Summer 2012 SynBio Project (Davidson and MWSU)

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Summer 2012 Synthetic Biology Project: MWSU and Davidson College


  1. Davidson Protocols
  2. MWSU_protocols
  3. GCAT-alog Freezer Stocks
  4. Laboratory_Notebooks
  5. Golden Gate
  6. Philosophy and Ethics of our Project


Student Proposals from Ind. Studies

-I think the use of Phytochromes might be a good way to have either a continual stimulus that would repress/express certain genes that could be turned off and on depending on what we want them to do. There are other aspects of the research in this proposal that if not used outright, could be adapted to our continuing projects as either controls or feedback mechanisms. As for the proposed Salis RBS sites, I would like to see more information in the efficacy of the predicted RBS sequence. Possibly if we could use some of the C-Dog information based on a few known sequences to determine if the computer can predict those RBS's we know to be effective then we might be able to count on the calculator as a tool for our experimental design. -Caleb Carr



PPT Presentations

  • This PPT file contains all the slides from student presentations addressing the idea proposed by MWSU.

Media:Reports_on_Circuits.pptx

  • This PPT contains slides summarizing some of the best and most complicated papers from Week 11.

Media:Week_11.pptx

Papers

Methods Papers

  • DNA assembly for synthetic biology: from parts to pathways and beyond

Tom Ellis, Tom Adieac and Geoff S. Baldwin
Integr. Biol., 2011, 3, 109–118

  • Everyone should watch this 5 minute video on optogenetics. Combine that video with the 2010 champoinship iGEM invention of E. glowi.


Older Lab Papers

  • Engineering bacteria to solve the Burnt Pancake Problem.

Haynes, Karmella, et al.
Journal of Biological Engineering. Vol. 2(8): 1 – 12.

  • Solving a Hamiltonian Path Problem with a Bacterial Computer.

Baumgardner, Jordan et al.
Journal of Biological Engineering. Vol. 3:11

  • Bacterial Hash Function Using DNA-Based XOR Logic Reveals Unexpected Behavior of the LuxR Promoter.

Brianna Pearson*, Kin H. Lau* et al.
Interdisciplinary Bio Central. Vol. 3, article no. 10.
Time Delayed Growth Movie


Network Papers

Jonathan M. Raser and Erin K. O’Shea
Science. Vol. 309, page 2010

Please post pdf.

Nagarajan Nandagopal and Michael B. Elowitz
Science. Vol. 333, page 1244.

Please post pdf.

R. Milo, S. Shen-Orr, et al
Science. Vol. 298, page 824.

Please post pdf.

Yang-Yu Liu, Jean-Jacques Slotine, & Albert-La ́szlo ́ Baraba ́si
Nature. 2011. Vol. 473, page 167.

Please post pdf.


Ethics Papers

Colin Mcilswain
Nature. Vol 465, page 867.

-This paper does a great job at highlighting the importance of socio-political legitimation in the funding of science. It seems that all new sciences must survive a period during which their only funding comes from public sources under the condition that those conducting it can make some kind of promises of future benefit to the society as a whole. After proving itself not only useful but also profitable, private money may then start flowing in, though by that point, the nature of that field may arguably have changed for better or worse. I think we would all agree that synthetic biology holds more promise than we can currently even imagine, both for advancing the public good and for providing opportunity for profit (in more than just pharmaceuticals), but it's not enough for us to believe it. Those of us who will someday pursue grants and/or private investments in synthetic biology must learn to speak not only the rational language of the science of synthetic biology but also the politically-driven language of the social benefits of synthetic biology, the socially conscious language of the ethics of synthetic biology, and the profit-driven language of the (future) business of synthetic biology (and possibly others). -Eddie Miles

Questions to Consider About Network Pathways

  • Are they naturally occurring or synthetic?
  • Do they involve screening or selection?
  • Are they anabolic or catabolic?
  • How many steps are in each pathway?
  • How can they relate to cell fitness?
  • What specific challenges would need to be addressed if we worked with the pathway?

