactosidase activity in the presence of theophylline, caffeine, or no ligand for cells without a riboswitch did not significantly vary, the increase in beta-galactosidase activity in the presence of theophylline for cells with a riboswitch most likely resulted from the theophylline binding to the riboswitch and allowing translation.

[[Image:Plate.JPG]][[Image:Beta.JPG]][[Image:Beta_bar.JPG]]

(Desai and Gallivan 2004)

'''Figure 14:''' '''a)''' On the plate are cells with the riboswitch that were grown in three different conditions: theophylline present, caffeine present, and no ligand present. Cells that were grown in the presence of theophylline have a distinct blue/green color, which indicates beta-galactosidase activity, compared to the cells grown with caffeine or without ligand. The lack of color in the cells grown with caffeine or without ligand indicates that the riboswitch most likely prevents translation of the mRNA encoding beta-galactosidase. The blue color of the cells grown with theophylline indicates that theophylline probably is binding to the riboswitch and allowing for translation.''' b)''' The green line represents cells that have had theophylline added to them while the red line represents cells that had caffeine added. Beta-galactosidase activity's increasing, as measured by Miller Units, significantly only in the presence of high enough concentrations of theophylline but not caffeine suggests that the riboswitch is highly specific for its ligand. '''c)''' When no riboswitch existed in the beta-galactosidase transcript, beta-galactosidase activity was not significantly different for cells grown in the presence of theophylline, caffeine, or no ligand. When a riboswitch existed in the beta-galactosidase transcript, cells grown in the presence of theophylline exhibited significantly greater beta-galactosidase activity than cells grown in the presence of caffeine or no ligand. Beta-galactosidase activity for cells grown in the presence of caffeine did not vary significantly from activity for cells grown without any ligand present.

To further support that the theophylline was interacting with the aptamer and not just increasing protein translation through another route, a single point mutation that ''in vitro'' decreases affinity for theophylline and increases affinity for 3-methylxanthine was introduced to the aptamer. When using this mutant riboswitch, the beta-galactosidase activity, in the presence of theophylline now was almost the same as beta-galactosidase activity without any small molecule while beta-galactosidase in the presence of 3-methylxanthine was significantly higher than base-line. These results suggest that the change in translation as indicated by the increase of beta-galactosidase is controlled by the riboswitch and not some other mechanism.

[[Image:Mutation.JPG]]

(Desai and Gallivan 2004)

'''Figure 15:''' Beta-galactosidase activity was low and basically the same for cells with the mutant riboswitch that had no molecule (black), caffeine (red), or theophylline (green) added. Cells with the mutant riboswitch showed significant increase in beta-galactosidase activity when 3-methylxanthine (blue) was added.

In synthetic biology, parts are often tweaked until the efficiency, detectability, and other properties are enhanced to work as a part in a device. Gallivan's lab worked on optimization of the riboswitch by changing the position of the aptamer in relation to the RBS. The riboswitch showed a greater increase in beta-galactosidase activity when the aptamer was 8 base pairs upstream of the RBS than when the aptamer was either 2 or 5 base pairs upstream. This result could be because of the surrounding bases being purines in this particular transcript or the actual distance.

[[Image:Optimization.JPG]]

(Desai and Gallivan 2004)

'''Figure 16:''' Cells exposed to theophylline (green) showed greater beta-galactosidase activity than cells exposed to caffeine (red) or cells exposed to nothing (black) no matter the distance of the riboswitch from the RBS, but the greatest increase was experienced by cells with the riboswitch 8 base pairs (the farthest tested distance) from the RBS exposed to theophylline.

==Modularity==

An advantage of riboswitches is that they are modular. The aptamer that is in front of the RBS does not have the restriction that it only works in front of specific sequences. A riboswitch can technically be integrated into any mRNA without redesigning the whole riboswitch component unlike with antiswitches. Because riboswitches do not have a complement to any specific RBS, they can function in front of RBS's that vary drastically from each other while Isaac's riboregulators may need to be redesigned to accomodate the different RBS's.

==References==

Desai SK and Gallivan JP. Genetic Screens and Selections for Small Molecules Based on a Synthetic Riboswitch That Activates Protein Translation. J. Am. Chem. Soc.(2004) 126:13247-54.

[[Post-transcriptional Regulation Technologies - Erin Zwack|Return to Post-transcriptional Regulation Technologies]]

Riboswitches

2007-12-06T18:27:56Z

Erzwack: /* From Concept to Wet Lab */

All information on Riboswitches came from Desai and Gallivan (2004).

Riboswitches are small sequences in mRNA that bind small molecules to regulate translation and occasionally transcription. Riboswitches occur naturally in both eukaryotes and prokaryotes. Desai and Gallivan hoped to find new synthetic riboswitches (riboswitches with new ligand specificities) by creating libraries of mutant riboswitches and using genetic selection to pick the functional ones of interest. Desai and Gallivan also employed riboswitches to screen for the presence of specific small molecules. In theory riboswitches are perfect because the number of aptamers already in existence and our capbaility to engineer new aptamers through rational design.

==Design==

Reviews of previous research showed that the theophylline aptamer worked in riboswitches in wheat germ, a eukaryote, and ''Bacillus subtilis'', a gram positive bacterium. Desai and Gallivan decided to translate the technology to gram negative bacteria. To do this they cloned the theophylline aptamer five base pairs upstream of the RBS for ''lacZ''. The gene is controlled by a weak promoter and a weak RBS allowing for sensitivity to changes in translation because of the presence of theophylline. The construct was then transformed into ''E. coli''. When theophylline is added, translation should be turned on again.

==From Concept to Wet Lab==

The aptamer for theophylline was inserted in front of the gene for beta-galactosidase gene to create an mRNA with a riboswitch. Beta-dalactosidase activity is dependent on the amount of the enzyme beta-galactosidase; therefore, measurements of beta-galactosidase indicate whether translation of the mRNA is taking place. Testing and comparing beta-galactosidase activity for cells with the riboswitch in the presence of theophylline, caffeine, and no ligand as well as testing and comparing beta-galactosidase activity for cells without the riboswitch under the same conditions demonstrated that the riboswitch does control gene expression in response to theophylline (figure 14). Significant increases in beta-galactosidase activity in cells with the riboswitch were only found when theophylline was added. Because beta-galactosidase activity in the presence of theophylline, caffeine, or no ligand for cells without a riboswitch did not significantly vary, the increase in beta-galactosidase activity in the presence of theophylline for cells with a riboswitch most likely resulted from the theophylline binding to the riboswitch and allowing translation.

[[Image:Plate.JPG]][[Image:Beta.JPG]][[Image:Beta_bar.JPG]]

(Desai and Gallivan 2004)

'''Figure 14:''' '''a)''' On the plate are cells with the riboswitch that were grown in three different conditions: theophylline present, caffeine present, and no ligand present. Cells that were grown in the presence of theophylline have a distinct blue/green color, which indicates beta-galactosidase activity, compared to the cells grown with caffeine or without ligand. The lack of color in the cells grown with caffeine or without ligand indicates that the riboswitch most likely prevents translation of the mRNA encoding beta-galactosidase. The blue color of the cells grown with theophylline indicates that theophylline probably is binding to the riboswitch and allowing for translation.''' b)''' The green line represents cells that have had theophylline added to them while the red line represents cells that had caffeine added. Beta-galactosidase activity's increasing, as measured by Miller Units, significantly only in the presence of high enough concentrations of theophylline but not caffeine suggests that the riboswitch is highly specific for its ligand. '''c)''' When no riboswitch existed in the beta-galactosidase transcript, beta-galactosidase activity was not significantly different for cells grown in the presence of theophylline, caffeine, or no ligand. When a riboswitch existed in the beta-galactosidase transcript, cells grown in the presence of theophylline exhibited significantly greater beta-galactosidase activity than cells grown in the presence of caffeine or no ligand. Beta-galactosidase activity for cells grown in the presence of caffeine did not vary significantly from activity for cells grown without any ligand present.

To further support that the theophylline was interacting with the aptamer and not just increasing protein translation through another route, a single point mutation that ''in vitro'' decreases affinity for theophylline and increases affinity for 3-methylxanthine was introduced to the aptamer. When using this mutant riboswitch, the beta-galactosidase activity, in the presence of theophylline now was almost the same as beta-galactosidase activity without any small molecule while beta-galactosidase in the presence of 3-methylxanthine was significantly higher than base-line. These results suggest that the change in translation as indicated by the increase of beta-galactosidase is controlled by the riboswitch and not some other mechanism.

[[Image:Mutation.JPG]]

(Desai and Gallivan 2004)

'''Figure 15:''' Beta-galactosidase activity was low and basically the same for cells with the mutant riboswitch that had no molecule (black), caffeine (red), or theophylline (green) added. Cells with the mutant riboswitch showed significant increase in beta-galactosidase activity when 3-methylxanthine (blue) was added.

In synthetic biology, parts are often tweaked until the efficiency, detectability, and other properties are enhanced to work as a part in a device. Gallivan's lab worked on optimization of the riboswitch by changing the position of the aptamer in relation to the RBS. The riboswitch showed a greater increase in beta-galactosidase activity when the aptamer was 8 base pairs upstream of the RBS than when the aptamer was either 2 or 5 base pairs upstream. This result could be because of the surrounding bases being purines in this particular transcript or the actual distance.

[[Image:Optimization.JPG]]

(Desai and Gallivan 2004)

'''Figure 16:''' Cells exposed to theophylline (green) showed greater beta-galactosidase activity than cells exposed to caffeine (red) or cells exposed to nothing (black) no matter the distance of the riboswitch from the RBS, but the greatest increase was experienced by cells with the riboswitch 8 base pairs (the farthest tested distance) from the RBS exposed to theophylline.

An advantage of riboswitches is that they are modular. The aptamer that is in front of the RBS does not have the restriction that it only works in front of specific sequences. A riboswitch can technically be integrated into any mRNA without redesigning the whole riboswitch component unlike with antiswitches. Because riboswitches do not have a complement to any specific RBS, they can function in front of RBS's that vary drastically from each other while Isaac's riboregulators may need to be redesigned to accomodate the different RBS's.

==References==

Desai SK and Gallivan JP. Genetic Screens and Selections for Small Molecules Based on a Synthetic Riboswitch That Activates Protein Translation. J. Am. Chem. Soc.(2004) 126:13247-54.

[[Post-transcriptional Regulation Technologies - Erin Zwack|Return to Post-transcriptional Regulation Technologies]]

Riboswitches

2007-12-06T17:36:46Z

Erzwack: /* From Concept to Wet Lab */

All information on Riboswitches came from Desai and Gallivan (2004).

Riboswitches are small sequences in mRNA that bind small molecules to regulate translation and occasionally transcription. Riboswitches occur naturally in both eukaryotes and prokaryotes. Desai and Gallivan hoped to find new synthetic riboswitches (riboswitches with new ligand specificities) by creating libraries of mutant riboswitches and using genetic selection to pick the functional ones of interest. Desai and Gallivan also employed riboswitches to screen for the presence of specific small molecules. In theory riboswitches are perfect because the number of aptamers already in existence and our capbaility to engineer new aptamers through rational design.

==Design==

Reviews of previous research showed that the theophylline aptamer worked in riboswitches in wheat germ, a eukaryote, and ''Bacillus subtilis'', a gram positive bacterium. Desai and Gallivan decided to translate the technology to gram negative bacteria. To do this they cloned the theophylline aptamer five base pairs upstream of the RBS for ''lacZ''. The gene is controlled by a weak promoter and a weak RBS allowing for sensitivity to changes in translation because of the presence of theophylline. The construct was then transformed into ''E. coli''. When theophylline is added, translation should be turned on again.

==From Concept to Wet Lab==

The aptamer for theophylline was inserted in front of the gene for beta-galactosidase gene to create an mRNA with a riboswitch. Beta-dalactosidase activity is dependent on the amount of the enzyme beta-galactosidase; therefore, measurements of beta-galactosidase indicate whether translation of the mRNA is taking place. Testing and comparing beta-galactosidase activity for cells with the riboswitch in the presence of theophylline, caffeine, and no ligand as well as testing and comparing beta-galactosidase activity for cells without the riboswitch under the same conditions demonstrated that the riboswitch does control gene expression in response to theophylline (figure 14). Significant increases in beta-galactosidase activity in cells with the riboswitch were only found when theophylline was added. Because beta-galactosidase activity in the presence of theophylline, caffeine, or no ligand for cells without a riboswitch did not significantly vary, the increase in beta-galactosidase activity in the presence of theophylline for cells with a riboswitch most likely resulted from the theophylline binding to the riboswitch and allowing translation.

[[Image:Plate.JPG]][[Image:Beta.JPG]][[Image:Beta_bar.JPG]]

(Desai and Gallivan 2004)

'''Figure 14:''' '''a)''' On the plate are cells with the riboswitch that were grown in three different conditions: theophylline present, caffeine present, and no ligand present. Cells that were grown in the presence of theophylline have a distinct blue/green color, which indicates beta-galactosidase activity, compared to the cells grown with caffeine or without ligand. The lack of color in the cells grown with caffeine or without ligand indicates that the riboswitch most likely prevents translation of the mRNA encoding beta-galactosidase. The blue color of the cells grown with theophylline indicates that theophylline probably is binding to the riboswitch and allowing for translation.''' b)''' The green line represents cells that have had theophylline added to them while the red line represents cells that had caffeine added. Beta-galactosidase activity's increasing, as measured by Miller Units, significantly only in the presence of high enough concentrations of theophylline but not caffeine suggests that the riboswitch is highly specific for its ligand. '''c)''' When no riboswitch existed in the beta-galactosidase transcript, beta-galactosidase activity was not significantly different for cells grown in the presence of theophylline, caffeine, or no ligand. When a riboswitch existed in the beta-galactosidase transcript, cells grown in the presence of theophylline exhibited significantly greater beta-galactosidase activity than cells grown in the presence of caffeine or no ligand. Beta-galactosidase activity for cells grown in the presence of caffeine did not vary significantly from activity for cells grown without any ligand present.

To further support that the theophylline was interacting with the aptamer and not just increasing protein translation through another route, a single point mutation that ''in vitro'' decreases affinity for theophylline and increases affinity for 3-methylxanthine was introduced to the aptamer. When using this mutant riboswitch, the beta-galactosidase activity, in the presence of theophylline now was almost the same as beta-galactosidase activity without any small molecule while beta-galactosidase in the presence of 3-methylxanthine was significantly higher than base-line. These results suggest that the change in translation as indicated by the increase of beta-galactosidase is controlled by the riboswitch and not some other mechanism.

[[Image:Mutation.JPG]]

(Desai and Gallivan 2004)

'''Figure 15:''' Beta-galactosidase activity was low and basically the same for cells with the mutant riboswitch that had no molecule (black), caffeine (red), or theophylline (green) added. Cells with the mutant riboswitch showed significant increase in beta-galactosidase activity when 3-methylxanthine (blue) was added.

In synthetic biology, parts are often tweaked until the efficiency, detectability, and other properties are enhanced to work as a part in a device. Gallivan's lab worked on optimization of the riboswitch by changing the position of the aptamer in relation to the RBS. The riboswitch showed a greater increase in beta-galactosidase activity when the aptamer was 8 base pairs upstream of the RBS than when the aptamer was either 2 or 5 base pairs upstream. This result could be because of the surrounding bases being purines in this particular transcript or the actual distance.

[[Image:Optimization.JPG]]

(Desai and Gallivan 2004)

'''Figure 16:''' Cells exposed to theophylline (green) showed greater beta-galactosidase activity than cells exposed to caffeine (red) or cells exposed to nothing (black) no matter the distance of the riboswitch from the RBS, but the greatest increase was experienced by cells with the riboswitch 8 base pairs (the farthest tested distance) from the RBS exposed to theophylline.

==References==

Desai SK and Gallivan JP. Genetic Screens and Selections for Small Molecules Based on a Synthetic Riboswitch That Activates Protein Translation. J. Am. Chem. Soc.(2004) 126:13247-54.

[[Post-transcriptional Regulation Technologies - Erin Zwack|Return to Post-transcriptional Regulation Technologies]]

Post-transcriptional Regulation Technologies - Erin Zwack

2007-12-06T17:36:01Z

Erzwack: /* References */

==Post-transcriptional Regulation Technologies==

==Overview:==

Regulation of translation provides an excellent tool for research on metabolic and other pathways in organisms, and for the production of different sensors by controlling the translation of specific genes depending on cellular conditions. Further development of these types of technology could provide a “knock-down” equivalent to RNAi, which exist in some eukaryotes. A gene of interest could be expressed normally at all times when the regulator is not active; thus, no ill effects will result before the pathway activates if the gene has another purpose as well. Other sythetic biologists could use these technologies to engineer fast-responding, RNA-based biological sensors for environmental chemicals, or novel pathways.

Using RNA regulatory molecules instead of regulatory proteins to control gene expression provides several benefits to synthetic biologists. Regulatory proteins bind to specific sites such as sites on the promoter or sites upstream of the promoter called operators. Control by these proteins can rely heavily on [[CellularMemory:Mathematical Models#Cooperativity_and_Bistability| cooperativity]] (Gardner et al. 2000), that is multiple proteins binding to one site, in order to see an effect. Regulatory RNA molecules on the other hand need a one to one ratio of regulatory molecule to target. As long as the molecule is expressed in an equal or greater amount than the target, the regulatory RNAs will normally be able to bind to their targets and control transcription.

While synthetic biologists could use the regulatory proteins and their binding sites that are found in nature, rational design of more new regulatory proteins is difficult. With the oligo and gene sythesis technology in existence today, RNA can be engineered that complements and thus targets any other RNA sequence. Regulatory proteins are also controlled mainly by promoter when determining whether they are active or not. As only so many inducible promoters exist, their is a small number of stimuli that can be used to determine under what conditions the gene under a regulatory protein's control will be expressed or repressed. An aptamer, RNA sequence that binds to a small molecule such as theophylline, can be used not only to regulate gene expression but can also regulate under what conditions an RNA regulatory molecule is active. New aptamers are easily developed through rational design, and the number in existence is continually increasing and providing new molecules that can act as ligands.

Finally, regulatory proteins stop gene expression before transcription. When the stimulus changes and the gene is expressed (either because a regulatory protein has now bound to or has released its site), the time it takes for the phenotype to be expressed is longer because both transcription and translation must occur instead of just translation. With RNA, the gene expression is halted after transcription. Once the stimulus is removed, the RNA already produced by the gene simply need to be translated.

==Development of Systems==

In most cases, post-transcriptional regulatory mechanisms that were developed and worked in eukaryotes cannot be directly transferred to prokaryotes. Modifications are necessary because eukaryotic and prokaryotic transcription and translation do not follow the exact same path. In eukaryotes, mRNA must have introns spliced out before translation begins; thus, any mechanism that regulates translation has time to bind or manipulate the mRNA (figure 1). In prokaryotes, translation begins as soon as the ribosomal binding site (RBS) is transcribed and accessible to a ribosome (figure 2).

[[Image:Eukaryotic.jpg]]

'''Figure 1:''' Inside of the nucleus of the eukaryote, the gene is transcribed into pre-mRNA, which contain both introns (orange) and exons (red). The pre-mRNA is then modified so that the introns are spliced out and the exons are put together. Finally the mRNA are translated by the ribosome in the cytoplasm.

