Difference between revisions of "Team:XMU-China/Proof Of Concept"

1、The toxicity of MazF

The premise of our kill switch is to verify the toxicity of MazF. The toxicity of MazF cannot be reflected by OD₆₀₀, viability count of colony-forming unit (CFU) was employed to investigate if MazF can kill E. coli.

pBAD/araC is chosen to express MazF. the plasmid carrying pBAD/araC-RBS-MazF-Terminator (BBa_K3332083) were transformed into E. coli BL21 (DE3) and 0.2% arabinose was added into the culture medium as an inducer in the induction group. Compared with that in the non-induction group (Fig. 1 A1-4), MazF inhibited the growth of E. coli stronger from 6 hours to 12 hours in the induction group (Fig. 1 B1-4). However, the phenomenon described above was not obvious 12 hours later.

Fig. 1 The picture of CFU of pBAD/araC-RBS-MazF-Terminator (A is the non-induction group and B is the induction group)

Fig. 2 The results of CFU of pBAD/araC-RBS-MazF-Terminator

In order to make sure that the engineered E. coli was killed in the environment without arabinose, Inverter was inserted into the circuit. Similarly, the viability count of CFU was chosen to verify the performance of our suicide switch. The plasmid carrying pBAD/araC-Inverter-RBS-MazF-Terminator (BBa_K3332081) were transformed into E. coli BL21 (DE3) and 0.2% arabinose was added into the culture medium as an inducer in the induction group. As shown in Fig. 3, the growth of E. coli in the non-induction group (Fig. 3 C1-4) was inhibited significantly after 6 hours. What’s more, there were no colonies left on the petri plate after 15 hours. That is to say, our kill switch has a good performance in killing E. coli when there is no arabinose. Also, we should pay attention to the reduction of the number of E. coli in the induction group (Fig. 3 D1-4), which indicated the leakage of MazF. Although the leakage at a low level.

Fig. 3 The picture of CFU of pBAD/araC-Inverter-RBS-MazF-Terminator (C is the non-induction group and D is the induction group)

Fig. 4 The result of CFU of pBAD/araC-Inverter-RBS-MazF-Terminator