Network Pathways Chart

Cellular Automata

  • [1] General CA introduction
  • [2], [3] Elementary Cellular Automata
  • [4] Good explanation of how elementary CAs work
  • [5] Rule 110

Peptides

Environmental factors that enhance the action of the cell penetrating peptide pep-1 - A spectroscopic study using lipidic vesicles [[7]]

Assembly

[8]iGEM Introduction to Gibson Assembly

[9]Enzymatic assembly of DNA molecules up to several hundred kilobases

[10] Supplemental Methods for Enzymatic assembly of DNA molecules up to several hundred kilobases

[11]Assembly of BioBricks by the Gibson Method

[12] Properties of Exonuclease

[13]Tool for using Gibson Assembly Method

Library of Parts

Research Papers, Articles & Manuscripts--all inclusive and in regards to any and all parts that are listed, or wish to be listed

  • [14] gene-specific promoter element is required for optimal expression of the histone H1 gene in S-phase.
  • [15] Multiple Sigma Factors

Promoters Section

  • 6 possible promoters for project 3 constitutive, 3 inducible - (Word file not yet saved on wiki)

C-Dog Section

Degradation Tag Section

Selection Modules

Bad-ish genes/proteins

Good genes/proteins

CRISPR process

Regulated Biosynthesis Pathways

http://cat.inist.fr/?aModele=afficheN&cpsidt=6828850

Aptamers

Gas-Phase Communication

Light

Pump phR MscL NpHR e-BO/e-BR/h-BR PR
wavelength max absorbance at 578-599 open with 366 nm, close with visible light (>466 nm) 578 nm (with NaCl in media) 550-560 nm ~525 nm
particles that can travel through it Chloride ions non-selective, 3-nm diameter anions protons protons
pump/channel? pump channel pump pump pump
type of protein halorhodopsin n/a halorhodopsin BR=bacteriorhodopsin, BO=bacterio-opsin proteorhodopsin
direction into cell n/a into cell into cell out of cell

Blue Light Regulated Promoter YgcF

  • Articles/ References:
    • [18] The BLUF-EAL protein YgcF acts as a direct anti-repressor in a blue-light response of E.coli
    • [19] Light induced structural changes of a full-length protein and its BLUF domain in YcgF(Blrp), a blue-light sensing protein that uses FAD (BLUF)
    • [20] Group: iGEM09_KULeuven (2009-08-02)
  • Proposed Pathway:
    • text
      Relationship to Selection Module










  • Parts to Build:
    • K238013
    • gnl|ECOLI|G6603
    • gnl|ECOLI|G6602
  • Additional Parts:
    • Vector: PSB1A2 (Isolated and purified from a gel)
    • Insert with plac promoter and RFP gene: J04450

Maths

  • Agent Based Models/Complex Adaptive Systems
    • [26] Set of lecture slides on chaos, including one on ABMs.
    • [27] Stuart Kauffman on emergence
    • [28] Good slide-show covering ideas of ABM
    • [29] Sante Fe Institute Agent-Based Modeling links
    • [30] Slideshow on modeling intercell stuff via AMB
  • Real Computing/Complexity
    • [31] Lecture transcripts from two MIT courses on compleity by a very smart guy in the field
    • [32] Review of physical computing by the same researcher
    • [33] Part of a textbook on computation theory
    • [34] Harvard analog computing
    • [35] Free draft of Princeton text on computational complexity
    • [36] Paper written by one of the authors of Complexity and Real Computation that contains the same basic ideas
    • [37] An analog computer museum and information site run by a Dr. Bernd Ulmann, who did his doctoral thesis on analog computing
    • [38] Abstract of a 1964 study that used analog computers to model a bacterial cell
    • [39] Paper on combined use of analog and digital computation
    • [40] First 28 pages of Neural Networks and Analog Computing: Beyond the Turing Limit
  • Neural Networks
    • [41]
    • [42] Neural networks in plain English; seems to be a basic of how to programming guide for them as well
    • [43] Paper on neural networks in bacteria

Communication

General


GGA for College Teaching Labs

6/21/12 Experiment

Objective: The goal is for the cell colonies to exhibit a visible amount of GFP in the least amount of time possible.