[[Image:Prokaryote.jpg]]

'''Figure 2:''' In a prokaryotic cell the DNA is transcribed by RNA polymerase. As soon as the polymerase transcribes the ribosomal binding site, a ribosome binds and begins translating the sequence into protein. There is no modification step between transcription and translation.

==Eukaryotes:==

[[Antiswitches]]

==Prokaryotes:==

[[Riboregulators]]

[[Riboswitches]]

==Use of Post-transciptional Regulatory Technologies==

Regulation of translation provides an excellent tool for research on metabolic and other pathways in organisms, and for the production of different sensors by controlling the translation of specific genes depending on cellular conditions. Researchers can turn-off translation of certain genes in response to different pathways being activated, such as metabolic pathways. If a researcher wanted to know if a particular gene was necessary to proper function of a pathway, the aptamer of the antiswitch or riboswitch could be designed to have a molecule produced in the pathway to be its ligand. The gene would be expressed normally at all times when the pathway is not active; thus, no ill effects will result before the pathway activates if the gene has another purpose as well. This would provide a “knock-down” equivalent to RNAi available in some eukaryotes.

Synthetic biologists could engineer fast-responding, RNA-based biological sensors for environmental chemicals, or novel pathways that only activate when the environmental conditions are favorable.

==The Labs==

[http://www.che.caltech.edu/groups/cds/index.htm Smolke]

[http://www.bu.edu/abl/ Collins]

[http://www.bu.edu/abl/files/naturebiotech_isaacs.pdf Dr. Isaacs's Review of RNA Synthetic Biology]

[http://gallivan1.chem.emory.edu/Gallivan%20Lab/Home.html Gallivan]

==References==

[http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=Retrieve&db=pubmed&dopt=AbstractPlus&list_uids=15723047 Bayer TS and Smolke CD. Programmable ligand-controlled riboregulators of eukaryotic gene expression. ''Nat Biotechnol''. (2005) 3:337-43.]

[http://pubs.acs.org/cgi-bin/article.cgi/jacsat/2004/126/i41/pdf/ja048634j.pdf Desai SK and Gallivan JP. Genetic Screens and Selections for Small Molecules Based on a Synthetic Riboswitch That Activates Protein Translation. ''J. Am. Chem. Soc.''(2004) 126:13247-54.]

[http://www.bio.davidson.edu/courses/synthetic/papers/Collins_ToggleSwitch.pdf Gardner, T.S., Cantor, C.R., and Collins, J.J. Construction of a genetic toggle switch in Eschreichia coli. ''Nature'' (2000) 403: 339-342.]

[http://www.bio.davidson.edu/Courses/Synthetic/papers/RNA_Regulation.pdf Isaacs FJ, et al. Engineered riboregulators enable post-transcriptional control of gene expression. ''Nat Biotechnol.'' (2004) 22:841-47.]

[[A Review of Synthetic Biology| Return to Main Page]]

Post-transcriptional Regulation Technologies - Erin Zwack

2007-12-06T17:35:06Z

Erzwack: /* References */

==Post-transcriptional Regulation Technologies==

==Overview:==

Regulation of translation provides an excellent tool for research on metabolic and other pathways in organisms, and for the production of different sensors by controlling the translation of specific genes depending on cellular conditions. Further development of these types of technology could provide a “knock-down” equivalent to RNAi, which exist in some eukaryotes. A gene of interest could be expressed normally at all times when the regulator is not active; thus, no ill effects will result before the pathway activates if the gene has another purpose as well. Other sythetic biologists could use these technologies to engineer fast-responding, RNA-based biological sensors for environmental chemicals, or novel pathways.

Using RNA regulatory molecules instead of regulatory proteins to control gene expression provides several benefits to synthetic biologists. Regulatory proteins bind to specific sites such as sites on the promoter or sites upstream of the promoter called operators. Control by these proteins can rely heavily on [[CellularMemory:Mathematical Models#Cooperativity_and_Bistability| cooperativity]] (Gardner et al. 2000), that is multiple proteins binding to one site, in order to see an effect. Regulatory RNA molecules on the other hand need a one to one ratio of regulatory molecule to target. As long as the molecule is expressed in an equal or greater amount than the target, the regulatory RNAs will normally be able to bind to their targets and control transcription.

While synthetic biologists could use the regulatory proteins and their binding sites that are found in nature, rational design of more new regulatory proteins is difficult. With the oligo and gene sythesis technology in existence today, RNA can be engineered that complements and thus targets any other RNA sequence. Regulatory proteins are also controlled mainly by promoter when determining whether they are active or not. As only so many inducible promoters exist, their is a small number of stimuli that can be used to determine under what conditions the gene under a regulatory protein's control will be expressed or repressed. An aptamer, RNA sequence that binds to a small molecule such as theophylline, can be used not only to regulate gene expression but can also regulate under what conditions an RNA regulatory molecule is active. New aptamers are easily developed through rational design, and the number in existence is continually increasing and providing new molecules that can act as ligands.

Finally, regulatory proteins stop gene expression before transcription. When the stimulus changes and the gene is expressed (either because a regulatory protein has now bound to or has released its site), the time it takes for the phenotype to be expressed is longer because both transcription and translation must occur instead of just translation. With RNA, the gene expression is halted after transcription. Once the stimulus is removed, the RNA already produced by the gene simply need to be translated.

==Development of Systems==

In most cases, post-transcriptional regulatory mechanisms that were developed and worked in eukaryotes cannot be directly transferred to prokaryotes. Modifications are necessary because eukaryotic and prokaryotic transcription and translation do not follow the exact same path. In eukaryotes, mRNA must have introns spliced out before translation begins; thus, any mechanism that regulates translation has time to bind or manipulate the mRNA (figure 1). In prokaryotes, translation begins as soon as the ribosomal binding site (RBS) is transcribed and accessible to a ribosome (figure 2).

[[Image:Eukaryotic.jpg]]

'''Figure 1:''' Inside of the nucleus of the eukaryote, the gene is transcribed into pre-mRNA, which contain both introns (orange) and exons (red). The pre-mRNA is then modified so that the introns are spliced out and the exons are put together. Finally the mRNA are translated by the ribosome in the cytoplasm.

[[Image:Prokaryote.jpg]]

'''Figure 2:''' In a prokaryotic cell the DNA is transcribed by RNA polymerase. As soon as the polymerase transcribes the ribosomal binding site, a ribosome binds and begins translating the sequence into protein. There is no modification step between transcription and translation.

==Eukaryotes:==

[[Antiswitches]]

==Prokaryotes:==

[[Riboregulators]]

[[Riboswitches]]

==Use of Post-transciptional Regulatory Technologies==

Regulation of translation provides an excellent tool for research on metabolic and other pathways in organisms, and for the production of different sensors by controlling the translation of specific genes depending on cellular conditions. Researchers can turn-off translation of certain genes in response to different pathways being activated, such as metabolic pathways. If a researcher wanted to know if a particular gene was necessary to proper function of a pathway, the aptamer of the antiswitch or riboswitch could be designed to have a molecule produced in the pathway to be its ligand. The gene would be expressed normally at all times when the pathway is not active; thus, no ill effects will result before the pathway activates if the gene has another purpose as well. This would provide a “knock-down” equivalent to RNAi available in some eukaryotes.

Synthetic biologists could engineer fast-responding, RNA-based biological sensors for environmental chemicals, or novel pathways that only activate when the environmental conditions are favorable.

==The Labs==

[http://www.che.caltech.edu/groups/cds/index.htm Smolke]

[http://www.bu.edu/abl/ Collins]

[http://www.bu.edu/abl/files/naturebiotech_isaacs.pdf Dr. Isaacs's Review of RNA Synthetic Biology]

[http://gallivan1.chem.emory.edu/Gallivan%20Lab/Home.html Gallivan]

==References==

[http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=Retrieve&db=pubmed&dopt=AbstractPlus&list_uids=15723047 Bayer TS and Smolke CD. Programmable ligand-controlled riboregulators of eukaryotic gene expression. ''Nat Biotechnol''. (2005) 3:337-43.]

[http://pubs.acs.org/cgi-bin/article.cgi/jacsat/2004/126/i41/pdf/ja048634j.pdf Desai SK and Gallivan JP. Genetic Screens and Selections for Small Molecules Based on a Synthetic Riboswitch That Activates Protein Translation. ''J. Am. Chem. Soc.''(2004) 126:13247-54.]

[http://www.bio.davidson.edu/courses/synthetic/papers/Collins_ToggleSwitch.pdf Gardner, T.S., Cantor, C.R., and Collins, J.J. Construction of a genetic toggle switch in Eschreichia coli. ''Nature'' (2000) 403: 339-342.]

[http://www.bio.davidson.edu/Courses/Synthetic/papers/RNA_Regulation.pdf Isaacs FJ, et al. Engineered riboregulators enable post-transcriptional control of gene expression. ''Nat Biotechnol.'' (2004) 22:841-47.]

[[http://gcat.davidson.edu/GcatWiki/index.php/A_Review_of_Synthetic_Biology| Return to Main Page]]

Riboregulators

2007-12-06T16:05:56Z

Erzwack: /* From Concept to Wet Lab */

All information on riboregulators came from Isaacs et al. 2004.

After observing the many and varied naturally occuring post-transcriptional, regulatory RNA systems, Isaacs et al. designed and engineered a modular synthetic system where RNA turns on and off gene expression by controlling translation. The modularity of their system allows any gene to be regulated instead of only a specific gene to which the riboregulator is targeted.

==Design==

The design itself has two components: a short cis-repressed RNA sequence (crRNA) that is inserted upstream of the gene and a transactivating RNA sequence (taRNA) that targets the crRNA and is not attached to the target. The crRNA sequence contains two fundamental components: the complement of the ribosomal binding site (RBS) and a pyrimidine-uracil-nucleotide-purine (YUNR) sequence (figure 9). When not interacting with the taRNA, the complement of the RBS binds to the RBS, causing the crRNA to loop and block the ribosome's access to the RBS. When the RBS is blocked, translation does not occur; the gene expression is off. The YUNR sequence has a complement on the taRNA. When the taRNA finds a crRNA, the interaction with the YUNR sequence begins pulling the crRNA off the RBS.

[[Image:CrRNA.JPG ]]

(Isaacs et al. 2004)

'''Figure 9:''' Cis repressing RNA, cr-RNA, sequence is inserted upstream of the RBS. Part of the cr-RNA (red) complements the RBS and a few bases on either side. This section of the cr-RNA is not an exact complement; thus, the cr-RNA can be peeled off the RBS by the trans activating RNA, ta-RNA. The complementary sequence to YUNR is found in the ta-RNA and begins peeling off the cr-RNA by binding to it (figure 11).

The taRNA is free floating and regulated by an inducible promoter. The inducible promoter allows the researcher to determine under what conditions translation will be allowed. Besides the complement to the YUNR sequence, the taRNA contains a section of high complementarity to section of the crRNA that folds over to cover the RBS, which allows the taRNA to keep the crRNA sequestered from the RBS (figure 10).

[[Image:TaRNA.JPG]]

(Isaacs et al. 2004)

'''Figure 10:''' ta-RNA when not in contact with cr-RNA sequesters the complement to the cr-RNA as it is extremely close to the RBS sequence. When the YUNR complement binds to the YUNR sequence in the cr-RNA, the binding of the cr-RNA to the rest of its complement in the ta-RNA begins, and the cr-RNA is pulled off the RBS (figure 11).

To recap, gene expression is off when there is crRNA upstream of the gene but no taRNA is in the system. In the presence of taRNA, gene expression is turned back on.

[[Image:Riboregulator system.JPG ]]

(Isaacs et al. 2004)

'''Figure 11:''' In a. the cr-RNA is bound to the RBS and blocks ribosomes from translating the mRNA into protein. In b. the ta-RNA comes in contact with the YUNR sequence on the cr-RNA and begins to bind to the rest of the cr-RNA. In c. the ta-RNA fully bound to the cr-RNA and peeled the cr-RNA off the RBS. The RBS is now free for the ribosome to bind to and begin translation.

==From Concept to Wet Lab==

In wet lab, the crRNA sequence was placed in front of the GFP gene and introduced to cells. Using flow cytometry, Isaacs et al. measured fluorescence of cells that had just the GFP gene, the GFP gene with crRNA upstream, the GFP gene with crRNA upstream and taRNA, and no GFP at all. The crRNA decreased fluorescence to near basal levels. When taRNA was present in the system, fluorescence increased by approximately a power of ten. While the fluorescence did not equal fluorescence of cells with just GFP, the repression of gene expression with crRNA and return of expression with taRNA are strong enough to suggest the riboregulator system works.

[[Image:experimental_taRNA.JPG]]

(Isaacs et al. 2004)

'''Figure 12:''' The black curve represents fluoresnce in cells that do not have a GFP gene (ie. the cells natural autofluoresence). The red curve represents fluoresence in cells that have GFP with crRNA upstream. The green curve represents fluoresence in cells that have GFP with crRNA upstream and produce the taRNA. The blue curve represents the fluorescence of cells that have normal GFP minus the cells' autofluoresence.

These experiments were done with pBad and pLac controlling the production of taRNA. As the system worked when the taRNA was under control of either promoter and the crRNA can be inserted upstream of any gene, this system is considered modular.

Finally, riboregulators can control different genes in response to different stinuli by using different crRNA/taRNA pairs as the pairs are specific to each other.

[[Image: Specific.JPG]]

(Isaacs et al. 2004)

'''Figure 13:''' The graph shows both GFP fluorescence (black and white bars) when the ta-RNA promoter, pBad, is off (- arabinose) and on (+ arabinose). All data is normalized to + arabinose GFP an RNA levels. The low level GFP fluorescence when no arabinose is present shows that the efficiency of cr-RNA is not 100% but is still high. As high GFP fluorescence is seen only when high amounts of the matching ta-RNA is present and not simply when any ta-RNA variant is present, this data shows that riboregulator pairs are specific and multiple pairs can be used to regulate multiple genes without fear that any ta-RNA produced would activated all the RNAs and not just its target RNA.

While all of these experiments were done with the GFP gene, Isaacs et al. designed their riboregulator to be modular, capable of being used with any gene (Isaacs et al. 2004). As the taRNA simply targets the crRNA and the crRNA can be placed in front of any gene, it can be considered modular. The only caveat is that the crRNA construct added to the gene will need to contain the RBS unless the gene's RBS is close enough to the complement to bind to it. Even small changes to a ribosomal binding site can can change the transcription rate of genes (Gardner et al. 2000). If the original RBS is not close enough to the complement in the crRNA and you desire to keep the original transcriptional rate and level, you would have to redesign the crRNA and the taRNA as the complement to the RBS in the crRNA and the complement to the crRNA in the taRNA would need to be different.

==Further Work==

As mentioned in the overview, using regulatory proteins or inducible promoters limits the number of stimuli (molecules) that can be used to determine when expression should occur. Generating taRNA that is ligand controlled like an antiswitch or riboswitch would provide a greater versatility for riboregulator use. Ligand controlled riboregulators may also be more effective as control by a ligand may decrease leaky activity, which occurs with promoters as they always allow for some basal level of transcription. Isaacs has continued working to engineer a ligand controlled version of the riboregulator.

==References==

Gardner, T.S., Cantor, C.R., and Collins, J.J. Construction of a genetic toggle switch in Eschreichia coli. Nature (2000) 403: 339-342.

Isaacs FJ, et al. Engineered riboregulators enable post-transcriptional control of gene expression. Nat Biotechnol. (2004) 22:841-47.

[[Post-transcriptional Regulation Technologies - Erin Zwack|Return to Post-transcriptional Regulation Technologies]]

Riboregulators

2007-12-06T16:05:36Z

Erzwack: /* References */

All information on riboregulators came from Isaacs et al. 2004.

After observing the many and varied naturally occuring post-transcriptional, regulatory RNA systems, Isaacs et al. designed and engineered a modular synthetic system where RNA turns on and off gene expression by controlling translation. The modularity of their system allows any gene to be regulated instead of only a specific gene to which the riboregulator is targeted.

==Design==

The design itself has two components: a short cis-repressed RNA sequence (crRNA) that is inserted upstream of the gene and a transactivating RNA sequence (taRNA) that targets the crRNA and is not attached to the target. The crRNA sequence contains two fundamental components: the complement of the ribosomal binding site (RBS) and a pyrimidine-uracil-nucleotide-purine (YUNR) sequence (figure 9). When not interacting with the taRNA, the complement of the RBS binds to the RBS, causing the crRNA to loop and block the ribosome's access to the RBS. When the RBS is blocked, translation does not occur; the gene expression is off. The YUNR sequence has a complement on the taRNA. When the taRNA finds a crRNA, the interaction with the YUNR sequence begins pulling the crRNA off the RBS.

[[Image:CrRNA.JPG ]]

(Isaacs et al. 2004)

'''Figure 9:''' Cis repressing RNA, cr-RNA, sequence is inserted upstream of the RBS. Part of the cr-RNA (red) complements the RBS and a few bases on either side. This section of the cr-RNA is not an exact complement; thus, the cr-RNA can be peeled off the RBS by the trans activating RNA, ta-RNA. The complementary sequence to YUNR is found in the ta-RNA and begins peeling off the cr-RNA by binding to it (figure 11).

The taRNA is free floating and regulated by an inducible promoter. The inducible promoter allows the researcher to determine under what conditions translation will be allowed. Besides the complement to the YUNR sequence, the taRNA contains a section of high complementarity to section of the crRNA that folds over to cover the RBS, which allows the taRNA to keep the crRNA sequestered from the RBS (figure 10).

[[Image:TaRNA.JPG]]

(Isaacs et al. 2004)

'''Figure 10:''' ta-RNA when not in contact with cr-RNA sequesters the complement to the cr-RNA as it is extremely close to the RBS sequence. When the YUNR complement binds to the YUNR sequence in the cr-RNA, the binding of the cr-RNA to the rest of its complement in the ta-RNA begins, and the cr-RNA is pulled off the RBS (figure 11).

To recap, gene expression is off when there is crRNA upstream of the gene but no taRNA is in the system. In the presence of taRNA, gene expression is turned back on.

[[Image:Riboregulator system.JPG ]]

(Isaacs et al. 2004)

'''Figure 11:''' In a. the cr-RNA is bound to the RBS and blocks ribosomes from translating the mRNA into protein. In b. the ta-RNA comes in contact with the YUNR sequence on the cr-RNA and begins to bind to the rest of the cr-RNA. In c. the ta-RNA fully bound to the cr-RNA and peeled the cr-RNA off the RBS. The RBS is now free for the ribosome to bind to and begin translation.

==From Concept to Wet Lab==

In wet lab, the crRNA sequence was placed in front of the GFP gene and introduced to cells. Using flow cytometry, Isaacs et al. measured fluorescence of cells that had just the GFP gene, the GFP gene with crRNA upstream, the GFP gene with crRNA upstream and taRNA, and no GFP at all. The crRNA decreased fluorescence to near basal levels. When taRNA was present in the system, fluorescence increased by approximately a power of ten. While the fluorescence did not equal fluorescence of cells with just GFP, the repression of gene expression with crRNA and return of expression with taRNA are strong enough to suggest the riboregulator system works.

