Parts

Here is a list of all the parts that we submitted to the registry this year. Click on a link to learn more

<groupparts>iGEM014 UChicago</groupparts>

Below we will give more detailed explanations for the purposes behind each of the following plasmids, explaining the function of every component of each plasmid and how we did them.

Plasmids:
IPTG inducible promoter - RBS - GFP - Mutator
ParoF--RBS--mCherry--RBS--mutator
Generic Promoter--RBS--mutated TyrR

IPTG+GFP Mutator:

(IPTG inducible promoter - RBS - Mutator) The IPTG promoter is used to quantify the mutation rate of each mutator via rifampicin (rif) fluctuation analysis. The reason that IPTG was used was so that the expression of the gene of interest could be controlled and be turned on only during the gene during the fluctuation analysis (but not during the cloning of the RBS and Mutator parts). Ergo, an unaltered copy of the RBS and mutator gene could be cloned for later use.

GFP (or Green Fluorescent Protein) was used primarily to visualize cells that had successfully gained the insert. The presence or absence of fluorescence under blue light could instantly give an indication as to if the cells contained the insert without having to either use some of the sample or wait for a lengthy procedure to determine the same thing.

The mutators were first cloned into the IPTG-GFP backbone in order to effectively assay their mutation rates. The expression of the mutators on this backbone is activated by the presence of IPTG in the growth medium, allowing us to effectively control the expression of the mutator gene to prevent any unnecessary mutations in the cell prior to cloning into the main ParoF-mCherry backbone for FREP. Plasmids that had already undergone the fluctuation analysis were then transferred to the ParoF+mCherry backbone.

ParoF--RBS--mCherry--RBS--mutator

The actuator module involves an optimized promoter, a reporter, and one or more mutators.

As the mutation rate of our construct increases, the rate of tyrosine production increases. However, in a feedback-regulated system, the transcription factor will bind to the desired molecule, in this case tyrosine, and cause the transcription-factor-regulated promoter to decrease expression. Thus, the mutation rate will decrease and the plasmid will decreasingly express a reporter gene. In order to observe low levels of expression, we chose mCherry, a strong variation of RFP. Human vision is most sensitive to red light, so using RFP would allow us to scan for low fluorescence and we would also be able to eye-ball whether or not the construct was working. We used part BBa_J06505.

For our promoter, we chose to use ParoF, a TyrR-regulated promoter that was found most-effective in response to tyrosine concentrations by Keasling and Chou (not sure if we’re doing footnotes, internal citations, etc). We received primers from Keasling and PCRed the promoter from wildtype E. coli. We designed universal primers to PCR-out the RBS--mutators from the IPTG--GFP--Mutator construct. By PCR-ing out active mutators, we reduce the amount of site-directed mutations we had to make. Additionally, by creating universal primers that can PCR-out an RBS and any of our mutators, it is easier to ligate additional mutators into our ParoF--mCherry construct to create combinations of mutators, our ultimate goal.

Generic Promoter--RBS--mutated TyrR

The sensor module requires a generic promoter and a tyrosine-sensitive transcription factor

TyrR is a transcription factor that binds to tyrosine. As the tyrosine levels increase, the mutation rate will begin to decrease because the ParoF promoter in the actuator module is regulated by TyrR dimers. We optimized TyrR through site-directed mutagenesis. Point mutations E274Q and N316K have been shown to increase TyrR’s ability to repress ParoF in the presence of tyrosine and thus increased the effectiveness of our system. We chose an IPTG-inducible promoter to allow us to control the expression of TyrR and thus control the efficiency level of our sensor module. We used part BBa_J04500.

Full citations for citing E and N point mutations.

1. Kwok, T., Yang, J., Pittard, A. J., Wilson, T. J. & Davidson, B. E. Analysis of an Escherichia coli mutant TyrR protein with impaired capacity for tyrosine mediated repression, but still able to activate at sigma 70 promoters. Mol. Micro. 17, 471–481 (1995).
2. Koyanagi, T., Katayama, T., Suzuki, H. & Kumagai, H. Altered oligomerization properties of N316 mutants of Escherichia coli TyrR. J. Bacteriol. 190, 8238–8243 (2008).