Process: Previously, I had seen red fluorescence in the plate of the solution that included J119022 plasmids and was in the PCR machine for 30 cycles. So, Dr. Campbell and I decided to try the same, but with 5 or 10 cycles. We also tried 5 or 10 cycles with cleaned PCR product (J23100 insert), as opposed to the entire 022 plasmid. This left us with four experimental plates and two negative controls (without enzymes; one for 5 cycles, one for 10 cycles).

Results: After letting the plates incubate overnight, we saw the results. Initially, only the 5- and 10-cycle plasmid plates produced red colonies. However, as the day went on, the PCR product plates showed fluorescence, too. The plates then sat out for the weekend, and by Tuesday morning, three of the four experimental plates were obviously red. These three were the 10-cycle 022 plasmid plate, the 10-cycle PCR product plate, and the 5-cycle PCR product plate. The 5-cycle 022 plasmid plate had a few red colonies, but not nearly as many as the other three. We then took pictures under UV light (seen below) of the three that showed fluorescence plus a negative control plate (10 cycles) as a comparison. The 5-cycle PCR product plate displays the least amount of RFP of the three shown, with roughly half of the colonies glowing red.

These inserts (J23100) were cloned into the plasmid J119044.

4plates.jpeg
Figure 1. Photograph of 4 plates containing transformed E. coli (JM109) used in this GGA experiment. Clockwise from the top: Negative control, 10-cycle 022 plasmid, 10-cycle PCR product, 5-cycle PCR product
Negcontrol.jpeg
Figure 2. Photograph of the negative control plate

Plasmid10.jpeg
Figure 3. Photograph of the 10-cycle plasmid plate

PCR10.jpeg
Figure 4. Photograph of the 10-cycle PCR product plate

PCR5.jpeg
Figure 5. Photograph of the 5-cycle PCR product plate





6/28/12 Experiment

Objective: The goal of this experiment was to see if unclean PCR product works as efficiently in Golden Gate Assembly (GGA) as the clean PCR product. If it does, this would eliminate the step of cleaning the PCR product, thereby making GGA a quicker process.

Process: I had eight experimental plates and three control plates. For the experimental plates, I made the GGA solution with different volumes of unclean PCR product instead of J119022 plasmid. I used 1, 5, 10, and 20 µL of unclean PCR product, and one of each solution went through either 5 or 10 cycles in the PCR machine.

I then compared these experimental plates to the positive control (the same solution, but with 44 ng of clean PCR product; I know from a previous experiment that this should produce RFP) to see what volume (if any) of unclean PCR product would produce similar results to the clean PCR product. In the set of 10-cycle plates, there were two positive controls (one was 25 µL total, and the other was 10 µL; this was to see if adding more water would affect the results).

I only had one positive control in the 5-cycle set because there was not enough clean PCR product to have two. I chose to keep the 10 µL control because I knew that it should definitely work (because of a previous successful experiment), and I didn’t know yet if a 25 µL solution would produce RFP (the 5-cycle plates experiment was performed a day earlier than that of the 10-cycle plates).

There was one negative control in each of the 5- and 10-cycle experiments; the solutions were the same, just without the insert (PCR product—clean or unclean).

Results: The 10-cycle positive controls (with different amounts of water) produced the same amount of colonies (and also the same amount of red colonies), so I can conclude that the amount of water in the solutions does not affect the effectiveness of GGA.

The two 10-cycle positive controls (25 µL and 10 µL) both had about 50% of their colonies glowing (see Figures 2 and 3). The 5-cycle control, however, had a lawn of colonies in the middle (that did not exhibit RFP) and had red colonies on the periphery of the plate (see Figure 1). This was confusing because last week I performed an identical experiment, and red colonies were across the entire plate, not just the periphery. I’m assuming this is a result of faulty spreading of the cells, because the colonies along the edge of the plate show a significant amount of RFP, indicating that GGA was successful.