[[Image:experimental_taRNA.JPG]]

(Isaacs et al. 2004)

'''Figure 12:''' The black curve represents fluoresnce in cells that do not have a GFP gene (ie. the cells natural autofluoresence). The red curve represents fluoresence in cells that have GFP with crRNA upstream. The green curve represents fluoresence in cells that have GFP with crRNA upstream and produce the taRNA. The blue curve represents the fluorescence of cells that have normal GFP minus the cells' autofluoresence.

These experiments were done with pBad and pLac controlling the production of taRNA. As the system worked when the taRNA was under control of either promoter and the crRNA can be inserted upstream of any gene, this system is considered modular.

Finally, riboregulators can control different genes in response to different stinuli by using different crRNA/taRNA pairs as the pairs are specific to each other.

[[Image: Specific.JPG]]

(Isaacs et al. 2004)

'''Figure 13:''' The graph shows both GFP fluorescence (black and white bars) when the ta-RNA promoter, pBad, is off (- arabinose) and on (+ arabinose). All data is normalized to + arabinose GFP an RNA levels. The low level GFP fluorescence when no arabinose is present shows that the efficiency of cr-RNA is not 100% but is still high. As high GFP fluorescence is seen only when high amounts of the matching ta-RNA is present and not simply when any ta-RNA variant is present, this data shows that riboregulator pairs are specific and multiple pairs can be used to regulate multiple genes without fear that any ta-RNA produced would activated all the RNAs and not just its target RNA.

While all of these experiments were done with the GFP gene, Isaacs et al. designed their riboregulator to be modular, capable of being used with any gene (Isaacs et al. 2004). As the taRNA simply targets the crRNA and the crRNA can be placed in front of any gene, it can be considered modular. The only caveat is that the crRNA construct added to the gene will need to contain the RBS unless the gene's RBS is close enough to the complement to bind to it. Even small changes to a ribosomal binding site can can change the transcription rate of genes (Gardner et al. 2004). If the original RBS is not close enough to the complement in the crRNA and you desire to keep the original transcriptional rate and level, you would have to redesign the crRNA and the taRNA as the complement to the RBS in the crRNA and the complement to the crRNA in the taRNA would need to be different.

==Further Work==

As mentioned in the overview, using regulatory proteins or inducible promoters limits the number of stimuli (molecules) that can be used to determine when expression should occur. Generating taRNA that is ligand controlled like an antiswitch or riboswitch would provide a greater versatility for riboregulator use. Ligand controlled riboregulators may also be more effective as control by a ligand may decrease leaky activity, which occurs with promoters as they always allow for some basal level of transcription. Isaacs has continued working to engineer a ligand controlled version of the riboregulator.

==References==

Gardner, T.S., Cantor, C.R., and Collins, J.J. Construction of a genetic toggle switch in Eschreichia coli. Nature (2000) 403: 339-342.

Isaacs FJ, et al. Engineered riboregulators enable post-transcriptional control of gene expression. Nat Biotechnol. (2004) 22:841-47.

[[Post-transcriptional Regulation Technologies - Erin Zwack|Return to Post-transcriptional Regulation Technologies]]

Riboregulators

2007-12-06T16:05:16Z

Erzwack: /* From Concept to Wet Lab */

All information on riboregulators came from Isaacs et al. 2004.

After observing the many and varied naturally occuring post-transcriptional, regulatory RNA systems, Isaacs et al. designed and engineered a modular synthetic system where RNA turns on and off gene expression by controlling translation. The modularity of their system allows any gene to be regulated instead of only a specific gene to which the riboregulator is targeted.

==Design==

The design itself has two components: a short cis-repressed RNA sequence (crRNA) that is inserted upstream of the gene and a transactivating RNA sequence (taRNA) that targets the crRNA and is not attached to the target. The crRNA sequence contains two fundamental components: the complement of the ribosomal binding site (RBS) and a pyrimidine-uracil-nucleotide-purine (YUNR) sequence (figure 9). When not interacting with the taRNA, the complement of the RBS binds to the RBS, causing the crRNA to loop and block the ribosome's access to the RBS. When the RBS is blocked, translation does not occur; the gene expression is off. The YUNR sequence has a complement on the taRNA. When the taRNA finds a crRNA, the interaction with the YUNR sequence begins pulling the crRNA off the RBS.

[[Image:CrRNA.JPG ]]

(Isaacs et al. 2004)

'''Figure 9:''' Cis repressing RNA, cr-RNA, sequence is inserted upstream of the RBS. Part of the cr-RNA (red) complements the RBS and a few bases on either side. This section of the cr-RNA is not an exact complement; thus, the cr-RNA can be peeled off the RBS by the trans activating RNA, ta-RNA. The complementary sequence to YUNR is found in the ta-RNA and begins peeling off the cr-RNA by binding to it (figure 11).

The taRNA is free floating and regulated by an inducible promoter. The inducible promoter allows the researcher to determine under what conditions translation will be allowed. Besides the complement to the YUNR sequence, the taRNA contains a section of high complementarity to section of the crRNA that folds over to cover the RBS, which allows the taRNA to keep the crRNA sequestered from the RBS (figure 10).

[[Image:TaRNA.JPG]]

(Isaacs et al. 2004)

'''Figure 10:''' ta-RNA when not in contact with cr-RNA sequesters the complement to the cr-RNA as it is extremely close to the RBS sequence. When the YUNR complement binds to the YUNR sequence in the cr-RNA, the binding of the cr-RNA to the rest of its complement in the ta-RNA begins, and the cr-RNA is pulled off the RBS (figure 11).

To recap, gene expression is off when there is crRNA upstream of the gene but no taRNA is in the system. In the presence of taRNA, gene expression is turned back on.

[[Image:Riboregulator system.JPG ]]

(Isaacs et al. 2004)

'''Figure 11:''' In a. the cr-RNA is bound to the RBS and blocks ribosomes from translating the mRNA into protein. In b. the ta-RNA comes in contact with the YUNR sequence on the cr-RNA and begins to bind to the rest of the cr-RNA. In c. the ta-RNA fully bound to the cr-RNA and peeled the cr-RNA off the RBS. The RBS is now free for the ribosome to bind to and begin translation.

==From Concept to Wet Lab==

In wet lab, the crRNA sequence was placed in front of the GFP gene and introduced to cells. Using flow cytometry, Isaacs et al. measured fluorescence of cells that had just the GFP gene, the GFP gene with crRNA upstream, the GFP gene with crRNA upstream and taRNA, and no GFP at all. The crRNA decreased fluorescence to near basal levels. When taRNA was present in the system, fluorescence increased by approximately a power of ten. While the fluorescence did not equal fluorescence of cells with just GFP, the repression of gene expression with crRNA and return of expression with taRNA are strong enough to suggest the riboregulator system works.

[[Image:experimental_taRNA.JPG]]

(Isaacs et al. 2004)

'''Figure 12:''' The black curve represents fluoresnce in cells that do not have a GFP gene (ie. the cells natural autofluoresence). The red curve represents fluoresence in cells that have GFP with crRNA upstream. The green curve represents fluoresence in cells that have GFP with crRNA upstream and produce the taRNA. The blue curve represents the fluorescence of cells that have normal GFP minus the cells' autofluoresence.

These experiments were done with pBad and pLac controlling the production of taRNA. As the system worked when the taRNA was under control of either promoter and the crRNA can be inserted upstream of any gene, this system is considered modular.

Finally, riboregulators can control different genes in response to different stinuli by using different crRNA/taRNA pairs as the pairs are specific to each other.

[[Image: Specific.JPG]]

(Isaacs et al. 2004)

'''Figure 13:''' The graph shows both GFP fluorescence (black and white bars) when the ta-RNA promoter, pBad, is off (- arabinose) and on (+ arabinose). All data is normalized to + arabinose GFP an RNA levels. The low level GFP fluorescence when no arabinose is present shows that the efficiency of cr-RNA is not 100% but is still high. As high GFP fluorescence is seen only when high amounts of the matching ta-RNA is present and not simply when any ta-RNA variant is present, this data shows that riboregulator pairs are specific and multiple pairs can be used to regulate multiple genes without fear that any ta-RNA produced would activated all the RNAs and not just its target RNA.

While all of these experiments were done with the GFP gene, Isaacs et al. designed their riboregulator to be modular, capable of being used with any gene (Isaacs et al. 2004). As the taRNA simply targets the crRNA and the crRNA can be placed in front of any gene, it can be considered modular. The only caveat is that the crRNA construct added to the gene will need to contain the RBS unless the gene's RBS is close enough to the complement to bind to it. Even small changes to a ribosomal binding site can can change the transcription rate of genes (Gardner et al. 2004). If the original RBS is not close enough to the complement in the crRNA and you desire to keep the original transcriptional rate and level, you would have to redesign the crRNA and the taRNA as the complement to the RBS in the crRNA and the complement to the crRNA in the taRNA would need to be different.

==Further Work==

As mentioned in the overview, using regulatory proteins or inducible promoters limits the number of stimuli (molecules) that can be used to determine when expression should occur. Generating taRNA that is ligand controlled like an antiswitch or riboswitch would provide a greater versatility for riboregulator use. Ligand controlled riboregulators may also be more effective as control by a ligand may decrease leaky activity, which occurs with promoters as they always allow for some basal level of transcription. Isaacs has continued working to engineer a ligand controlled version of the riboregulator.

==References==

Isaacs FJ, et al. Engineered riboregulators enable post-transcriptional control of gene expression. Nat Biotechnol. (2004) 22:841-47.

[[Post-transcriptional Regulation Technologies - Erin Zwack|Return to Post-transcriptional Regulation Technologies]]

Riboregulators

2007-12-06T15:57:41Z

Erzwack: /* From Concept to Wet Lab */

All information on riboregulators came from Isaacs et al. 2004.

After observing the many and varied naturally occuring post-transcriptional, regulatory RNA systems, Isaacs et al. designed and engineered a modular synthetic system where RNA turns on and off gene expression by controlling translation. The modularity of their system allows any gene to be regulated instead of only a specific gene to which the riboregulator is targeted.

==Design==

The design itself has two components: a short cis-repressed RNA sequence (crRNA) that is inserted upstream of the gene and a transactivating RNA sequence (taRNA) that targets the crRNA and is not attached to the target. The crRNA sequence contains two fundamental components: the complement of the ribosomal binding site (RBS) and a pyrimidine-uracil-nucleotide-purine (YUNR) sequence (figure 9). When not interacting with the taRNA, the complement of the RBS binds to the RBS, causing the crRNA to loop and block the ribosome's access to the RBS. When the RBS is blocked, translation does not occur; the gene expression is off. The YUNR sequence has a complement on the taRNA. When the taRNA finds a crRNA, the interaction with the YUNR sequence begins pulling the crRNA off the RBS.

[[Image:CrRNA.JPG ]]

(Isaacs et al. 2004)

'''Figure 9:''' Cis repressing RNA, cr-RNA, sequence is inserted upstream of the RBS. Part of the cr-RNA (red) complements the RBS and a few bases on either side. This section of the cr-RNA is not an exact complement; thus, the cr-RNA can be peeled off the RBS by the trans activating RNA, ta-RNA. The complementary sequence to YUNR is found in the ta-RNA and begins peeling off the cr-RNA by binding to it (figure 11).

The taRNA is free floating and regulated by an inducible promoter. The inducible promoter allows the researcher to determine under what conditions translation will be allowed. Besides the complement to the YUNR sequence, the taRNA contains a section of high complementarity to section of the crRNA that folds over to cover the RBS, which allows the taRNA to keep the crRNA sequestered from the RBS (figure 10).

[[Image:TaRNA.JPG]]

(Isaacs et al. 2004)

'''Figure 10:''' ta-RNA when not in contact with cr-RNA sequesters the complement to the cr-RNA as it is extremely close to the RBS sequence. When the YUNR complement binds to the YUNR sequence in the cr-RNA, the binding of the cr-RNA to the rest of its complement in the ta-RNA begins, and the cr-RNA is pulled off the RBS (figure 11).

To recap, gene expression is off when there is crRNA upstream of the gene but no taRNA is in the system. In the presence of taRNA, gene expression is turned back on.

[[Image:Riboregulator system.JPG ]]

(Isaacs et al. 2004)

'''Figure 11:''' In a. the cr-RNA is bound to the RBS and blocks ribosomes from translating the mRNA into protein. In b. the ta-RNA comes in contact with the YUNR sequence on the cr-RNA and begins to bind to the rest of the cr-RNA. In c. the ta-RNA fully bound to the cr-RNA and peeled the cr-RNA off the RBS. The RBS is now free for the ribosome to bind to and begin translation.

==From Concept to Wet Lab==

In wet lab, the crRNA sequence was placed in front of the GFP gene and introduced to cells. Using flow cytometry, Isaacs et al. measured fluorescence of cells that had just the GFP gene, the GFP gene with crRNA upstream, the GFP gene with crRNA upstream and taRNA, and no GFP at all. The crRNA decreased fluorescence to near basal levels. When taRNA was present in the system, fluorescence increased by approximately a power of ten. While the fluorescence did not equal fluorescence of cells with just GFP, the repression of gene expression with crRNA and return of expression with taRNA are strong enough to suggest the riboregulator system works.

[[Image:experimental_taRNA.JPG]]

(Isaacs et al. 2004)

'''Figure 12:''' The black curve represents fluoresnce in cells that do not have a GFP gene (ie. the cells natural autofluoresence). The red curve represents fluoresence in cells that have GFP with crRNA upstream. The green curve represents fluoresence in cells that have GFP with crRNA upstream and produce the taRNA. The blue curve represents the fluorescence of cells that have normal GFP minus the cells' autofluoresence.

These experiments were done with pBad and pLac controlling the production of taRNA. As the system worked when the taRNA was under control of either promoter and the crRNA can be inserted upstream of any gene, this system is considered modular.

Finally, riboregulators can control different genes in response to different stinuli by using different crRNA/taRNA pairs as the pairs are specific to each other.

[[Image: Specific.JPG]]

(Isaacs et al. 2004)

'''Figure 13:''' The graph shows both GFP fluorescence (black and white bars) when the ta-RNA promoter, pBad, is off (- arabinose) and on (+ arabinose). All data is normalized to + arabinose GFP an RNA levels. The low level GFP fluorescence when no arabinose is present shows that the efficiency of cr-RNA is not 100% but is still high. As high GFP fluorescence is seen only when high amounts of the matching ta-RNA is present and not simply when any ta-RNA variant is present, this data shows that riboregulator pairs are specific and multiple pairs can be used to regulate multiple genes without fear that any ta-RNA produced would activated all the RNAs and not just its target RNA.

While all of these experiments were done with the GFP gene, Isaacs et al. designed their riboregulator to be modular, capable of being used with any gene (Isaacs et al. 2004). As the taRNA simply targets the crRNA and the crRNA can be placed in front of any gene, it can be considered modular. The only caveat is that the crRNA construct added to the gene will need to contain the RBS unless the gene's RBS is close enough to the complement to bind to it. Even small changes to a ribosomal binding site can can change the transcription rate of genes. If the original RBS is not close enough to the complement in the crRNA and you desire to keep the original transcriptional rate and level, you would have to redesign the crRNA and the taRNA as the complement to the RBS in the crRNA and the complement to the crRNA in the taRNA would need to be different.

==Further Work==

As mentioned in the overview, using regulatory proteins or inducible promoters limits the number of stimuli (molecules) that can be used to determine when expression should occur. Generating taRNA that is ligand controlled like an antiswitch or riboswitch would provide a greater versatility for riboregulator use. Ligand controlled riboregulators may also be more effective as control by a ligand may decrease leaky activity, which occurs with promoters as they always allow for some basal level of transcription. Isaacs has continued working to engineer a ligand controlled version of the riboregulator.

==References==

Isaacs FJ, et al. Engineered riboregulators enable post-transcriptional control of gene expression. Nat Biotechnol. (2004) 22:841-47.

[[Post-transcriptional Regulation Technologies - Erin Zwack|Return to Post-transcriptional Regulation Technologies]]

Riboregulators

2007-12-06T15:35:24Z

Erzwack: /* Further Work */

All information on riboregulators came from Isaacs et al. 2004.

After observing the many and varied naturally occuring post-transcriptional, regulatory RNA systems, Isaacs et al. designed and engineered a modular synthetic system where RNA turns on and off gene expression by controlling translation. The modularity of their system allows any gene to be regulated instead of only a specific gene to which the riboregulator is targeted.

==Design==

The design itself has two components: a short cis-repressed RNA sequence (crRNA) that is inserted upstream of the gene and a transactivating RNA sequence (taRNA) that targets the crRNA and is not attached to the target. The crRNA sequence contains two fundamental components: the complement of the ribosomal binding site (RBS) and a pyrimidine-uracil-nucleotide-purine (YUNR) sequence (figure 9). When not interacting with the taRNA, the complement of the RBS binds to the RBS, causing the crRNA to loop and block the ribosome's access to the RBS. When the RBS is blocked, translation does not occur; the gene expression is off. The YUNR sequence has a complement on the taRNA. When the taRNA finds a crRNA, the interaction with the YUNR sequence begins pulling the crRNA off the RBS.

[[Image:CrRNA.JPG ]]

(Isaacs et al. 2004)

'''Figure 9:''' Cis repressing RNA, cr-RNA, sequence is inserted upstream of the RBS. Part of the cr-RNA (red) complements the RBS and a few bases on either side. This section of the cr-RNA is not an exact complement; thus, the cr-RNA can be peeled off the RBS by the trans activating RNA, ta-RNA. The complementary sequence to YUNR is found in the ta-RNA and begins peeling off the cr-RNA by binding to it (figure 11).

The taRNA is free floating and regulated by an inducible promoter. The inducible promoter allows the researcher to determine under what conditions translation will be allowed. Besides the complement to the YUNR sequence, the taRNA contains a section of high complementarity to section of the crRNA that folds over to cover the RBS, which allows the taRNA to keep the crRNA sequestered from the RBS (figure 10).

[[Image:TaRNA.JPG]]

(Isaacs et al. 2004)

'''Figure 10:''' ta-RNA when not in contact with cr-RNA sequesters the complement to the cr-RNA as it is extremely close to the RBS sequence. When the YUNR complement binds to the YUNR sequence in the cr-RNA, the binding of the cr-RNA to the rest of its complement in the ta-RNA begins, and the cr-RNA is pulled off the RBS (figure 11).

To recap, gene expression is off when there is crRNA upstream of the gene but no taRNA is in the system. In the presence of taRNA, gene expression is turned back on.

[[Image:Riboregulator system.JPG ]]

(Isaacs et al. 2004)

'''Figure 11:''' In a. the cr-RNA is bound to the RBS and blocks ribosomes from translating the mRNA into protein. In b. the ta-RNA comes in contact with the YUNR sequence on the cr-RNA and begins to bind to the rest of the cr-RNA. In c. the ta-RNA fully bound to the cr-RNA and peeled the cr-RNA off the RBS. The RBS is now free for the ribosome to bind to and begin translation.

==From Concept to Wet Lab==

In wet lab, the crRNA sequence was placed in front of the GFP gene and introduced to cells. Using flow cytometry, Isaacs et al. measured fluorescence of cells that had just the GFP gene, the GFP gene with crRNA upstream, the GFP gene with crRNA upstream and taRNA, and no GFP at all. The crRNA decreased fluorescence to near basal levels. When taRNA was present in the system, fluorescence increased by approximately a power of ten. While the fluorescence did not equal fluorescence of cells with just GFP, the repression of gene expression with crRNA and return of expression with taRNA are strong enough to suggest the riboregulator system works.

