Part:BBa_K2100055:Experience
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Applications of BBa_K2100055
pENTR L4_EGSH_4xk-turn_R1 can be cloned together with a pENTR gene vector into a backbone. The downstream is now under two regulation systems, inducible promoter pEGSH and RNA-regulation system L7Ae/k-turn.
For our project, we use this system to control expression of TP901, a serine recombinase. We performed LRs to create pEXPR EGSH_4xk-turn:TP901 and pEXPR EGSH_4xk-turn:mKate, and then conducted experiments with these two plasmids in HEK293 cell line to examine the repressing level of L7Ae/k-turn on the expression of TP901.
Purpose
Using recombinases as biological 'latches' gives our genetic circuit the 'memory' necessary for identifying the disease's temporal specificity. Since the recombinase is controlled by an inducible promoter, however, leaky expression of the promoter (activation without input signals - disease biomarkers) could lead to unwanted activation of the output gene and thus a false positive diagnosis. By putting k-turn motifs in front of the recombinase gene, we hope to eliminate leaky expression of the recombinase when the system is not activated.
We designed an experiment to examine the repression level of the L7Ae - kink turn system on the expression of an output gene (eYFP - enhanced yellow flourescent protein), which is regulated by TP901 (a serine recombinase).
Experimental Setup
We used two inducible promoter systems - pEGSH/PonA and pTRE/Dox - in this experiment to control the expression of two genes, L7Ae for tuning the repressing level and TP901, the recombinase. The pTRE/Dox system controled the expression of L7Ae. We induced the cells with Dox at the same time as transfection because the repressing system needed to be activated before TP901 recombinase was induced by PonA/pEGSH.
Additionally, including all the neccessary genes (TP901, flipped eYFP, L7Ae, VgEcr-RXR, and rtTA) and the reporter flourescent genes, the total number of plasmids went up to 7 (or 8 with the dummy DNA - pDONR). We were reaching the upper limit of the number of plasmids that can be cotransfected using lipofection. After asking for advice from other members of the Weiss lab, we increased the ratio of viafect:DNA from 1ul:500ng total DNA to 1.5ul:500ng total DNA.
For each well:
Total amount of DNA: 1500ng
Viafect transfection reagent: 4.5ul
Result
Testing the 2x k-turn L7Ae system with varied L7Ae expression level
These graphs show that as the amount of Dox increased, there was smaller amount of eYFP expressed when TP901 expression was induced. When [Dox] = 0uM, at a high plasmid copy number, the amount of activated eYFP in induced TP901 sample was 2-fold higher than in uninduced TP901 sample (y-axis is in log scale). However, when Dox = [1000nM], the amount of activated EYFP in uninduced and induced TP901 samples stayed the same for a larger range of plasmid copy numbers. At high copy numbers, however, the amount of eYFP produced was still higher for induced TP901 than uninduced, and the amount difference is larger at lower concentrations of Dox. Thus, the L7Ae/k-turn RNA-based gene regulation system could reduce the basal expression of inactivated TP901 while still allowing the recombinase to perform its function when the whole system is activated.
Testing the effect of varying k-turn sequences
When we compared varying numbers of k-turns with constant amounts of Dox and PonA, we saw that the amount repression of mKate expression (and thus TP901 expression) increased significantly as we increased the number of k-turns for both the uninduced and induced states of pEGSH. While the difference between the amount of yellow fluorescence (and thus the amount of recombination) for 2 k-turns and 4 k-turns was less pronounced, we still observed a noticeable decrease in recombination when k-turns were present compared to when they were not present. We concluded that increasing the number of k-turns over the range we considered led to tighter repression by L7Ae.
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