As for the experimental plates, only the 5- and 10-cycle plates containing 1 µL of unclean PCR product showed RFP, with the 10-cycle plate showing even more (see Figures 4 and 5). The plates containing 5, 10, and 20 µL of unclean PCR (Figures 6-11) product did not exhibit RFP at all in either the 5- or 10-cycle plates, and, in fact, the amount of colonies on the plates had a negative correlation with how much unclean PCR product was added. I assume that, because the positive controls are already glowing so brightly by this point, all of the plates would have exhibited some red by now if they were going to. Thus, the plates that don’t have any GFP (the 5- and 10-cycle plates of 5, 10, and 20 µL unclean PCR prodcut, and the negative controls) probably will not begin to glow if they have not already. Because the 1 µL solutions have shown RFP, I decided to go forward with these.

Next: I plan to ligate and transform solutions identical to those I used for the plates with 1 µL of unclean PCR product, except they will be in the PCR machine for 20 or 30 cycles. Because the 10-cycle plate with 1 µL of unclean PCR product produced more RFP than the 5-cycle plate of the same solution, I am guessing that the more cycles the solution goes through, the higher percentage of colonies will exhibit RFP. I will be transforming four experimental solutions: two will be in the PCR machine for 20 cycles, and two for 30 cycles. Two will have a total volume of 10 µL and two will have a total volume of 25µL (one of each will be in the PCR machine for either 20 or 30 cycles). I am testing two different volumes because I got positive results with a total of 25 µL, and I want to verify that the amount of water in the solution doesn't affect the solution with 1 µL unclean PCR product (just as I found it doesn't affect the clean PCR product).

If more cycles in the PCR machine do turn out to produce higher percentages of red fluorescent colonies in plates, then professors can have options of how to perform GGA in their classroom. They can choose to add the extra step of cleaning the PCR product (and then only have to put the solution in for 5 cycles in the PCR machine), or they can not clean the PCR product and put the solution in the PCR machine for more time (if this ends up being a viable option; I will find out soon).

These inserts (J23100) were cloned into the plasmid J119044.

Poscontrol5cycles.jpg
Figure 1: Photograph of a plate containing transformed E. coli (JM109) used in this GGA experiment; Positive control, 5 cycles.

10µLposcontrol10cycles.jpg
Figure 2: Photograph of positive control plate, 10 µL, 10 cycles
25µLposcontrol10cycles.jpg
Figure 3: Photograph of positive control plate, 25 µL, 10 cycles
Screen shot 2012-07-02 at 10.37.49 AM.png
Figure 4: Photograph of plate containing 1 µL unclean PCR product, 5 cycles; circles indicate some locations of colonies exhibiting RFP
1µL10cycles.jpg
Figure 5: Photograph of plate containing 1 µL unclean PCR product, 10 cycles
5µL5cycles.jpeg
Figure 6: Photograph of plate containing 5 µL unclean PCR product, 5 cycles
5µL10cycles.jpeg
Figure 7: Photograph of plate containing 5 µL unclean PCR product, 10 cycles
10µL5cycles.jpeg
Figure 8: Photograph of plate containing 10 µL unclean PCR product, 5 cycles
10µL10cycles.jpeg
Figure 9: Photograph of plate containing 10 µL unclean PCR product, 10 cycles
20µL5cycles.jpeg
Figure 10: Photograph of plate containing 20 µL unclean PCR product, 5 cycles
20µL10cycles.jpeg
Figure 11: Photograph of plate containing 20 µL unclean PCR product, 10 cycles
Negcontrol5cycles.JPG
Figure 12: Photograph of negative control plate, 5 cycles
Negcontrol10cycles.JPG

Figure 13: Photograph of negative control plate, 10 cycles
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