[[Image:experimental_taRNA.JPG]]

(Isaacs et al. 2004)

'''Figure 12:''' The black curve represents fluoresnce in cells that do not have a GFP gene (ie. the cells natural autofluoresence). The red curve represents fluoresence in cells that have GFP with crRNA upstream. The green curve represents fluoresence in cells that have GFP with crRNA upstream and produce the taRNA. The blue curve represents the fluorescence of cells that have normal GFP minus the cells' autofluoresence.

These experiments were done with pBad and pLac controlling the production of taRNA. As the system worked when the taRNA was under control of either promoter and the crRNA can be inserted upstream of any gene, this system is considered modular.

Finally, riboregulators can control different genes in response to different stinuli by using different crRNA/taRNA pairs as the pairs are specific to each other.

[[Image: Specific.JPG]]

(Isaacs et al. 2004)

'''Figure 13:''' The graph shows both GFP fluorescence (black and white bars) when the ta-RNA promoter, pBad, is off (- arabinose) and on (+ arabinose). All data is normalized to + arabinose GFP an RNA levels. The low level GFP fluorescence when no arabinose is present shows that the efficiency of cr-RNA is not 100% but is still high. As high GFP fluorescence is seen only when high amounts of the matching ta-RNA is present and not simply when any ta-RNA variant is present, this data shows that riboregulator pairs are specific and multiple pairs can be used to regulate multiple genes without fear that any ta-RNA produced would activated all the RNAs and not just its target RNA.

==Further Work==

As mentioned in the overview, using regulatory proteins or inducible promoters limits the number of stimuli (molecules) that can be used to determine when expression should occur. Generating taRNA that is ligand controlled like an antiswitch or riboswitch would provide a greater versatility for riboregulator use. Ligand controlled riboregulators may also be more effective as control by a ligand may decrease leaky activity, which occurs with promoters as they always allow for some basal level of transcription. Isaacs has continued working to engineer a ligand controlled version of the riboregulator.

==References==

Isaacs FJ, et al. Engineered riboregulators enable post-transcriptional control of gene expression. Nat Biotechnol. (2004) 22:841-47.

[[Post-transcriptional Regulation Technologies - Erin Zwack|Return to Post-transcriptional Regulation Technologies]]

Riboswitches

2007-12-06T15:33:44Z

Erzwack:

All information on Riboswitches came from Desai and Gallivan (2004).

Riboswitches are small sequences in mRNA that bind small molecules to regulate translation and occasionally transcription. Riboswitches occur naturally in both eukaryotes and prokaryotes. Desai and Gallivan hoped to find new synthetic riboswitches (riboswitches with new ligand specificities) by creating libraries of mutant riboswitches and using genetic selection to pick the functional ones of interest. Desai and Gallivan also employed riboswitches to screen for the presence of specific small molecules. In theory riboswitches are perfect because the number of aptamers already in existence and our capbaility to engineer new aptamers through rational design.

==Design==

Reviews of previous research showed that the theophylline aptamer worked in riboswitches in wheat germ, a eukaryote, and ''Bacillus subtilis'', a gram positive bacterium. Desai and Gallivan decided to translate the technology to gram negative bacteria. To do this they cloned the theophylline aptamer five base pairs upstream of the RBS for ''lacZ''. The gene is controlled by a weak promoter and a weak RBS allowing for sensitivity to changes in translation because of the presence of theophylline. The construct was then transformed into ''E. coli''. When theophylline is added, translation should be turned on again.

==From Concept to Wet Lab==

The aptamer for theophylline was inserted in front of the gene for beta-galactosidase gene to create an mRNA with a riboswitch. Beta-dalactosidase activity is dependent on the amount of the enzyme beta-galactosidase; therefore, measurements of beta-galactosidase indicate whether translation of the mRNA is taking place. Testing and comparing beta-galactosidase activity for cells with the riboswitch in the presence of theophylline, caffeine, and no ligand as well as testing and comparing beta-galactosidase activity for cells without the riboswitch under the same conditions demonstrated that the riboswitch does control gene expression in response to theophylline (figure 14). Significant increases in beta-galactosidase activity in cells with the riboswitch were only found when theophylline was added. Because beta-galactosidase activity in the presence of theophylline, caffeine, or no ligand for cells without a riboswitch did not significantly vary, the increase in beta-galactosidase activity in the presence of theophylline for cells with a riboswitch most likely resulted from the theophylline binding to the riboswitch and allowing translation.

[[Image:Plate.JPG]][[Image:Beta.JPG]][[Image:Beta_bar.JPG]]

(Desai and Gallivan 2004 Permission Pending)

'''Figure 14:''' '''a)''' On the plate are cells with the riboswitch that were grown in three different conditions: theophylline present, caffeine present, and no ligand present. Cells that were grown in the presence of theophylline have a distinct blue/green color, which indicates beta-galactosidase activity, compared to the cells grown with caffeine or without ligand. The lack of color in the cells grown with caffeine or without ligand indicates that the riboswitch most likely prevents translation of the mRNA encoding beta-galactosidase. The blue color of the cells grown with theophylline indicates that theophylline probably is binding to the riboswitch and allowing for translation.''' b)''' The green line represents cells that have had theophylline added to them while the red line represents cells that had caffeine added. Beta-galactosidase activity's increasing, as measured by Miller Units, significantly only in the presence of high enough concentrations of theophylline but not caffeine suggests that the riboswitch is highly specific for its ligand. '''c)''' When no riboswitch existed in the beta-galactosidase transcript, beta-galactosidase activity was not significantly different for cells grown in the presence of theophylline, caffeine, or no ligand. When a riboswitch existed in the beta-galactosidase transcript, cells grown in the presence of theophylline exhibited significantly greater beta-galactosidase activity than cells grown in the presence of caffeine or no ligand. Beta-galactosidase activity for cells grown in the presence of caffeine did not vary significantly from activity for cells grown without any ligand present.

To further support that the theophylline was interacting with the aptamer and not just increasing protein translation through another route, a single point mutation that ''in vitro'' decreases affinity for theophylline and increases affinity for 3-methylxanthine was introduced to the aptamer. When using this mutant riboswitch, the beta-galactosidase activity, in the presence of theophylline now was almost the same as beta-galactosidase activity without any small molecule while beta-galactosidase in the presence of 3-methylxanthine was significantly higher than base-line. These results suggest that the change in translation as indicated by the increase of beta-galactosidase is controlled by the riboswitch and not some other mechanism.

[[Image:Mutation.JPG]]

(Desai and Gallivan 2004 Permission Pending)

'''Figure 15:''' Beta-galactosidase activity was low and basically the same for cells with the mutant riboswitch that had no molecule (black), caffeine (red), or theophylline (green) added. Cells with the mutant riboswitch showed significant increase in beta-galactosidase activity when 3-methylxanthine (blue) was added.

In synthetic biology, parts are often tweaked until the efficiency, detectability, and other properties are enhanced to work as a part in a device. Gallivan's lab worked on optimization of the riboswitch by changing the position of the aptamer in relation to the RBS. The riboswitch showed a greater increase in beta-galactosidase activity when the aptamer was 8 base pairs upstream of the RBS than when the aptamer was either 2 or 5 base pairs upstream. This result could be because of the surrounding bases being purines in this particular transcript or the actual distance.

[[Image:Optimization.JPG]]

(Desai and Gallivan 2004 Permission Pending)

'''Figure 16:''' Cells exposed to theophylline (green) showed greater beta-galactosidase activity than cells exposed to caffeine (red) or cells exposed to nothing (black) no matter the distance of the riboswitch from the RBS, but the greatest increase was experienced by cells with the riboswitch 8 base pairs (the farthest tested distance) from the RBS exposed to theophylline.

==References==

Desai SK and Gallivan JP. Genetic Screens and Selections for Small Molecules Based on a Synthetic Riboswitch That Activates Protein Translation. J. Am. Chem. Soc.(2004) 126:13247-54.

[[Post-transcriptional Regulation Technologies - Erin Zwack|Return to Post-transcriptional Regulation Technologies]]

Riboswitches

2007-12-06T15:33:33Z

Erzwack:

All information on Riboswitches came from Desai and Gallivan (2004).

Riboswitches are small sequences in mRNA that bind small molecules to regulate translation and occasionally transcription. Riboswitches occur naturally in both eukaryotes and prokaryotes. Desai and Gallivan hoped to find new synthetic riboswitches (riboswitches with new ligand specificities) by creating libraries of mutant riboswitches and using genetic selection to pick the functional ones of interest. Desai and Gallivan also employed riboswitches to screen for the presence of specific small molecules. In theory riboswitches are perfect because the number of aptamers already in existence and our capbaility to engineer new aptamers through rational design.

==Design==

Reviews of previous research showed that the theophylline aptamer worked in riboswitches in wheat germ, a eukaryote, and ''Bacillus subtilis'', a gram positive bacterium. Desai and Gallivan decided to translate the technology to gram negative bacteria. To do this they cloned the theophylline aptamer five base pairs upstream of the RBS for ''lacZ''. The gene is controlled by a weak promoter and a weak RBS allowing for sensitivity to changes in translation because of the presence of theophylline. The construct was then transformed into ''E. coli''. When theophylline is added, translation should be turned on again.

==From Concept to Wet Lab==

The aptamer for theophylline was inserted in front of the gene for beta-galactosidase gene to create an mRNA with a riboswitch. Beta-dalactosidase activity is dependent on the amount of the enzyme beta-galactosidase; therefore, measurements of beta-galactosidase indicate whether translation of the mRNA is taking place. Testing and comparing beta-galactosidase activity for cells with the riboswitch in the presence of theophylline, caffeine, and no ligand as well as testing and comparing beta-galactosidase activity for cells without the riboswitch under the same conditions demonstrated that the riboswitch does control gene expression in response to theophylline (figure 14). Significant increases in beta-galactosidase activity in cells with the riboswitch were only found when theophylline was added. Because beta-galactosidase activity in the presence of theophylline, caffeine, or no ligand for cells without a riboswitch did not significantly vary, the increase in beta-galactosidase activity in the presence of theophylline for cells with a riboswitch most likely resulted from the theophylline binding to the riboswitch and allowing translation.

[[Image:Plate.JPG]][[Image:Beta.JPG]][[Image:Beta_bar.JPG]]

(Desai and Gallivan 2004 Permission Pending)

'''Figure 14:''' '''a)''' On the plate are cells with the riboswitch that were grown in three different conditions: theophylline present, caffeine present, and no ligand present. Cells that were grown in the presence of theophylline have a distinct blue/green color, which indicates beta-galactosidase activity, compared to the cells grown with caffeine or without ligand. The lack of color in the cells grown with caffeine or without ligand indicates that the riboswitch most likely prevents translation of the mRNA encoding beta-galactosidase. The blue color of the cells grown with theophylline indicates that theophylline probably is binding to the riboswitch and allowing for translation.''' b)''' The green line represents cells that have had theophylline added to them while the red line represents cells that had caffeine added. Beta-galactosidase activity's increasing, as measured by Miller Units, significantly only in the presence of high enough concentrations of theophylline but not caffeine suggests that the riboswitch is highly specific for its ligand. '''c)''' When no riboswitch existed in the beta-galactosidase transcript, beta-galactosidase activity was not significantly different for cells grown in the presence of theophylline, caffeine, or no ligand. When a riboswitch existed in the beta-galactosidase transcript, cells grown in the presence of theophylline exhibited significantly greater beta-galactosidase activity than cells grown in the presence of caffeine or no ligand. Beta-galactosidase activity for cells grown in the presence of caffeine did not vary significantly from activity for cells grown without any ligand present.

To further support that the theophylline was interacting with the aptamer and not just increasing protein translation through another route, a single point mutation that ''in vitro'' decreases affinity for theophylline and increases affinity for 3-methylxanthine was introduced to the aptamer. When using this mutant riboswitch, the beta-galactosidase activity, in the presence of theophylline now was almost the same as beta-galactosidase activity without any small molecule while beta-galactosidase in the presence of 3-methylxanthine was significantly higher than base-line. These results suggest that the change in translation as indicated by the increase of beta-galactosidase is controlled by the riboswitch and not some other mechanism.

[[Image:Mutation.JPG]]

(Desai and Gallivan 2004 Permission Pending)

'''Figure 15:''' Beta-galactosidase activity was low and basically the same for cells with the mutant riboswitch that had no molecule (black), caffeine (red), or theophylline (green) added. Cells with the mutant riboswitch showed significant increase in beta-galactosidase activity when 3-methylxanthine (blue) was added.

In synthetic biology, parts are often tweaked until the efficiency, detectability, and other properties are enhanced to work as a part in a device. Gallivan's lab worked on optimization of the riboswitch by changing the position of the aptamer in relation to the RBS. The riboswitch showed a greater increase in beta-galactosidase activity when the aptamer was 8 base pairs upstream of the RBS than when the aptamer was either 2 or 5 base pairs upstream. This result could be because of the surrounding bases being purines in this particular transcript or the actual distance.

[[Image:Optimization.JPG]]

(Desai and Gallivan 2004 Permission Pending)

'''Figure 16:''' Cells exposed to theophylline (green) showed greater beta-galactosidase activity than cells exposed to caffeine (red) or cells exposed to nothing (black) no matter the distance of the riboswitch from the RBS, but the greatest increase was experienced by cells with the riboswitch 8 base pairs (the farthest tested distance) from the RBS exposed to theophylline.

==References==

Desai SK and Gallivan JP. Genetic Screens and Selections for Small Molecules Based on a Synthetic Riboswitch That Activates Protein Translation. J. Am. Chem. Soc.(2004) 126:13247-54.

[[Post-transcriptional Regulation Technologies - Erin Zwack|Return to Post-transcriptional Regulation Technologies]]

Riboregulators

2007-12-06T15:32:08Z

Erzwack:

All information on riboregulators came from Isaacs et al. 2004.

After observing the many and varied naturally occuring post-transcriptional, regulatory RNA systems, Isaacs et al. designed and engineered a modular synthetic system where RNA turns on and off gene expression by controlling translation. The modularity of their system allows any gene to be regulated instead of only a specific gene to which the riboregulator is targeted.

==Design==

The design itself has two components: a short cis-repressed RNA sequence (crRNA) that is inserted upstream of the gene and a transactivating RNA sequence (taRNA) that targets the crRNA and is not attached to the target. The crRNA sequence contains two fundamental components: the complement of the ribosomal binding site (RBS) and a pyrimidine-uracil-nucleotide-purine (YUNR) sequence (figure 9). When not interacting with the taRNA, the complement of the RBS binds to the RBS, causing the crRNA to loop and block the ribosome's access to the RBS. When the RBS is blocked, translation does not occur; the gene expression is off. The YUNR sequence has a complement on the taRNA. When the taRNA finds a crRNA, the interaction with the YUNR sequence begins pulling the crRNA off the RBS.

[[Image:CrRNA.JPG ]]

(Isaacs et al. 2004)

'''Figure 9:''' Cis repressing RNA, cr-RNA, sequence is inserted upstream of the RBS. Part of the cr-RNA (red) complements the RBS and a few bases on either side. This section of the cr-RNA is not an exact complement; thus, the cr-RNA can be peeled off the RBS by the trans activating RNA, ta-RNA. The complementary sequence to YUNR is found in the ta-RNA and begins peeling off the cr-RNA by binding to it (figure 11).

The taRNA is free floating and regulated by an inducible promoter. The inducible promoter allows the researcher to determine under what conditions translation will be allowed. Besides the complement to the YUNR sequence, the taRNA contains a section of high complementarity to section of the crRNA that folds over to cover the RBS, which allows the taRNA to keep the crRNA sequestered from the RBS (figure 10).

[[Image:TaRNA.JPG]]

(Isaacs et al. 2004)

'''Figure 10:''' ta-RNA when not in contact with cr-RNA sequesters the complement to the cr-RNA as it is extremely close to the RBS sequence. When the YUNR complement binds to the YUNR sequence in the cr-RNA, the binding of the cr-RNA to the rest of its complement in the ta-RNA begins, and the cr-RNA is pulled off the RBS (figure 11).

To recap, gene expression is off when there is crRNA upstream of the gene but no taRNA is in the system. In the presence of taRNA, gene expression is turned back on.

[[Image:Riboregulator system.JPG ]]

(Isaacs et al. 2004)

'''Figure 11:''' In a. the cr-RNA is bound to the RBS and blocks ribosomes from translating the mRNA into protein. In b. the ta-RNA comes in contact with the YUNR sequence on the cr-RNA and begins to bind to the rest of the cr-RNA. In c. the ta-RNA fully bound to the cr-RNA and peeled the cr-RNA off the RBS. The RBS is now free for the ribosome to bind to and begin translation.

==From Concept to Wet Lab==

In wet lab, the crRNA sequence was placed in front of the GFP gene and introduced to cells. Using flow cytometry, Isaacs et al. measured fluorescence of cells that had just the GFP gene, the GFP gene with crRNA upstream, the GFP gene with crRNA upstream and taRNA, and no GFP at all. The crRNA decreased fluorescence to near basal levels. When taRNA was present in the system, fluorescence increased by approximately a power of ten. While the fluorescence did not equal fluorescence of cells with just GFP, the repression of gene expression with crRNA and return of expression with taRNA are strong enough to suggest the riboregulator system works.

[[Image:experimental_taRNA.JPG]]

(Isaacs et al. 2004)

'''Figure 12:''' The black curve represents fluoresnce in cells that do not have a GFP gene (ie. the cells natural autofluoresence). The red curve represents fluoresence in cells that have GFP with crRNA upstream. The green curve represents fluoresence in cells that have GFP with crRNA upstream and produce the taRNA. The blue curve represents the fluorescence of cells that have normal GFP minus the cells' autofluoresence.

These experiments were done with pBad and pLac controlling the production of taRNA. As the system worked when the taRNA was under control of either promoter and the crRNA can be inserted upstream of any gene, this system is considered modular.

Finally, riboregulators can control different genes in response to different stinuli by using different crRNA/taRNA pairs as the pairs are specific to each other.

[[Image: Specific.JPG]]

(Isaacs et al. 2004)

'''Figure 13:''' The graph shows both GFP fluorescence (black and white bars) when the ta-RNA promoter, pBad, is off (- arabinose) and on (+ arabinose). All data is normalized to + arabinose GFP an RNA levels. The low level GFP fluorescence when no arabinose is present shows that the efficiency of cr-RNA is not 100% but is still high. As high GFP fluorescence is seen only when high amounts of the matching ta-RNA is present and not simply when any ta-RNA variant is present, this data shows that riboregulator pairs are specific and multiple pairs can be used to regulate multiple genes without fear that any ta-RNA produced would activated all the RNAs and not just its target RNA.

==Further Work==

As mentioned in the overview, using regulatory proteins or inducible promoters limits the number of stimuli (molecules) that can be used to determine when expression should occur. Generating taRNA that is ligand controlled like an antiswitch or riboswitch would provide a greater versatility for riboregulator use. Ligand controlled riboregulators may also be more effective as control by a ligand may decrease leaky activity, which occurs with promoters as they always allow for some basal level of transcription. Isaacs has continued work to engineer a ligand controlled version of the riboregulator.

==References==

Isaacs FJ, et al. Engineered riboregulators enable post-transcriptional control of gene expression. Nat Biotechnol. (2004) 22:841-47.

