Difference between revisions of "Team:Vanderbilt/Project/Circuit"

Homologous elements in a genetic circuit pose high risks for destabilizing genomic rearrangements. Unlike sources of genomic instability that primarily generate point mutations, which in many circumstances may not produce meaningful functional changes, these rearrangements will typically generate considerable functional consequences. This issue of recombination has, among other challenges, posed a barrier to implementing redundancy as a means of decreasing the probability of loss of function. Were it possible to stably introduce two copies of a given gene, the probability for that gene to be rendered nonfunctional would be greatly reduced, by a factor of that probability squared.

One function of our circuit optimization program is to eliminate the amount of homology between two sequences. We were interested in the possibility of using this optimization strategy to minimize homology to the point where it may become possible to integrate multiple copies of certain genes as a viable strategy to increase their evolutionary longevity. Using a set of repair endonucleases (T7 Endonuclease, for use with our "Incorruptible Cell") as our model, we sought to develop a methodology for quantifying how well our homology optimization reduces rates of recombination.

Using our algorithm, we were able to generate a second copy of the T7 repair enzyme that has the number homologies greater than five bases reduced by 78.8% compared to the alternative approach of repeating the same sequence twice. To quantify the effect this optimization has on recombination, we placed our two T7 sequences flanking a portion of the yeast genome including the ura3 gene. One version would have the same native T7 sequence repeated at both ends, while the other would have the native sequence with the homology-optimized sequence. Once integrated into the genome of uracil auxotrophic yeast, if a recombination event occurred between the two T7 sequences, it would excise ura3 as a byproduct. Selection on 5-FOA media, which is lethal to yeast expressing uracil, would then produce a colony for every cell in which recombination occurred.

The sequences with ura3 and T7 were first extracted and modified by extension PCR. Following extraction, the PCR fragments were again amplified by extension PCR, in the process introducing homology regions for Gibson and assembly and gal1 homology arms for integration into the yeast genome. Following Gibson assembly, the three fragments (two T7 and one ura3) were placed into a shuttle vector. Transformed cells with the correct ampicillin resistance were found for both the assembly of the control cassette and the homology optimized cassette. Next, these assembled vectors will be purified and introduced into yeast for experiments.

In parallel, we have been developing a bidirectional promoter system. Although our methods are effective at lowering the mutation rates of genes, the promoters of these genes remain vulnerable targets for mutations. We paired our bidirectional promoter, based on a modified IPTG-inducible promoter developed by Yang et al 2012, with an ampicillin gene transcribed in the reverse direction. This pairing allows us to select against mutations in the promoter sequence, by making any promoter mutation lethal when the cells are in the presence of ampicillin. We demonstrated our bidirectional promoter functions as expected by incubating our cells in ampicillin.

To integrate our circuit optimization strategies, we used Visualizing Evolution in Real Time (VERT). We incorporated the various stability optimization strategies that our team has developed into a single circuit with three fluorescent proteins. We synthesized the components of the optimized circuit into three parts, which were then assembled into pSB1C3 using Gibson assembly. A colony containing a correctly-sized construct was found by screening and purified. For the control circuit, based on traditional circuit design principles, we extracted genes from parts in the Registry using extension PCR, in preparation for Gibson assembly.