When it is applied to our circuit, P1 is inverted in regular working condition where inducer is present, recombinase catalyzes the cleavage of each cognate recognition sites attB and attP, inverting the orientation of P1 and rejoining the circuit at attL and attR respectively. Due to the fact that irreversible recombinase can only flip the DNA in one direction, the inverted P1 which is inhibited in working condition where inducer is present remains inverted even when the recombinase is not expressed as inducer is absent. When the inducer is absent, R1 doesn’t inhibit the expression of the inverted P1 anymore, the toxin in the second layer can be then expressed and conduct suicide. The inversion is a permanent effect on the bistable system, that is to say even if we stimulate the whole system again by adding inducer, P1 will not switch its orientation back.

III.Experiment & Results

We used pBAD and PLtetO as promoters to regulate the expression of the three recombinases in order to test the effect flipping over the promoter J23119 (P1) by the three recombinases and to see if there is expression leakage.

Theoretically speaking, when inducer is present, recombinase should be expressed and successfully reverts the promoter P1 in-between, the bacterium will express mRFP, displaying red color; while if no inducer is present, the inhibitory effect the transcriptional factor exerts on P2 will stop the expression of recombinase, P1 fails to reverse, the bacterium will therefore express sfGFP, displaying green color.

In the plating medium with no inducer, the leaked expression of recombinase reverted the promoter J23119, displaying red color. Comparing the three sets of recombinases (Fig. 3&4&5), we found that TP901 has the lowest expression leakage.

Figure 3: Bxb1(TetR) and Bxb1(pBAD) colony

Figure 5: TP901(TetR) andTP901(pBAD) colony

Figure 4: PhiC31(TetR) and PhiC31(pBAD) colony

Then we used pBAD to test the recombinases’ working efficiency in inducer’s presence through flow cytometry. Results are quantitatively shown in the diagram below.

Figure 6: The x-axis of each small square represents the fluorescence of GFP, while the y-axis represents the fluorescence of RFP. All of them show expression leakage in inducer present and absent. (e.g. for PhiC31, according figures in the second row, dispersed high fluorescence of both RFP and GFP in most of the cells show that there is expression leakage of the recombinase both before and after flipping over the recombinase).

IV.Discussion

Our recombinases can effectively invert the in-between promoter under our design and experimental validation. This feature works well as a reactor for changes in the external environment. It permanently changes the sequence of genes and thus the direction of gene expression, which implements our goal of connecting two bistable system and exerting permanent effect on the circuit.

Sequence and Features