[[Post-transcriptional Regulation Technologies - Erin Zwack|Return to Post-transcriptional Regulation Technologies]]

Riboregulators

2007-12-06T15:31:56Z

Erzwack:

=NOTOC=

All information on riboregulators came from Isaacs et al. 2004.

After observing the many and varied naturally occuring post-transcriptional, regulatory RNA systems, Isaacs et al. designed and engineered a modular synthetic system where RNA turns on and off gene expression by controlling translation. The modularity of their system allows any gene to be regulated instead of only a specific gene to which the riboregulator is targeted.

==Design==

The design itself has two components: a short cis-repressed RNA sequence (crRNA) that is inserted upstream of the gene and a transactivating RNA sequence (taRNA) that targets the crRNA and is not attached to the target. The crRNA sequence contains two fundamental components: the complement of the ribosomal binding site (RBS) and a pyrimidine-uracil-nucleotide-purine (YUNR) sequence (figure 9). When not interacting with the taRNA, the complement of the RBS binds to the RBS, causing the crRNA to loop and block the ribosome's access to the RBS. When the RBS is blocked, translation does not occur; the gene expression is off. The YUNR sequence has a complement on the taRNA. When the taRNA finds a crRNA, the interaction with the YUNR sequence begins pulling the crRNA off the RBS.

[[Image:CrRNA.JPG ]]

(Isaacs et al. 2004)

'''Figure 9:''' Cis repressing RNA, cr-RNA, sequence is inserted upstream of the RBS. Part of the cr-RNA (red) complements the RBS and a few bases on either side. This section of the cr-RNA is not an exact complement; thus, the cr-RNA can be peeled off the RBS by the trans activating RNA, ta-RNA. The complementary sequence to YUNR is found in the ta-RNA and begins peeling off the cr-RNA by binding to it (figure 11).

The taRNA is free floating and regulated by an inducible promoter. The inducible promoter allows the researcher to determine under what conditions translation will be allowed. Besides the complement to the YUNR sequence, the taRNA contains a section of high complementarity to section of the crRNA that folds over to cover the RBS, which allows the taRNA to keep the crRNA sequestered from the RBS (figure 10).

[[Image:TaRNA.JPG]]

(Isaacs et al. 2004)

'''Figure 10:''' ta-RNA when not in contact with cr-RNA sequesters the complement to the cr-RNA as it is extremely close to the RBS sequence. When the YUNR complement binds to the YUNR sequence in the cr-RNA, the binding of the cr-RNA to the rest of its complement in the ta-RNA begins, and the cr-RNA is pulled off the RBS (figure 11).

To recap, gene expression is off when there is crRNA upstream of the gene but no taRNA is in the system. In the presence of taRNA, gene expression is turned back on.

[[Image:Riboregulator system.JPG ]]

(Isaacs et al. 2004)

'''Figure 11:''' In a. the cr-RNA is bound to the RBS and blocks ribosomes from translating the mRNA into protein. In b. the ta-RNA comes in contact with the YUNR sequence on the cr-RNA and begins to bind to the rest of the cr-RNA. In c. the ta-RNA fully bound to the cr-RNA and peeled the cr-RNA off the RBS. The RBS is now free for the ribosome to bind to and begin translation.

==From Concept to Wet Lab==

In wet lab, the crRNA sequence was placed in front of the GFP gene and introduced to cells. Using flow cytometry, Isaacs et al. measured fluorescence of cells that had just the GFP gene, the GFP gene with crRNA upstream, the GFP gene with crRNA upstream and taRNA, and no GFP at all. The crRNA decreased fluorescence to near basal levels. When taRNA was present in the system, fluorescence increased by approximately a power of ten. While the fluorescence did not equal fluorescence of cells with just GFP, the repression of gene expression with crRNA and return of expression with taRNA are strong enough to suggest the riboregulator system works.

[[Image:experimental_taRNA.JPG]]

(Isaacs et al. 2004)

'''Figure 12:''' The black curve represents fluoresnce in cells that do not have a GFP gene (ie. the cells natural autofluoresence). The red curve represents fluoresence in cells that have GFP with crRNA upstream. The green curve represents fluoresence in cells that have GFP with crRNA upstream and produce the taRNA. The blue curve represents the fluorescence of cells that have normal GFP minus the cells' autofluoresence.

These experiments were done with pBad and pLac controlling the production of taRNA. As the system worked when the taRNA was under control of either promoter and the crRNA can be inserted upstream of any gene, this system is considered modular.

Finally, riboregulators can control different genes in response to different stinuli by using different crRNA/taRNA pairs as the pairs are specific to each other.

[[Image: Specific.JPG]]

(Isaacs et al. 2004)

'''Figure 13:''' The graph shows both GFP fluorescence (black and white bars) when the ta-RNA promoter, pBad, is off (- arabinose) and on (+ arabinose). All data is normalized to + arabinose GFP an RNA levels. The low level GFP fluorescence when no arabinose is present shows that the efficiency of cr-RNA is not 100% but is still high. As high GFP fluorescence is seen only when high amounts of the matching ta-RNA is present and not simply when any ta-RNA variant is present, this data shows that riboregulator pairs are specific and multiple pairs can be used to regulate multiple genes without fear that any ta-RNA produced would activated all the RNAs and not just its target RNA.

==Further Work==

As mentioned in the overview, using regulatory proteins or inducible promoters limits the number of stimuli (molecules) that can be used to determine when expression should occur. Generating taRNA that is ligand controlled like an antiswitch or riboswitch would provide a greater versatility for riboregulator use. Ligand controlled riboregulators may also be more effective as control by a ligand may decrease leaky activity, which occurs with promoters as they always allow for some basal level of transcription. Isaacs has continued work to engineer a ligand controlled version of the riboregulator.

==References==

Isaacs FJ, et al. Engineered riboregulators enable post-transcriptional control of gene expression. Nat Biotechnol. (2004) 22:841-47.

[[Post-transcriptional Regulation Technologies - Erin Zwack|Return to Post-transcriptional Regulation Technologies]]

Riboregulators

2007-12-06T15:31:33Z

Erzwack:

==NOTOC==

All information on riboregulators came from Isaacs et al. 2004.

After observing the many and varied naturally occuring post-transcriptional, regulatory RNA systems, Isaacs et al. designed and engineered a modular synthetic system where RNA turns on and off gene expression by controlling translation. The modularity of their system allows any gene to be regulated instead of only a specific gene to which the riboregulator is targeted.

==Design==

The design itself has two components: a short cis-repressed RNA sequence (crRNA) that is inserted upstream of the gene and a transactivating RNA sequence (taRNA) that targets the crRNA and is not attached to the target. The crRNA sequence contains two fundamental components: the complement of the ribosomal binding site (RBS) and a pyrimidine-uracil-nucleotide-purine (YUNR) sequence (figure 9). When not interacting with the taRNA, the complement of the RBS binds to the RBS, causing the crRNA to loop and block the ribosome's access to the RBS. When the RBS is blocked, translation does not occur; the gene expression is off. The YUNR sequence has a complement on the taRNA. When the taRNA finds a crRNA, the interaction with the YUNR sequence begins pulling the crRNA off the RBS.

[[Image:CrRNA.JPG ]]

(Isaacs et al. 2004)

'''Figure 9:''' Cis repressing RNA, cr-RNA, sequence is inserted upstream of the RBS. Part of the cr-RNA (red) complements the RBS and a few bases on either side. This section of the cr-RNA is not an exact complement; thus, the cr-RNA can be peeled off the RBS by the trans activating RNA, ta-RNA. The complementary sequence to YUNR is found in the ta-RNA and begins peeling off the cr-RNA by binding to it (figure 11).

The taRNA is free floating and regulated by an inducible promoter. The inducible promoter allows the researcher to determine under what conditions translation will be allowed. Besides the complement to the YUNR sequence, the taRNA contains a section of high complementarity to section of the crRNA that folds over to cover the RBS, which allows the taRNA to keep the crRNA sequestered from the RBS (figure 10).

[[Image:TaRNA.JPG]]

(Isaacs et al. 2004)

'''Figure 10:''' ta-RNA when not in contact with cr-RNA sequesters the complement to the cr-RNA as it is extremely close to the RBS sequence. When the YUNR complement binds to the YUNR sequence in the cr-RNA, the binding of the cr-RNA to the rest of its complement in the ta-RNA begins, and the cr-RNA is pulled off the RBS (figure 11).

To recap, gene expression is off when there is crRNA upstream of the gene but no taRNA is in the system. In the presence of taRNA, gene expression is turned back on.

[[Image:Riboregulator system.JPG ]]

(Isaacs et al. 2004)

'''Figure 11:''' In a. the cr-RNA is bound to the RBS and blocks ribosomes from translating the mRNA into protein. In b. the ta-RNA comes in contact with the YUNR sequence on the cr-RNA and begins to bind to the rest of the cr-RNA. In c. the ta-RNA fully bound to the cr-RNA and peeled the cr-RNA off the RBS. The RBS is now free for the ribosome to bind to and begin translation.

==From Concept to Wet Lab==

In wet lab, the crRNA sequence was placed in front of the GFP gene and introduced to cells. Using flow cytometry, Isaacs et al. measured fluorescence of cells that had just the GFP gene, the GFP gene with crRNA upstream, the GFP gene with crRNA upstream and taRNA, and no GFP at all. The crRNA decreased fluorescence to near basal levels. When taRNA was present in the system, fluorescence increased by approximately a power of ten. While the fluorescence did not equal fluorescence of cells with just GFP, the repression of gene expression with crRNA and return of expression with taRNA are strong enough to suggest the riboregulator system works.

[[Image:experimental_taRNA.JPG]]

(Isaacs et al. 2004)

'''Figure 12:''' The black curve represents fluoresnce in cells that do not have a GFP gene (ie. the cells natural autofluoresence). The red curve represents fluoresence in cells that have GFP with crRNA upstream. The green curve represents fluoresence in cells that have GFP with crRNA upstream and produce the taRNA. The blue curve represents the fluorescence of cells that have normal GFP minus the cells' autofluoresence.

These experiments were done with pBad and pLac controlling the production of taRNA. As the system worked when the taRNA was under control of either promoter and the crRNA can be inserted upstream of any gene, this system is considered modular.

Finally, riboregulators can control different genes in response to different stinuli by using different crRNA/taRNA pairs as the pairs are specific to each other.

[[Image: Specific.JPG]]

(Isaacs et al. 2004)

'''Figure 13:''' The graph shows both GFP fluorescence (black and white bars) when the ta-RNA promoter, pBad, is off (- arabinose) and on (+ arabinose). All data is normalized to + arabinose GFP an RNA levels. The low level GFP fluorescence when no arabinose is present shows that the efficiency of cr-RNA is not 100% but is still high. As high GFP fluorescence is seen only when high amounts of the matching ta-RNA is present and not simply when any ta-RNA variant is present, this data shows that riboregulator pairs are specific and multiple pairs can be used to regulate multiple genes without fear that any ta-RNA produced would activated all the RNAs and not just its target RNA.

==Further Work==

As mentioned in the overview, using regulatory proteins or inducible promoters limits the number of stimuli (molecules) that can be used to determine when expression should occur. Generating taRNA that is ligand controlled like an antiswitch or riboswitch would provide a greater versatility for riboregulator use. Ligand controlled riboregulators may also be more effective as control by a ligand may decrease leaky activity, which occurs with promoters as they always allow for some basal level of transcription. Isaacs has continued work to engineer a ligand controlled version of the riboregulator.

==References==

Isaacs FJ, et al. Engineered riboregulators enable post-transcriptional control of gene expression. Nat Biotechnol. (2004) 22:841-47.

[[Post-transcriptional Regulation Technologies - Erin Zwack|Return to Post-transcriptional Regulation Technologies]]

Riboregulators

2007-12-06T15:31:18Z

Erzwack:

==NOTOC==

All information on riboregulators came from Isaacs et al. 2004.

After observing the many and varied naturally occuring post-transcriptional, regulatory RNA systems, Isaacs et al. designed and engineered a modular synthetic system where RNA turns on and off gene expression by controlling translation. The modularity of their system allows any gene to be regulated instead of only a specific gene to which the riboregulator is targeted.

==NOTOC==

==Design==

The design itself has two components: a short cis-repressed RNA sequence (crRNA) that is inserted upstream of the gene and a transactivating RNA sequence (taRNA) that targets the crRNA and is not attached to the target. The crRNA sequence contains two fundamental components: the complement of the ribosomal binding site (RBS) and a pyrimidine-uracil-nucleotide-purine (YUNR) sequence (figure 9). When not interacting with the taRNA, the complement of the RBS binds to the RBS, causing the crRNA to loop and block the ribosome's access to the RBS. When the RBS is blocked, translation does not occur; the gene expression is off. The YUNR sequence has a complement on the taRNA. When the taRNA finds a crRNA, the interaction with the YUNR sequence begins pulling the crRNA off the RBS.

[[Image:CrRNA.JPG ]]

(Isaacs et al. 2004)

'''Figure 9:''' Cis repressing RNA, cr-RNA, sequence is inserted upstream of the RBS. Part of the cr-RNA (red) complements the RBS and a few bases on either side. This section of the cr-RNA is not an exact complement; thus, the cr-RNA can be peeled off the RBS by the trans activating RNA, ta-RNA. The complementary sequence to YUNR is found in the ta-RNA and begins peeling off the cr-RNA by binding to it (figure 11).

The taRNA is free floating and regulated by an inducible promoter. The inducible promoter allows the researcher to determine under what conditions translation will be allowed. Besides the complement to the YUNR sequence, the taRNA contains a section of high complementarity to section of the crRNA that folds over to cover the RBS, which allows the taRNA to keep the crRNA sequestered from the RBS (figure 10).

[[Image:TaRNA.JPG]]

(Isaacs et al. 2004)

'''Figure 10:''' ta-RNA when not in contact with cr-RNA sequesters the complement to the cr-RNA as it is extremely close to the RBS sequence. When the YUNR complement binds to the YUNR sequence in the cr-RNA, the binding of the cr-RNA to the rest of its complement in the ta-RNA begins, and the cr-RNA is pulled off the RBS (figure 11).

To recap, gene expression is off when there is crRNA upstream of the gene but no taRNA is in the system. In the presence of taRNA, gene expression is turned back on.

[[Image:Riboregulator system.JPG ]]

(Isaacs et al. 2004)

'''Figure 11:''' In a. the cr-RNA is bound to the RBS and blocks ribosomes from translating the mRNA into protein. In b. the ta-RNA comes in contact with the YUNR sequence on the cr-RNA and begins to bind to the rest of the cr-RNA. In c. the ta-RNA fully bound to the cr-RNA and peeled the cr-RNA off the RBS. The RBS is now free for the ribosome to bind to and begin translation.

==From Concept to Wet Lab==

In wet lab, the crRNA sequence was placed in front of the GFP gene and introduced to cells. Using flow cytometry, Isaacs et al. measured fluorescence of cells that had just the GFP gene, the GFP gene with crRNA upstream, the GFP gene with crRNA upstream and taRNA, and no GFP at all. The crRNA decreased fluorescence to near basal levels. When taRNA was present in the system, fluorescence increased by approximately a power of ten. While the fluorescence did not equal fluorescence of cells with just GFP, the repression of gene expression with crRNA and return of expression with taRNA are strong enough to suggest the riboregulator system works.

[[Image:experimental_taRNA.JPG]]

(Isaacs et al. 2004)

'''Figure 12:''' The black curve represents fluoresnce in cells that do not have a GFP gene (ie. the cells natural autofluoresence). The red curve represents fluoresence in cells that have GFP with crRNA upstream. The green curve represents fluoresence in cells that have GFP with crRNA upstream and produce the taRNA. The blue curve represents the fluorescence of cells that have normal GFP minus the cells' autofluoresence.

These experiments were done with pBad and pLac controlling the production of taRNA. As the system worked when the taRNA was under control of either promoter and the crRNA can be inserted upstream of any gene, this system is considered modular.

Finally, riboregulators can control different genes in response to different stinuli by using different crRNA/taRNA pairs as the pairs are specific to each other.

[[Image: Specific.JPG]]

(Isaacs et al. 2004)

'''Figure 13:''' The graph shows both GFP fluorescence (black and white bars) when the ta-RNA promoter, pBad, is off (- arabinose) and on (+ arabinose). All data is normalized to + arabinose GFP an RNA levels. The low level GFP fluorescence when no arabinose is present shows that the efficiency of cr-RNA is not 100% but is still high. As high GFP fluorescence is seen only when high amounts of the matching ta-RNA is present and not simply when any ta-RNA variant is present, this data shows that riboregulator pairs are specific and multiple pairs can be used to regulate multiple genes without fear that any ta-RNA produced would activated all the RNAs and not just its target RNA.

==Further Work==

As mentioned in the overview, using regulatory proteins or inducible promoters limits the number of stimuli (molecules) that can be used to determine when expression should occur. Generating taRNA that is ligand controlled like an antiswitch or riboswitch would provide a greater versatility for riboregulator use. Ligand controlled riboregulators may also be more effective as control by a ligand may decrease leaky activity, which occurs with promoters as they always allow for some basal level of transcription. Isaacs has continued work to engineer a ligand controlled version of the riboregulator.

==References==

Isaacs FJ, et al. Engineered riboregulators enable post-transcriptional control of gene expression. Nat Biotechnol. (2004) 22:841-47.

[[Post-transcriptional Regulation Technologies - Erin Zwack|Return to Post-transcriptional Regulation Technologies]]

Riboregulators

2007-12-06T15:31:00Z

Erzwack:

All information on riboregulators came from Isaacs et al. 2004.

After observing the many and varied naturally occuring post-transcriptional, regulatory RNA systems, Isaacs et al. designed and engineered a modular synthetic system where RNA turns on and off gene expression by controlling translation. The modularity of their system allows any gene to be regulated instead of only a specific gene to which the riboregulator is targeted.

==NOTOC==

==Design==

The design itself has two components: a short cis-repressed RNA sequence (crRNA) that is inserted upstream of the gene and a transactivating RNA sequence (taRNA) that targets the crRNA and is not attached to the target. The crRNA sequence contains two fundamental components: the complement of the ribosomal binding site (RBS) and a pyrimidine-uracil-nucleotide-purine (YUNR) sequence (figure 9). When not interacting with the taRNA, the complement of the RBS binds to the RBS, causing the crRNA to loop and block the ribosome's access to the RBS. When the RBS is blocked, translation does not occur; the gene expression is off. The YUNR sequence has a complement on the taRNA. When the taRNA finds a crRNA, the interaction with the YUNR sequence begins pulling the crRNA off the RBS.

[[Image:CrRNA.JPG ]]

(Isaacs et al. 2004)

'''Figure 9:''' Cis repressing RNA, cr-RNA, sequence is inserted upstream of the RBS. Part of the cr-RNA (red) complements the RBS and a few bases on either side. This section of the cr-RNA is not an exact complement; thus, the cr-RNA can be peeled off the RBS by the trans activating RNA, ta-RNA. The complementary sequence to YUNR is found in the ta-RNA and begins peeling off the cr-RNA by binding to it (figure 11).

The taRNA is free floating and regulated by an inducible promoter. The inducible promoter allows the researcher to determine under what conditions translation will be allowed. Besides the complement to the YUNR sequence, the taRNA contains a section of high complementarity to section of the crRNA that folds over to cover the RBS, which allows the taRNA to keep the crRNA sequestered from the RBS (figure 10).

