Difference between revisions of "Part:BBa K3594002"

Latest revision as of 05:45, 24 October 2020

Quorum Sensing Self-lysis Circuit

Usage and Biology

This composite part is used as the synchronized self-lysis device in SLC. It contains a PhiX174E lysis gene sequence, a LuxR gene sequence and BBa_K3594009. When the concentration of signal molecule AHL reaches threshold, LuxR-AHL functions as the promotor to promote the transcription of the downstream gene PhiX174E. As a result, the plasma membrane of the bacteria are destroyed, which means that they commit suicide.

the design of our bacterial synchronized lysis circuit

Plasmid construction: pSHS7_ pSB1C3_pTac_LuxR_Plux_Phix174E: Gibson assembly Ligate pTac_LuxR_Plux_Phix174E (~2kb), and backbone PSB1C3（~2Kb） This plasmid is the combination of LuxR, plux promoter and PhiX174E sequences, which is the final set up in our design.

Verify the function of LuxR and plux

We ligated the BBa_K3594002 plasmids successfully

Sequencing result of pSHS7

Verify the function of Phix174E lysis protein

In this summer, we did a lot of Gibson assembly experiments to ligate the fragments with PhiX174E. However, we didn’t get one living E. coli colony yet. We thought the reason might be that all the colonies with right sequence of pTac-PhiX174E die due to the leaked expression of pTac promoter. Though we didn’t do the experiments, the clear LB plates verified the lysis function of PhiX174E from the side of the “dead” colonies.

The result of BBa K3594002

If the OD value in the tube increases with time, and the OD value in the tube with IPTG and atc is lower than no inducer or only one inducer, it proves that the bacteria lyse and die after reaching the threshold, so the experiment success. In our experiments, the results are different from the ideal ones. The possible reason for the unsuccessful experiment is that we built the plasmid on pSB4K5 before, the copy number is relatively low, it is difficult to produce sufficient amounts of LuxI, LuxR, cannot perform effective Quorum sensing reaction.

Combine the Quorum Sensing System and PhiX174E and verify the funciton of SLC

Though we didn’t get good results of PhiX174E and the quorum sensing system in this summer, we also designed the experiments about how to test if SLC could work. We used the MATLAB software to model the SLC circuit and the results supports our SLC is OK and our design can work quite well. In our design, after verifying the effectiveness of PhiX174E, LuxR and plux, we will assemble the gene circuit shown in Figure 9, and verify whether E. coli can show the oscillatory growth effect of SLC like Figure 14 shown, and whether it can release degradation enzymes.

The oscillating effect of bacterial growth

The predicted and ideal results presented in Figure 16. When the number of bacteria reaches the threshold, they will produce green fluorescence, and the PhiX174E expresses, too, which induces cell lysis to death. After a period of time, they will grow again, showing an oscillating effect.

Sequence and Features