CHE.496/2009/Schedule/Oral presentations of part design/Group 3

Group 3

• Members: Joe Bozzay and Brandon Freshcorn

Parts

• Part 1
• Description

A genetic oscillator would be designed that would utilize negative feedback and the quorum sensing mechanism to simultaneously express a gene at a certain time, based on the presence of an inducer. This type of oscillator is driven mainly by two elements: the concentration of a repressor protein and the dynamics of an activator protein forming an inactive complex with the repressor. Either protein would serve as an inducer, so that quorum sensing would be used to sync the output of the bacteria. Since simultaneous activation is threshold based, the period of oscillation could be used to account for the synthesis of the activator and repressor proteins. Eventually, the concentrations would reach a certain threshold and simultaneous gene expression would occur.

• Sources & References

http://ieeexplore.ieee.org/iel5/4290605/4290606/04290624.pdf?arnumber=4290624 http://www.pnas.org/content/99/9/5988.full

• Part 2
• Description

A band pass filter would be constructed that utilizes feedback to regulate the expression of a gene based on input concentrations. An inducer would be used that codes for two genes, and the product of one of these genes would act to suppress the expression of the other gene. The suppressed gene would act on a reporter gene (like GFP) so that expression of this gene would be observed. Once a certain level of expression was reached, based on the concentration of the input inducer, the output would be sufficiently visible, but as the concentration of the input inducer increased, eventually the negative feedback by the suppressor gene would overwhelm the signaling gene and decrease the output. So, it would take a certain level of inducer for any output to be observed, and eventually the negative feedback would suppress the output at a certain (higher) inducer level, so that a band pass filter would be achieved.

• Source & References

Genetic parts to program bacteria link

• Part 3
• Description

In patients with drug ODs, in which the drug is lipid soluble and therefore protein-bound, the drug can remain in the body for hours, days, or even months. It is possible, therefore, to treat the OD by destroying the albumin which transports it. A proteolytic enzyme known as plasma protease could accomplish this. By decreasing the body's capacity for the drug, the drug could be forced out of the system into the plasma to be excreted by the kidneys.

• Part 4
• Description

There is a severe lack of post-translational enzymes in the IGEM parts registry. This dramatically limits the complexity (and thus the utility) of the proteins which we intend to create- the very essence of what makes genetic engineering useful. If we could synthesize a few evolutionarily primitive post-translation enzymes, we could open up a whole new side to the IGEM competition- the ability to synthesize eukaryotic genes. In doing so, these primitive post-translational enzymes could be used to create other, more complex post-translational enzymes which require their own post-translational modifications.

Part 5

Purpose

To enable qualitative detection of an antibody by E coli. This would be useful for detecting a disease based on antibodies present in a sample.

Description

Competitive binding by an antigen would be used with the quorum sensing mechanism and florescent protein expression to devise a disease detection system. In quorum sensing, transcription of a group of bacteria can be turned on simultaneously when a threshold level of inducer is reached. Bacteria will not recognize their own inducers, so the presence of inducers that induce gene expression must come from other bacteria. Ideally, the GFP promoter would be modified to contain tet operator elements, allowing repression by TetR. Presence of an antigen would promote the expression of the SSK1 transcription factor, which would suppress the TetR repressor, thus activating the GFP operon and promoting expression of GFP. The presence of this inducer would promote other bacteria to express GFP. The antigen would need to be detected, possibly by using a tag protein.

Source & References

1. Cell-to-cell signalling in Escherichia coli and Salmonella enterica. Ahmer BM. Mol Microbiol. 2004 May;52(4):933-45. Review.
2. Hense BA, Kuttler C, Müller J, Rothballer M, Hartmann A and Kreft JU (2007). "Does efficiency sensing unify diffusion and quorum sensing?". Nature Reviews Microbiology 5: 230–239.
3. http://2008.igem.org/Team:University_of_Ottawa/Project (modifing GFP for repression by TetR)

Part 6

Purpose

To demonstrate the synthesis of a eukaryotic protein in a prokaryotic system.

Description

Factor VII is a clotting protein in the coagulation cascade. The protein requires an external factor in order to activate and therefore can be used as a treatment for internal hemorrhage when surgical aide is not immediately available. We currently have the gene; it has already been synthesized from the BIOM200 class. However, factor VII relies on a few post-translational modifications in order to function. I therefore propose that we focus on getting this gene to work in a prokaryotic system based on the parts mentioned previously. If nothing else, it could demonstrate a proof-of-concept.