[[Image:TaRNA.JPG]]

(Isaacs et al. 2004)

'''Figure 10:''' ta-RNA when not in contact with cr-RNA sequesters the complement to the cr-RNA as it is extremely close to the RBS sequence. When the YUNR complement binds to the YUNR sequence in the cr-RNA, the binding of the cr-RNA to the rest of its complement in the ta-RNA begins, and the cr-RNA is pulled off the RBS (figure 11).

To recap, gene expression is off when there is crRNA upstream of the gene but no taRNA is in the system. In the presence of taRNA, gene expression is turned back on.

[[Image:Riboregulator system.JPG ]]

(Isaacs et al. 2004)

'''Figure 11:''' In a. the cr-RNA is bound to the RBS and blocks ribosomes from translating the mRNA into protein. In b. the ta-RNA comes in contact with the YUNR sequence on the cr-RNA and begins to bind to the rest of the cr-RNA. In c. the ta-RNA fully bound to the cr-RNA and peeled the cr-RNA off the RBS. The RBS is now free for the ribosome to bind to and begin translation.

==From Concept to Wet Lab==

In wet lab, the crRNA sequence was placed in front of the GFP gene and introduced to cells. Using flow cytometry, Isaacs et al. measured fluorescence of cells that had just the GFP gene, the GFP gene with crRNA upstream, the GFP gene with crRNA upstream and taRNA, and no GFP at all. The crRNA decreased fluorescence to near basal levels. When taRNA was present in the system, fluorescence increased by approximately a power of ten. While the fluorescence did not equal fluorescence of cells with just GFP, the repression of gene expression with crRNA and return of expression with taRNA are strong enough to suggest the riboregulator system works.

[[Image:experimental_taRNA.JPG]]

(Isaacs et al. 2004)

'''Figure 12:''' The black curve represents fluoresnce in cells that do not have a GFP gene (ie. the cells natural autofluoresence). The red curve represents fluoresence in cells that have GFP with crRNA upstream. The green curve represents fluoresence in cells that have GFP with crRNA upstream and produce the taRNA. The blue curve represents the fluorescence of cells that have normal GFP minus the cells' autofluoresence.

These experiments were done with pBad and pLac controlling the production of taRNA. As the system worked when the taRNA was under control of either promoter and the crRNA can be inserted upstream of any gene, this system is considered modular.

Finally, riboregulators can control different genes in response to different stinuli by using different crRNA/taRNA pairs as the pairs are specific to each other.

[[Image: Specific.JPG]]

(Isaacs et al. 2004)

'''Figure 13:''' The graph shows both GFP fluorescence (black and white bars) when the ta-RNA promoter, pBad, is off (- arabinose) and on (+ arabinose). All data is normalized to + arabinose GFP an RNA levels. The low level GFP fluorescence when no arabinose is present shows that the efficiency of cr-RNA is not 100% but is still high. As high GFP fluorescence is seen only when high amounts of the matching ta-RNA is present and not simply when any ta-RNA variant is present, this data shows that riboregulator pairs are specific and multiple pairs can be used to regulate multiple genes without fear that any ta-RNA produced would activated all the RNAs and not just its target RNA.

==Further Work==

As mentioned in the overview, using regulatory proteins or inducible promoters limits the number of stimuli (molecules) that can be used to determine when expression should occur. Generating taRNA that is ligand controlled like an antiswitch or riboswitch would provide a greater versatility for riboregulator use. Ligand controlled riboregulators may also be more effective as control by a ligand may decrease leaky activity, which occurs with promoters as they always allow for some basal level of transcription. Isaacs has continued work to engineer a ligand controlled version of the riboregulator.

==References==

Isaacs FJ, et al. Engineered riboregulators enable post-transcriptional control of gene expression. Nat Biotechnol. (2004) 22:841-47.

[[Post-transcriptional Regulation Technologies - Erin Zwack|Return to Post-transcriptional Regulation Technologies]]

Riboregulators

2007-12-06T15:30:41Z

Erzwack:

All information on riboregulators came from Isaacs et al. 2004.

After observing the many and varied naturally occuring post-transcriptional, regulatory RNA systems, Isaacs et al. designed and engineered a modular synthetic system where RNA turns on and off gene expression by controlling translation. The modularity of their system allows any gene to be regulated instead of only a specific gene to which the riboregulator is targeted.

==Design==

The design itself has two components: a short cis-repressed RNA sequence (crRNA) that is inserted upstream of the gene and a transactivating RNA sequence (taRNA) that targets the crRNA and is not attached to the target. The crRNA sequence contains two fundamental components: the complement of the ribosomal binding site (RBS) and a pyrimidine-uracil-nucleotide-purine (YUNR) sequence (figure 9). When not interacting with the taRNA, the complement of the RBS binds to the RBS, causing the crRNA to loop and block the ribosome's access to the RBS. When the RBS is blocked, translation does not occur; the gene expression is off. The YUNR sequence has a complement on the taRNA. When the taRNA finds a crRNA, the interaction with the YUNR sequence begins pulling the crRNA off the RBS.

[[Image:CrRNA.JPG ]]

(Isaacs et al. 2004)

'''Figure 9:''' Cis repressing RNA, cr-RNA, sequence is inserted upstream of the RBS. Part of the cr-RNA (red) complements the RBS and a few bases on either side. This section of the cr-RNA is not an exact complement; thus, the cr-RNA can be peeled off the RBS by the trans activating RNA, ta-RNA. The complementary sequence to YUNR is found in the ta-RNA and begins peeling off the cr-RNA by binding to it (figure 11).

The taRNA is free floating and regulated by an inducible promoter. The inducible promoter allows the researcher to determine under what conditions translation will be allowed. Besides the complement to the YUNR sequence, the taRNA contains a section of high complementarity to section of the crRNA that folds over to cover the RBS, which allows the taRNA to keep the crRNA sequestered from the RBS (figure 10).

[[Image:TaRNA.JPG]]

(Isaacs et al. 2004)

'''Figure 10:''' ta-RNA when not in contact with cr-RNA sequesters the complement to the cr-RNA as it is extremely close to the RBS sequence. When the YUNR complement binds to the YUNR sequence in the cr-RNA, the binding of the cr-RNA to the rest of its complement in the ta-RNA begins, and the cr-RNA is pulled off the RBS (figure 11).

To recap, gene expression is off when there is crRNA upstream of the gene but no taRNA is in the system. In the presence of taRNA, gene expression is turned back on.

[[Image:Riboregulator system.JPG ]]

(Isaacs et al. 2004)

'''Figure 11:''' In a. the cr-RNA is bound to the RBS and blocks ribosomes from translating the mRNA into protein. In b. the ta-RNA comes in contact with the YUNR sequence on the cr-RNA and begins to bind to the rest of the cr-RNA. In c. the ta-RNA fully bound to the cr-RNA and peeled the cr-RNA off the RBS. The RBS is now free for the ribosome to bind to and begin translation.

==From Concept to Wet Lab==

In wet lab, the crRNA sequence was placed in front of the GFP gene and introduced to cells. Using flow cytometry, Isaacs et al. measured fluorescence of cells that had just the GFP gene, the GFP gene with crRNA upstream, the GFP gene with crRNA upstream and taRNA, and no GFP at all. The crRNA decreased fluorescence to near basal levels. When taRNA was present in the system, fluorescence increased by approximately a power of ten. While the fluorescence did not equal fluorescence of cells with just GFP, the repression of gene expression with crRNA and return of expression with taRNA are strong enough to suggest the riboregulator system works.

[[Image:experimental_taRNA.JPG]]

(Isaacs et al. 2004)

'''Figure 12:''' The black curve represents fluoresnce in cells that do not have a GFP gene (ie. the cells natural autofluoresence). The red curve represents fluoresence in cells that have GFP with crRNA upstream. The green curve represents fluoresence in cells that have GFP with crRNA upstream and produce the taRNA. The blue curve represents the fluorescence of cells that have normal GFP minus the cells' autofluoresence.

These experiments were done with pBad and pLac controlling the production of taRNA. As the system worked when the taRNA was under control of either promoter and the crRNA can be inserted upstream of any gene, this system is considered modular.

Finally, riboregulators can control different genes in response to different stinuli by using different crRNA/taRNA pairs as the pairs are specific to each other.

[[Image: Specific.JPG]]

(Isaacs et al. 2004)

'''Figure 13:''' The graph shows both GFP fluorescence (black and white bars) when the ta-RNA promoter, pBad, is off (- arabinose) and on (+ arabinose). All data is normalized to + arabinose GFP an RNA levels. The low level GFP fluorescence when no arabinose is present shows that the efficiency of cr-RNA is not 100% but is still high. As high GFP fluorescence is seen only when high amounts of the matching ta-RNA is present and not simply when any ta-RNA variant is present, this data shows that riboregulator pairs are specific and multiple pairs can be used to regulate multiple genes without fear that any ta-RNA produced would activated all the RNAs and not just its target RNA.

==Further Work==

As mentioned in the overview, using regulatory proteins or inducible promoters limits the number of stimuli (molecules) that can be used to determine when expression should occur. Generating taRNA that is ligand controlled like an antiswitch or riboswitch would provide a greater versatility for riboregulator use. Ligand controlled riboregulators may also be more effective as control by a ligand may decrease leaky activity, which occurs with promoters as they always allow for some basal level of transcription. Isaacs has continued work to engineer a ligand controlled version of the riboregulator.

==References==

Isaacs FJ, et al. Engineered riboregulators enable post-transcriptional control of gene expression. Nat Biotechnol. (2004) 22:841-47.

[[Post-transcriptional Regulation Technologies - Erin Zwack|Return to Post-transcriptional Regulation Technologies]]

Antiswitches

2007-12-06T15:29:27Z

Erzwack: /* References */

All information about antiswitches came from Bayer and Smolke (2005).

Antiswitches, trans-RNA molecules that regulate translation of mRNA based on ligands, were first developed by Smolke and Bayer (2005) in Saccharomyces cerevisiae. Two types of antiswitches were engineered: on-switches and off-switches. On-switches turn on gene expression in the presence of the ligand while off-switches turn off gene expression in the presence of ligand. Control by the ligand allows researchers to regulate pathways’ protein production with less leakiness,low level transcription even when “off”, than using a specific promoter.

==Design==

Antiswitches are made of an aptamer and two stems: the aptamer stem and the antisense stem (figure 3).

[[Image:Switch.JPG]]

(Bayer and Smolke 2005 Permission Pending)

'''Figure 3:''' The antisense stem can bind to itself (duplex). The antisense stem also contains the complement to the RNA that a researcher desires to regulate. The aptamer stem swings either toward the antisense stem and disrupts the duplex or swings away from the antisense stem and allows the antisense stem to duplex depending on whether the aptamer is bound to its ligand.

The aptamer is the sequence that binds the ligand and causes a conformational (shape) change of the antiswitch molecule. Aptamers can be highly specific for their particular molecules. The theophylline aptamer used by Smolke and Bayer can distinguish between caffeine and theophylline, which differ by a single methyl.

[[Image:Caffeine.JPG]]

'''Figure 4.''' Caffeine (a) and Theophylline (b) differ by one methyl group (circled in red).

While Smolke and Bayer mainly used the aptamer for the ligand theophylline, aptamers for many other molecules are now being generated using rational design. The large number of possible aptamers provides versatility in what will control gene expression.

The antisense stem contains a sequence that complements a targeted RNA transcript and a second sequence that sequesters this complementary sequence to keep it from binding the transcript. When the antisense stem is not duplexed with itself, it prevents translation by binding to the complementary mRNA.

The aptamer stem is a short sequence that complements a portion of the sequestering sequence of the antisense stem. In an off-switch, the aptamer stem swings towards the antisense stem and displaces the portion that complements the targeted transcript when the liand binds to the aptamer. In an on-switch, the aptamer swings away from the antisense stem when the ligand bids to the aptamer; thus, the antisense stem can duplex and is no longer free to bind to the transcript.

[[Image:ANTI.jpg]]

(Bayer and Smolke 2005 Permission Pending)

'''Figure 5:''' a) An inactive off-switch. The antisense stem is duplexed with itself. b) The same switch after being activated by theophylline (blue ellipse). The antisense stem is duplexed with the mRNA; thus, translation is stopped.

==From Concept to Wet Lab==

Smolke and Bayer synthesized genes for antiswitches with antisense stems that contained complements to either GFP or YFP transcripts and the aptamer to either theophylline or tetracycline. These genes were then transformed into '''Sacchromyces cerevisea'''. Several experiments, including dose-response curves to the appropriate ligand and fluoresence measurements of cells when exposed to both the appropriate ligand and wrong ligand, demonstrated that these antiswitches effectively regulate translation of their specific target in response to only the correct ligand.

In the dose-response experiments for off-switches when enough ligand (~ 1 mM theophylline or tetracycline depending on the switch) was present, the inactive off-switches became active and relative GFP expression dropped to almost zero. These experimental results support the antiswitch technology being functional. As off-switches with theophylline and tetracycline binding aptamers worked, the versatility of antiswitches is supported (figure 6).

[[Image:Offswitch_experiment.JPG]]

(Bayer and Smolke 2005 Permission Pending)

'''Figure 6:''' The red line represents the tetracycline controlled switch while blue line represents the theophylline controlled switch. When the concentration of ligand is high enough, enough switches are active and binding to mRNA to stop translation of the gene.

In the dose-response experiments for the on-switches when ~ 1 mM of theophylline was added to cells containing the inactive on-switch, the cells relative expression of GFP jumped from near zero to approximately 0.9. This data show that the on-switch is also functional (figure 7).

[[Image:Onswitch_experiment.JPG]]

(Bayer and Smolke 2005 Permission Pending)

'''Figure 7:''' The red line represents the onswitch while the blue Line represents the offswitch. When a high enough concentration of ligand is present, enough on-switches are activated and have thus released their target mRNA for detectable near normal translation to occur. The off-switch follows the opposite path as in figure 6.

Finally, antiswitches in eukaryotes can be combined to regulate multiple genes for different conditions like the taRNA/crRNA system in prokaryotes. By simply switching the aptamer and antisense stem targeting sequence, the switch now controls a different gene by a different stimulus. Off-switch experiments using a theophylline aptamer with a GFP targeting antisense stem and a tetracycline aptamer with a YFP targeting antisense stem showed that the switches regulate the genes independently and can be used in combination to create complex regulatory mechanisms (figure 8).

[[Image:Combinatorial.JPG]]

(Bayer and Smolke 2005 Permission Pending)

'''Figure 8:''' a) On the left, theophylline activates the antiswitch's aptamer, which then binds to the GFP transcript and prevents translation. On the right, tetracycline binds to the antiswitch's aptamer, which then binds to the YFP transcript and prevents translation. If only one ligand is present (either theophylline or tetracycline), only one gene's transcripts (either GFP or YFP)is not translated. b) When no theophylline or tetracycline is present, both GFP and YFP are expressed at near normal levels. When only tetracycline is present, GFP is expressed at near normal levels while YFP expression is close to zero. When only theophylline is present, YFP is expressed at near normal levels while GFP expression is close to zero.
When both tetracycline and theophylline are present, GFP and YFP expression are both close to zero.

While antiswitches have many advantages, they do lack modularity, the ability to be integrated with any gene without redesigning a new sequence specific part (Isaacs et al. 2004). If you want to regulate a new gene, you have to synthesize a whole new antiswitch because the antisense stem is specific to a particular gene and the aptamer stem is specific to a particular antisense stem.

==References==

Bayer TS and Smolke CD. Programmable ligand-controlled riboregulators of eukaryotic gene expression. Nat Biotechnol. (2005) 3:337-43.

Isaacs FJ, et al. Engineered riboregulators enable post-transcriptional control of gene expression. Nat Biotechnol. (2004) 22:841-47.

[[Post-transcriptional Regulation Technologies - Erin Zwack|Return to Post-transcriptional Regulation Technologies]]

Antiswitches

2007-12-06T15:28:47Z

Erzwack: /* From Concept to Wet Lab */

All information about antiswitches came from Bayer and Smolke (2005).

Antiswitches, trans-RNA molecules that regulate translation of mRNA based on ligands, were first developed by Smolke and Bayer (2005) in Saccharomyces cerevisiae. Two types of antiswitches were engineered: on-switches and off-switches. On-switches turn on gene expression in the presence of the ligand while off-switches turn off gene expression in the presence of ligand. Control by the ligand allows researchers to regulate pathways’ protein production with less leakiness,low level transcription even when “off”, than using a specific promoter.

==Design==

Antiswitches are made of an aptamer and two stems: the aptamer stem and the antisense stem (figure 3).

[[Image:Switch.JPG]]

(Bayer and Smolke 2005 Permission Pending)

'''Figure 3:''' The antisense stem can bind to itself (duplex). The antisense stem also contains the complement to the RNA that a researcher desires to regulate. The aptamer stem swings either toward the antisense stem and disrupts the duplex or swings away from the antisense stem and allows the antisense stem to duplex depending on whether the aptamer is bound to its ligand.

The aptamer is the sequence that binds the ligand and causes a conformational (shape) change of the antiswitch molecule. Aptamers can be highly specific for their particular molecules. The theophylline aptamer used by Smolke and Bayer can distinguish between caffeine and theophylline, which differ by a single methyl.

[[Image:Caffeine.JPG]]

'''Figure 4.''' Caffeine (a) and Theophylline (b) differ by one methyl group (circled in red).

While Smolke and Bayer mainly used the aptamer for the ligand theophylline, aptamers for many other molecules are now being generated using rational design. The large number of possible aptamers provides versatility in what will control gene expression.

The antisense stem contains a sequence that complements a targeted RNA transcript and a second sequence that sequesters this complementary sequence to keep it from binding the transcript. When the antisense stem is not duplexed with itself, it prevents translation by binding to the complementary mRNA.

The aptamer stem is a short sequence that complements a portion of the sequestering sequence of the antisense stem. In an off-switch, the aptamer stem swings towards the antisense stem and displaces the portion that complements the targeted transcript when the liand binds to the aptamer. In an on-switch, the aptamer swings away from the antisense stem when the ligand bids to the aptamer; thus, the antisense stem can duplex and is no longer free to bind to the transcript.

[[Image:ANTI.jpg]]

(Bayer and Smolke 2005 Permission Pending)

'''Figure 5:''' a) An inactive off-switch. The antisense stem is duplexed with itself. b) The same switch after being activated by theophylline (blue ellipse). The antisense stem is duplexed with the mRNA; thus, translation is stopped.

==From Concept to Wet Lab==

Smolke and Bayer synthesized genes for antiswitches with antisense stems that contained complements to either GFP or YFP transcripts and the aptamer to either theophylline or tetracycline. These genes were then transformed into '''Sacchromyces cerevisea'''. Several experiments, including dose-response curves to the appropriate ligand and fluoresence measurements of cells when exposed to both the appropriate ligand and wrong ligand, demonstrated that these antiswitches effectively regulate translation of their specific target in response to only the correct ligand.

In the dose-response experiments for off-switches when enough ligand (~ 1 mM theophylline or tetracycline depending on the switch) was present, the inactive off-switches became active and relative GFP expression dropped to almost zero. These experimental results support the antiswitch technology being functional. As off-switches with theophylline and tetracycline binding aptamers worked, the versatility of antiswitches is supported (figure 6).

[[Image:Offswitch_experiment.JPG]]

(Bayer and Smolke 2005 Permission Pending)

'''Figure 6:''' The red line represents the tetracycline controlled switch while blue line represents the theophylline controlled switch. When the concentration of ligand is high enough, enough switches are active and binding to mRNA to stop translation of the gene.

In the dose-response experiments for the on-switches when ~ 1 mM of theophylline was added to cells containing the inactive on-switch, the cells relative expression of GFP jumped from near zero to approximately 0.9. This data show that the on-switch is also functional (figure 7).

[[Image:Onswitch_experiment.JPG]]

(Bayer and Smolke 2005 Permission Pending)

'''Figure 7:''' The red line represents the onswitch while the blue Line represents the offswitch. When a high enough concentration of ligand is present, enough on-switches are activated and have thus released their target mRNA for detectable near normal translation to occur. The off-switch follows the opposite path as in figure 6.

Finally, antiswitches in eukaryotes can be combined to regulate multiple genes for different conditions like the taRNA/crRNA system in prokaryotes. By simply switching the aptamer and antisense stem targeting sequence, the switch now controls a different gene by a different stimulus. Off-switch experiments using a theophylline aptamer with a GFP targeting antisense stem and a tetracycline aptamer with a YFP targeting antisense stem showed that the switches regulate the genes independently and can be used in combination to create complex regulatory mechanisms (figure 8).

[[Image:Combinatorial.JPG]]

(Bayer and Smolke 2005 Permission Pending)

'''Figure 8:''' a) On the left, theophylline activates the antiswitch's aptamer, which then binds to the GFP transcript and prevents translation. On the right, tetracycline binds to the antiswitch's aptamer, which then binds to the YFP transcript and prevents translation. If only one ligand is present (either theophylline or tetracycline), only one gene's transcripts (either GFP or YFP)is not translated. b) When no theophylline or tetracycline is present, both GFP and YFP are expressed at near normal levels. When only tetracycline is present, GFP is expressed at near normal levels while YFP expression is close to zero. When only theophylline is present, YFP is expressed at near normal levels while GFP expression is close to zero.
When both tetracycline and theophylline are present, GFP and YFP expression are both close to zero.

While antiswitches have many advantages, they do lack modularity, the ability to be integrated with any gene without redesigning a new sequence specific part (Isaacs et al. 2004). If you want to regulate a new gene, you have to synthesize a whole new antiswitch because the antisense stem is specific to a particular gene and the aptamer stem is specific to a particular antisense stem.

==References==

Bayer TS and Smolke CD. Programmable ligand-controlled riboregulators of eukaryotic gene expression. Nat Biotechnol. (2005) 3:337-43.

[[Post-transcriptional Regulation Technologies - Erin Zwack|Return to Post-transcriptional Regulation Technologies]]

Antiswitches

2007-12-06T15:22:47Z

Erzwack:

All information about antiswitches came from Bayer and Smolke (2005).

Antiswitches, trans-RNA molecules that regulate translation of mRNA based on ligands, were first developed by Smolke and Bayer (2005) in Saccharomyces cerevisiae. Two types of antiswitches were engineered: on-switches and off-switches. On-switches turn on gene expression in the presence of the ligand while off-switches turn off gene expression in the presence of ligand. Control by the ligand allows researchers to regulate pathways’ protein production with less leakiness,low level transcription even when “off”, than using a specific promoter.

==Design==

Antiswitches are made of an aptamer and two stems: the aptamer stem and the antisense stem (figure 3).

[[Image:Switch.JPG]]

(Bayer and Smolke 2005 Permission Pending)

'''Figure 3:''' The antisense stem can bind to itself (duplex). The antisense stem also contains the complement to the RNA that a researcher desires to regulate. The aptamer stem swings either toward the antisense stem and disrupts the duplex or swings away from the antisense stem and allows the antisense stem to duplex depending on whether the aptamer is bound to its ligand.

The aptamer is the sequence that binds the ligand and causes a conformational (shape) change of the antiswitch molecule. Aptamers can be highly specific for their particular molecules. The theophylline aptamer used by Smolke and Bayer can distinguish between caffeine and theophylline, which differ by a single methyl.

[[Image:Caffeine.JPG]]

'''Figure 4.''' Caffeine (a) and Theophylline (b) differ by one methyl group (circled in red).

While Smolke and Bayer mainly used the aptamer for the ligand theophylline, aptamers for many other molecules are now being generated using rational design. The large number of possible aptamers provides versatility in what will control gene expression.

The antisense stem contains a sequence that complements a targeted RNA transcript and a second sequence that sequesters this complementary sequence to keep it from binding the transcript. When the antisense stem is not duplexed with itself, it prevents translation by binding to the complementary mRNA.

The aptamer stem is a short sequence that complements a portion of the sequestering sequence of the antisense stem. In an off-switch, the aptamer stem swings towards the antisense stem and displaces the portion that complements the targeted transcript when the liand binds to the aptamer. In an on-switch, the aptamer swings away from the antisense stem when the ligand bids to the aptamer; thus, the antisense stem can duplex and is no longer free to bind to the transcript.

[[Image:ANTI.jpg]]

(Bayer and Smolke 2005 Permission Pending)

'''Figure 5:''' a) An inactive off-switch. The antisense stem is duplexed with itself. b) The same switch after being activated by theophylline (blue ellipse). The antisense stem is duplexed with the mRNA; thus, translation is stopped.

==From Concept to Wet Lab==

Smolke and Bayer synthesized genes for antiswitches with antisense stems that contained complements to either GFP or YFP transcripts and the aptamer to either theophylline or tetracycline. These genes were then transformed into '''Sacchromyces cerevisea'''. Several experiments, including dose-response curves to the appropriate ligand and fluoresence measurements of cells when exposed to both the appropriate ligand and wrong ligand, demonstrated that these antiswitches effectively regulate translation of their specific target in response to only the correct ligand.

In the dose-response experiments for off-switches when enough ligand (~ 1 mM theophylline or tetracycline depending on the switch) was present, the inactive off-switches became active and relative GFP expression dropped to almost zero. These experimental results support the antiswitch technology being functional. As off-switches with theophylline and tetracycline binding aptamers worked, the versatility of antiswitches is supported (figure 6).

[[Image:Offswitch_experiment.JPG]]

(Bayer and Smolke 2005 Permission Pending)

'''Figure 6:''' The red line represents the tetracycline controlled switch while blue line represents the theophylline controlled switch. When the concentration of ligand is high enough, enough switches are active and binding to mRNA to stop translation of the gene.

In the dose-response experiments for the on-switches when ~ 1 mM of theophylline was added to cells containing the inactive on-switch, the cells relative expression of GFP jumped from near zero to approximately 0.9. This data show that the on-switch is also functional (figure 7).

[[Image:Onswitch_experiment.JPG]]

(Bayer and Smolke 2005 Permission Pending)

'''Figure 7:''' The red line represents the onswitch while the blue Line represents the offswitch. When a high enough concentration of ligand is present, enough on-switches are activated and have thus released their target mRNA for detectable near normal translation to occur. The off-switch follows the opposite path as in figure 6.

Finally, antiswitches in eukaryotes can be combined to regulate multiple genes for different conditions like the taRNA/crRNA system in prokaryotes. By simply switching the aptamer and antisense stem targeting sequence, the switch now controls a different gene by a different stimulus. Off-switch experiments using a theophylline aptamer with a GFP targeting antisense stem and a tetracycline aptamer with a YFP targeting antisense stem showed that the switches regulate the genes independently and can be used in combination to create complex regulatory mechanisms (figure 8).

[[Image:Combinatorial.JPG]]

(Bayer and Smolke 2005 Permission Pending)

'''Figure 8:''' a) On the left, theophylline activates the antiswitch's aptamer, which then binds to the GFP transcript and prevents translation. On the right, tetracycline binds to the antiswitch's aptamer, which then binds to the YFP transcript and prevents translation. If only one ligand is present (either theophylline or tetracycline), only one gene's transcripts (either GFP or YFP)is not translated. b) When no theophylline or tetracycline is present, both GFP and YFP are expressed at near normal levels. When only tetracycline is present, GFP is expressed at near normal levels while YFP expression is close to zero. When only theophylline is present, YFP is expressed at near normal levels while GFP expression is close to zero.
When both tetracycline and theophylline are present, GFP and YFP expression are both close to zero.

While antiswitches have many advantages, they do lack modularity. If you want to regulate a new gene, you have to synthesize a whole new antiswitch because the antisense stem is specific to a particular gene and the aptamer stem is specific to a particular antisense stem.

==References==

Bayer TS and Smolke CD. Programmable ligand-controlled riboregulators of eukaryotic gene expression. Nat Biotechnol. (2005) 3:337-43.

[[Post-transcriptional Regulation Technologies - Erin Zwack|Return to Post-transcriptional Regulation Technologies]]

Antiswitches

2007-12-06T14:47:14Z

Erzwack: /* From Concept to Wet Lab */

Antiswitches, trans-RNA molecules that regulate translation of mRNA based on ligands, were first developed by Smolke and Bayer (2005) in Saccharomyces cerevisiae. Two types of antiswitches were engineered: on-switches and off-switches. On-switches turn on gene expression in the presence of the ligand while off-switches turn off gene expression in the presence of ligand. Control by the ligand allows researchers to regulate pathways’ protein production with less leakiness,low level transcription even when “off”, than using a specific promoter.

==Design==

Antiswitches are made of an aptamer and two stems: the aptamer stem and the antisense stem (figure 3).

[[Image:Switch.JPG]]

(Bayer and Smolke 2005 Permission Pending)

'''Figure 3:''' The antisense stem can bind to itself (duplex). The antisense stem also contains the complement to the RNA that a researcher desires to regulate. The aptamer stem swings either toward the antisense stem and disrupts the duplex or swings away from the antisense stem and allows the antisense stem to duplex depending on whether the aptamer is bound to its ligand.

The aptamer is the sequence that binds the ligand and causes a conformational (shape) change of the antiswitch molecule. Aptamers can be highly specific for their particular molecules. The theophylline aptamer used by Smolke and Bayer can distinguish between caffeine and theophylline, which differ by a single methyl.

[[Image:Caffeine.JPG]]

'''Figure 4.''' Caffeine (a) and Theophylline (b) differ by one methyl group (circled in red).

While Smolke and Bayer mainly used the aptamer for the ligand theophylline, aptamers for many other molecules are now being generated using rational design. The large number of possible aptamers provides versatility in what will control gene expression.

The antisense stem contains a sequence that complements a targeted RNA transcript and a second sequence that sequesters this complementary sequence to keep it from binding the transcript. When the antisense stem is not duplexed with itself, it prevents translation by binding to the complementary mRNA.

The aptamer stem is a short sequence that complements a portion of the sequestering sequence of the antisense stem. In an off-switch, the aptamer stem swings towards the antisense stem and displaces the portion that complements the targeted transcript when the liand binds to the aptamer. In an on-switch, the aptamer swings away from the antisense stem when the ligand bids to the aptamer; thus, the antisense stem can duplex and is no longer free to bind to the transcript.

[[Image:ANTI.jpg]]

(Bayer and Smolke 2005 Permission Pending)

'''Figure 5:''' a) An inactive off-switch. The antisense stem is duplexed with itself. b) The same switch after being activated by theophylline (blue ellipse). The antisense stem is duplexed with the mRNA; thus, translation is stopped.

==From Concept to Wet Lab==

Smolke and Bayer synthesized genes for antiswitches with antisense stems that contained complements to either GFP or YFP transcripts and the aptamer to either theophylline or tetracycline. These genes were then transformed into '''Sacchromyces cerevisea'''. Several experiments, including dose-response curves to the appropriate ligand and fluoresence measurements of cells when exposed to both the appropriate ligand and wrong ligand, demonstrated that these antiswitches effectively regulate translation of their specific target in response to only the correct ligand.

In the dose-response experiments for off-switches when enough ligand (~ 1 mM theophylline or tetracycline depending on the switch) was present, the inactive off-switches became active and relative GFP expression dropped to almost zero. These experimental results support the antiswitch technology being functional. As off-switches with theophylline and tetracycline binding aptamers worked, the versatility of antiswitches is supported (figure 6).

[[Image:Offswitch_experiment.JPG]]

(Bayer and Smolke 2005 Permission Pending)

'''Figure 6:''' The red line represents the tetracycline controlled switch while blue line represents the theophylline controlled switch. When the concentration of ligand is high enough, enough switches are active and binding to mRNA to stop translation of the gene.

In the dose-response experiments for the on-switches when ~ 1 mM of theophylline was added to cells containing the inactive on-switch, the cells relative expression of GFP jumped from near zero to approximately 0.9. This data show that the on-switch is also functional (figure 7).

[[Image:Onswitch_experiment.JPG]]

(Bayer and Smolke 2005 Permission Pending)

'''Figure 7:''' The red line represents the onswitch while the blue Line represents the offswitch. When a high enough concentration of ligand is present, enough on-switches are activated and have thus released their target mRNA for detectable near normal translation to occur. The off-switch follows the opposite path as in figure 6.

Finally, antiswitches in eukaryotes can be combined to regulate multiple genes for different conditions like the taRNA/crRNA system in prokaryotes. By simply switching the aptamer and antisense stem targeting sequence, the switch now controls a different gene by a different stimulus. Off-switch experiments using a theophylline aptamer with a GFP targeting antisense stem and a tetracycline aptamer with a YFP targeting antisense stem showed that the switches regulate the genes independently and can be used in combination to create complex regulatory mechanisms (figure 8).

[[Image:Combinatorial.JPG]]

(Bayer and Smolke 2005 Permission Pending)

'''Figure 8:''' a) On the left, theophylline activates the antiswitch's aptamer, which then binds to the GFP transcript and prevents translation. On the right, tetracycline binds to the antiswitch's aptamer, which then binds to the YFP transcript and prevents translation. If only one ligand is present (either theophylline or tetracycline), only one gene's transcripts (either GFP or YFP)is not translated. b) When no theophylline or tetracycline is present, both GFP and YFP are expressed at near normal levels. When only tetracycline is present, GFP is expressed at near normal levels while YFP expression is close to zero. When only theophylline is present, YFP is expressed at near normal levels while GFP expression is close to zero.
When both tetracycline and theophylline are present, GFP and YFP expression are both close to zero.

While antiswitches have many advantages, they do lack modularity. If you want to regulate a new gene, you have to synthesize a whole new antiswitch because the antisense stem is specific to a particular gene and the aptamer stem is specific to a particular antisense stem.

==References==

Bayer TS and Smolke CD. Programmable ligand-controlled riboregulators of eukaryotic gene expression. Nat Biotechnol. (2005) 3:337-43.

[[Post-transcriptional Regulation Technologies - Erin Zwack|Return to Post-transcriptional Regulation Technologies]]

Post-transcriptional Regulation Technologies - Erin Zwack

2007-12-06T04:32:54Z

Erzwack: /* References */

==Post-transcriptional Regulation Technologies==

==Overview:==

Regulation of translation provides an excellent tool for research on metabolic and other pathways in organisms, and for the production of different sensors by controlling the translation of specific genes depending on cellular conditions. Further development of these types of technology could provide a “knock-down” equivalent to RNAi, which exist in some eukaryotes. A gene of interest could be expressed normally at all times when the regulator is not active; thus, no ill effects will result before the pathway activates if the gene has another purpose as well. Other sythetic biologists could use these technologies to engineer fast-responding, RNA-based biological sensors for environmental chemicals, or novel pathways.

Using RNA regulatory molecules instead of regulatory proteins to control gene expression provides several benefits to synthetic biologists. Regulatory proteins bind to specific sites such as sites on the promoter or sites upstream of the promoter called operators. Control by these proteins can rely heavily on [[CellularMemory:Mathematical Models#Cooperativity_and_Bistability| cooperativity]] (Gardner et al. 2000), that is multiple proteins binding to one site, in order to see an effect. Regulatory RNA molecules on the other hand need a one to one ratio of regulatory molecule to target. As long as the molecule is expressed in an equal or greater amount than the target, the regulatory RNAs will normally be able to bind to their targets and control transcription.

While synthetic biologists could use the regulatory proteins and their binding sites that are found in nature, rational design of more new regulatory proteins is difficult. With the oligo and gene sythesis technology in existence today, RNA can be engineered that complements and thus targets any other RNA sequence. Regulatory proteins are also controlled mainly by promoter when determining whether they are active or not. As only so many inducible promoters exist, their is a small number of stimuli that can be used to determine under what conditions the gene under a regulatory protein's control will be expressed or repressed. An aptamer, RNA sequence that binds to a small molecule such as theophylline, can be used not only to regulate gene expression but can also regulate under what conditions an RNA regulatory molecule is active. New aptamers are easily developed through rational design, and the number in existence is continually increasing and providing new molecules that can act as ligands.

Finally, regulatory proteins stop gene expression before transcription. When the stimulus changes and the gene is expressed (either because a regulatory protein has now bound to or has released its site), the time it takes for the phenotype to be expressed is longer because both transcription and translation must occur instead of just translation. With RNA, the gene expression is halted after transcription. Once the stimulus is removed, the RNA already produced by the gene simply need to be translated.

==Development of Systems==

In most cases, post-transcriptional regulatory mechanisms that were developed and worked in eukaryotes cannot be directly transferred to prokaryotes. Modifications are necessary because eukaryotic and prokaryotic transcription and translation do not follow the exact same path. In eukaryotes, mRNA must have introns spliced out before translation begins; thus, any mechanism that regulates translation has time to bind or manipulate the mRNA (figure 1). In prokaryotes, translation begins as soon as the ribosomal binding site (RBS) is transcribed and accessible to a ribosome (figure 2).

[[Image:Eukaryotic.jpg]]

'''Figure 1:''' Inside of the nucleus of the eukaryote, the gene is transcribed into pre-mRNA, which contain both introns (orange) and exons (red). The pre-mRNA is then modified so that the introns are spliced out and the exons are put together. Finally the mRNA are translated by the ribosome in the cytoplasm.

[[Image:Prokaryote.jpg]]

'''Figure 2:''' In a prokaryotic cell the DNA is transcribed by RNA polymerase. As soon as the polymerase transcribes the ribosomal binding site, a ribosome binds and begins translating the sequence into protein. There is no modification step between transcription and translation.

==Eukaryotes:==

[[Antiswitches]]

==Prokaryotes:==

[[Riboregulators]]

[[Riboswitches]]

==Use of Post-transciptional Regulatory Technologies==

Regulation of translation provides an excellent tool for research on metabolic and other pathways in organisms, and for the production of different sensors by controlling the translation of specific genes depending on cellular conditions. Researchers can turn-off translation of certain genes in response to different pathways being activated, such as metabolic pathways. If a researcher wanted to know if a particular gene was necessary to proper function of a pathway, the aptamer of the antiswitch or riboswitch could be designed to have a molecule produced in the pathway to be its ligand. The gene would be expressed normally at all times when the pathway is not active; thus, no ill effects will result before the pathway activates if the gene has another purpose as well. This would provide a “knock-down” equivalent to RNAi available in some eukaryotes.

Synthetic biologists could engineer fast-responding, RNA-based biological sensors for environmental chemicals, or novel pathways that only activate when the environmental conditions are favorable.

==The Labs==

[http://www.che.caltech.edu/groups/cds/index.htm Smolke]

[http://www.bu.edu/abl/ Collins]

[http://www.bu.edu/abl/files/naturebiotech_isaacs.pdf Dr. Isaacs's Review of RNA Synthetic Biology]

[http://gallivan1.chem.emory.edu/Gallivan%20Lab/Home.html Gallivan]

==References==

[http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=Retrieve&db=pubmed&dopt=AbstractPlus&list_uids=15723047 Bayer TS and Smolke CD. Programmable ligand-controlled riboregulators of eukaryotic gene expression. ''Nat Biotechnol''. (2005) 3:337-43.]

[http://pubs.acs.org/cgi-bin/article.cgi/jacsat/2004/126/i41/pdf/ja048634j.pdf Desai SK and Gallivan JP. Genetic Screens and Selections for Small Molecules Based on a Synthetic Riboswitch That Activates Protein Translation. ''J. Am. Chem. Soc.''(2004) 126:13247-54.]

[http://www.bio.davidson.edu/courses/synthetic/papers/Collins_ToggleSwitch.pdf Gardner, T.S., Cantor, C.R., and Collins, J.J. Construction of a genetic toggle switch in Eschreichia coli. ''Nature'' (2000) 403: 339-342.]

[http://www.bio.davidson.edu/Courses/Synthetic/papers/RNA_Regulation.pdf Isaacs FJ, et al. Engineered riboregulators enable post-transcriptional control of gene expression. ''Nat Biotechnol.'' (2004) 22:841-47.]

[[Davidson College Synthetic Biology Seminar (Fall 2007)| Return to Main Page]]

Post-transcriptional Regulation Technologies - Erin Zwack

2007-12-06T04:31:54Z

Erzwack: /* References */

==Post-transcriptional Regulation Technologies==

==Overview:==

Regulation of translation provides an excellent tool for research on metabolic and other pathways in organisms, and for the production of different sensors by controlling the translation of specific genes depending on cellular conditions. Further development of these types of technology could provide a “knock-down” equivalent to RNAi, which exist in some eukaryotes. A gene of interest could be expressed normally at all times when the regulator is not active; thus, no ill effects will result before the pathway activates if the gene has another purpose as well. Other sythetic biologists could use these technologies to engineer fast-responding, RNA-based biological sensors for environmental chemicals, or novel pathways.

Using RNA regulatory molecules instead of regulatory proteins to control gene expression provides several benefits to synthetic biologists. Regulatory proteins bind to specific sites such as sites on the promoter or sites upstream of the promoter called operators. Control by these proteins can rely heavily on [[CellularMemory:Mathematical Models#Cooperativity_and_Bistability| cooperativity]] (Gardner et al. 2000), that is multiple proteins binding to one site, in order to see an effect. Regulatory RNA molecules on the other hand need a one to one ratio of regulatory molecule to target. As long as the molecule is expressed in an equal or greater amount than the target, the regulatory RNAs will normally be able to bind to their targets and control transcription.

While synthetic biologists could use the regulatory proteins and their binding sites that are found in nature, rational design of more new regulatory proteins is difficult. With the oligo and gene sythesis technology in existence today, RNA can be engineered that complements and thus targets any other RNA sequence. Regulatory proteins are also controlled mainly by promoter when determining whether they are active or not. As only so many inducible promoters exist, their is a small number of stimuli that can be used to determine under what conditions the gene under a regulatory protein's control will be expressed or repressed. An aptamer, RNA sequence that binds to a small molecule such as theophylline, can be used not only to regulate gene expression but can also regulate under what conditions an RNA regulatory molecule is active. New aptamers are easily developed through rational design, and the number in existence is continually increasing and providing new molecules that can act as ligands.

Finally, regulatory proteins stop gene expression before transcription. When the stimulus changes and the gene is expressed (either because a regulatory protein has now bound to or has released its site), the time it takes for the phenotype to be expressed is longer because both transcription and translation must occur instead of just translation. With RNA, the gene expression is halted after transcription. Once the stimulus is removed, the RNA already produced by the gene simply need to be translated.

==Development of Systems==

In most cases, post-transcriptional regulatory mechanisms that were developed and worked in eukaryotes cannot be directly transferred to prokaryotes. Modifications are necessary because eukaryotic and prokaryotic transcription and translation do not follow the exact same path. In eukaryotes, mRNA must have introns spliced out before translation begins; thus, any mechanism that regulates translation has time to bind or manipulate the mRNA (figure 1). In prokaryotes, translation begins as soon as the ribosomal binding site (RBS) is transcribed and accessible to a ribosome (figure 2).

[[Image:Eukaryotic.jpg]]

'''Figure 1:''' Inside of the nucleus of the eukaryote, the gene is transcribed into pre-mRNA, which contain both introns (orange) and exons (red). The pre-mRNA is then modified so that the introns are spliced out and the exons are put together. Finally the mRNA are translated by the ribosome in the cytoplasm.

[[Image:Prokaryote.jpg]]

'''Figure 2:''' In a prokaryotic cell the DNA is transcribed by RNA polymerase. As soon as the polymerase transcribes the ribosomal binding site, a ribosome binds and begins translating the sequence into protein. There is no modification step between transcription and translation.

==Eukaryotes:==

[[Antiswitches]]

==Prokaryotes:==

[[Riboregulators]]

[[Riboswitches]]

==Use of Post-transciptional Regulatory Technologies==

Regulation of translation provides an excellent tool for research on metabolic and other pathways in organisms, and for the production of different sensors by controlling the translation of specific genes depending on cellular conditions. Researchers can turn-off translation of certain genes in response to different pathways being activated, such as metabolic pathways. If a researcher wanted to know if a particular gene was necessary to proper function of a pathway, the aptamer of the antiswitch or riboswitch could be designed to have a molecule produced in the pathway to be its ligand. The gene would be expressed normally at all times when the pathway is not active; thus, no ill effects will result before the pathway activates if the gene has another purpose as well. This would provide a “knock-down” equivalent to RNAi available in some eukaryotes.

Synthetic biologists could engineer fast-responding, RNA-based biological sensors for environmental chemicals, or novel pathways that only activate when the environmental conditions are favorable.

==The Labs==

[http://www.che.caltech.edu/groups/cds/index.htm Smolke]

[http://www.bu.edu/abl/ Collins]

[http://www.bu.edu/abl/files/naturebiotech_isaacs.pdf Dr. Isaacs's Review of RNA Synthetic Biology]

[http://gallivan1.chem.emory.edu/Gallivan%20Lab/Home.html Gallivan]

==References==

[http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=Retrieve&db=pubmed&dopt=AbstractPlus&list_uids=15723047 Bayer TS and Smolke CD. Programmable ligand-controlled riboregulators of eukaryotic gene expression. ''Nat Biotechnol''. (2005) 3:337-43.]

[http://pubs.acs.org/cgi-bin/article.cgi/jacsat/2004/126/i41/pdf/ja048634j.pdf Desai SK and Gallivan JP. Genetic Screens and Selections for Small Molecules Based on a Synthetic Riboswitch That Activates Protein Translation. ''J. Am. Chem. Soc.''(2004) 126:13247-54.]

[http://www.bio.davidson.edu/courses/synthetic/papers/Collins_ToggleSwitch.pdf Gardner, T.S., Cantor, C.R., and Collins, J.J. 2000. Construction of a genetic toggle switch in Eschreichia coli. Nature 403: 339-342.]

[http://www.bio.davidson.edu/Courses/Synthetic/papers/RNA_Regulation.pdf Isaacs FJ, et al. Engineered riboregulators enable post-transcriptional control of gene expression. ''Nat Biotechnol.'' (2004) 22:841-47.]

[[Davidson College Synthetic Biology Seminar (Fall 2007)| Return to Main Page]]

Post-transcriptional Regulation Technologies - Erin Zwack

2007-12-06T04:31:12Z

Erzwack: /* Prokaryotes: */

==Post-transcriptional Regulation Technologies==

==Overview:==

Regulation of translation provides an excellent tool for research on metabolic and other pathways in organisms, and for the production of different sensors by controlling the translation of specific genes depending on cellular conditions. Further development of these types of technology could provide a “knock-down” equivalent to RNAi, which exist in some eukaryotes. A gene of interest could be expressed normally at all times when the regulator is not active; thus, no ill effects will result before the pathway activates if the gene has another purpose as well. Other sythetic biologists could use these technologies to engineer fast-responding, RNA-based biological sensors for environmental chemicals, or novel pathways.

Using RNA regulatory molecules instead of regulatory proteins to control gene expression provides several benefits to synthetic biologists. Regulatory proteins bind to specific sites such as sites on the promoter or sites upstream of the promoter called operators. Control by these proteins can rely heavily on [[CellularMemory:Mathematical Models#Cooperativity_and_Bistability| cooperativity]] (Gardner et al. 2000), that is multiple proteins binding to one site, in order to see an effect. Regulatory RNA molecules on the other hand need a one to one ratio of regulatory molecule to target. As long as the molecule is expressed in an equal or greater amount than the target, the regulatory RNAs will normally be able to bind to their targets and control transcription.

While synthetic biologists could use the regulatory proteins and their binding sites that are found in nature, rational design of more new regulatory proteins is difficult. With the oligo and gene sythesis technology in existence today, RNA can be engineered that complements and thus targets any other RNA sequence. Regulatory proteins are also controlled mainly by promoter when determining whether they are active or not. As only so many inducible promoters exist, their is a small number of stimuli that can be used to determine under what conditions the gene under a regulatory protein's control will be expressed or repressed. An aptamer, RNA sequence that binds to a small molecule such as theophylline, can be used not only to regulate gene expression but can also regulate under what conditions an RNA regulatory molecule is active. New aptamers are easily developed through rational design, and the number in existence is continually increasing and providing new molecules that can act as ligands.

Finally, regulatory proteins stop gene expression before transcription. When the stimulus changes and the gene is expressed (either because a regulatory protein has now bound to or has released its site), the time it takes for the phenotype to be expressed is longer because both transcription and translation must occur instead of just translation. With RNA, the gene expression is halted after transcription. Once the stimulus is removed, the RNA already produced by the gene simply need to be translated.

==Development of Systems==

In most cases, post-transcriptional regulatory mechanisms that were developed and worked in eukaryotes cannot be directly transferred to prokaryotes. Modifications are necessary because eukaryotic and prokaryotic transcription and translation do not follow the exact same path. In eukaryotes, mRNA must have introns spliced out before translation begins; thus, any mechanism that regulates translation has time to bind or manipulate the mRNA (figure 1). In prokaryotes, translation begins as soon as the ribosomal binding site (RBS) is transcribed and accessible to a ribosome (figure 2).

[[Image:Eukaryotic.jpg]]

'''Figure 1:''' Inside of the nucleus of the eukaryote, the gene is transcribed into pre-mRNA, which contain both introns (orange) and exons (red). The pre-mRNA is then modified so that the introns are spliced out and the exons are put together. Finally the mRNA are translated by the ribosome in the cytoplasm.

[[Image:Prokaryote.jpg]]

'''Figure 2:''' In a prokaryotic cell the DNA is transcribed by RNA polymerase. As soon as the polymerase transcribes the ribosomal binding site, a ribosome binds and begins translating the sequence into protein. There is no modification step between transcription and translation.

==Eukaryotes:==

[[Antiswitches]]

==Prokaryotes:==

[[Riboregulators]]

[[Riboswitches]]

==Use of Post-transciptional Regulatory Technologies==

Regulation of translation provides an excellent tool for research on metabolic and other pathways in organisms, and for the production of different sensors by controlling the translation of specific genes depending on cellular conditions. Researchers can turn-off translation of certain genes in response to different pathways being activated, such as metabolic pathways. If a researcher wanted to know if a particular gene was necessary to proper function of a pathway, the aptamer of the antiswitch or riboswitch could be designed to have a molecule produced in the pathway to be its ligand. The gene would be expressed normally at all times when the pathway is not active; thus, no ill effects will result before the pathway activates if the gene has another purpose as well. This would provide a “knock-down” equivalent to RNAi available in some eukaryotes.

Synthetic biologists could engineer fast-responding, RNA-based biological sensors for environmental chemicals, or novel pathways that only activate when the environmental conditions are favorable.

==The Labs==

[http://www.che.caltech.edu/groups/cds/index.htm Smolke]

[http://www.bu.edu/abl/ Collins]

[http://www.bu.edu/abl/files/naturebiotech_isaacs.pdf Dr. Isaacs's Review of RNA Synthetic Biology]

[http://gallivan1.chem.emory.edu/Gallivan%20Lab/Home.html Gallivan]

==References==

[http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=Retrieve&db=pubmed&dopt=AbstractPlus&list_uids=15723047 Bayer TS and Smolke CD. Programmable ligand-controlled riboregulators of eukaryotic gene expression. ''Nat Biotechnol''. (2005) 3:337-43.]

[http://www.bio.davidson.edu/courses/synthetic/papers/Collins_ToggleSwitch.pdf Gardner, T.S., Cantor, C.R., and Collins, J.J. 2000. Construction of a genetic toggle switch in Eschreichia coli. Nature 403: 339-342.]

[http://www.bio.davidson.edu/Courses/Synthetic/papers/RNA_Regulation.pdf Isaacs FJ, et al. Engineered riboregulators enable post-transcriptional control of gene expression. ''Nat Biotechnol.'' (2004) 22:841-47.]

[[Davidson College Synthetic Biology Seminar (Fall 2007)| Return to Main Page]]

Post-transcriptional Regulation Technologies - Erin Zwack

2007-12-06T04:30:55Z

Erzwack: /* References */

==Post-transcriptional Regulation Technologies==

==Overview:==

Regulation of translation provides an excellent tool for research on metabolic and other pathways in organisms, and for the production of different sensors by controlling the translation of specific genes depending on cellular conditions. Further development of these types of technology could provide a “knock-down” equivalent to RNAi, which exist in some eukaryotes. A gene of interest could be expressed normally at all times when the regulator is not active; thus, no ill effects will result before the pathway activates if the gene has another purpose as well. Other sythetic biologists could use these technologies to engineer fast-responding, RNA-based biological sensors for environmental chemicals, or novel pathways.

Using RNA regulatory molecules instead of regulatory proteins to control gene expression provides several benefits to synthetic biologists. Regulatory proteins bind to specific sites such as sites on the promoter or sites upstream of the promoter called operators. Control by these proteins can rely heavily on [[CellularMemory:Mathematical Models#Cooperativity_and_Bistability| cooperativity]] (Gardner et al. 2000), that is multiple proteins binding to one site, in order to see an effect. Regulatory RNA molecules on the other hand need a one to one ratio of regulatory molecule to target. As long as the molecule is expressed in an equal or greater amount than the target, the regulatory RNAs will normally be able to bind to their targets and control transcription.

While synthetic biologists could use the regulatory proteins and their binding sites that are found in nature, rational design of more new regulatory proteins is difficult. With the oligo and gene sythesis technology in existence today, RNA can be engineered that complements and thus targets any other RNA sequence. Regulatory proteins are also controlled mainly by promoter when determining whether they are active or not. As only so many inducible promoters exist, their is a small number of stimuli that can be used to determine under what conditions the gene under a regulatory protein's control will be expressed or repressed. An aptamer, RNA sequence that binds to a small molecule such as theophylline, can be used not only to regulate gene expression but can also regulate under what conditions an RNA regulatory molecule is active. New aptamers are easily developed through rational design, and the number in existence is continually increasing and providing new molecules that can act as ligands.

Finally, regulatory proteins stop gene expression before transcription. When the stimulus changes and the gene is expressed (either because a regulatory protein has now bound to or has released its site), the time it takes for the phenotype to be expressed is longer because both transcription and translation must occur instead of just translation. With RNA, the gene expression is halted after transcription. Once the stimulus is removed, the RNA already produced by the gene simply need to be translated.

==Development of Systems==

In most cases, post-transcriptional regulatory mechanisms that were developed and worked in eukaryotes cannot be directly transferred to prokaryotes. Modifications are necessary because eukaryotic and prokaryotic transcription and translation do not follow the exact same path. In eukaryotes, mRNA must have introns spliced out before translation begins; thus, any mechanism that regulates translation has time to bind or manipulate the mRNA (figure 1). In prokaryotes, translation begins as soon as the ribosomal binding site (RBS) is transcribed and accessible to a ribosome (figure 2).

[[Image:Eukaryotic.jpg]]

'''Figure 1:''' Inside of the nucleus of the eukaryote, the gene is transcribed into pre-mRNA, which contain both introns (orange) and exons (red). The pre-mRNA is then modified so that the introns are spliced out and the exons are put together. Finally the mRNA are translated by the ribosome in the cytoplasm.

[[Image:Prokaryote.jpg]]

'''Figure 2:''' In a prokaryotic cell the DNA is transcribed by RNA polymerase. As soon as the polymerase transcribes the ribosomal binding site, a ribosome binds and begins translating the sequence into protein. There is no modification step between transcription and translation.

==Eukaryotes:==

[[Antiswitches]]

==Prokaryotes:==

[[Riboregulators]]

[[Riboswitches]]

[http://pubs.acs.org/cgi-bin/article.cgi/jacsat/2004/126/i41/pdf/ja048634j.pdf Desai SK and Gallivan JP. Genetic Screens and Selections for Small Molecules Based on a Synthetic Riboswitch That Activates Protein Translation. ''J. Am. Chem. Soc.''(2004) 126:13247-54.]

==Use of Post-transciptional Regulatory Technologies==

Regulation of translation provides an excellent tool for research on metabolic and other pathways in organisms, and for the production of different sensors by controlling the translation of specific genes depending on cellular conditions. Researchers can turn-off translation of certain genes in response to different pathways being activated, such as metabolic pathways. If a researcher wanted to know if a particular gene was necessary to proper function of a pathway, the aptamer of the antiswitch or riboswitch could be designed to have a molecule produced in the pathway to be its ligand. The gene would be expressed normally at all times when the pathway is not active; thus, no ill effects will result before the pathway activates if the gene has another purpose as well. This would provide a “knock-down” equivalent to RNAi available in some eukaryotes.

Synthetic biologists could engineer fast-responding, RNA-based biological sensors for environmental chemicals, or novel pathways that only activate when the environmental conditions are favorable.

==The Labs==

[http://www.che.caltech.edu/groups/cds/index.htm Smolke]

[http://www.bu.edu/abl/ Collins]

[http://www.bu.edu/abl/files/naturebiotech_isaacs.pdf Dr. Isaacs's Review of RNA Synthetic Biology]

[http://gallivan1.chem.emory.edu/Gallivan%20Lab/Home.html Gallivan]

==References==

[http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=Retrieve&db=pubmed&dopt=AbstractPlus&list_uids=15723047 Bayer TS and Smolke CD. Programmable ligand-controlled riboregulators of eukaryotic gene expression. ''Nat Biotechnol''. (2005) 3:337-43.]

[http://www.bio.davidson.edu/courses/synthetic/papers/Collins_ToggleSwitch.pdf Gardner, T.S., Cantor, C.R., and Collins, J.J. 2000. Construction of a genetic toggle switch in Eschreichia coli. Nature 403: 339-342.]

[http://www.bio.davidson.edu/Courses/Synthetic/papers/RNA_Regulation.pdf Isaacs FJ, et al. Engineered riboregulators enable post-transcriptional control of gene expression. ''Nat Biotechnol.'' (2004) 22:841-47.]

[[Davidson College Synthetic Biology Seminar (Fall 2007)| Return to Main Page]]