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Applications of BBa_K302033

Test the toxin gene can be translated and play a role in situations The 2016 BIT-China iGEM Team equipped the bacteria with a plasmid-sensing logically adjustable cell killer(P-SLACKiller). To kill the slacker bacteria in time, we chose to construct circuits with toxin genes mazF and hokD. And here we designed two circuits to verify the function of toxin genes, one was araC+PBAD+B0032+mazFBBa_K2120002, another was araC+PBAD+B0032+hokDBBa_K2120003. The araC+PBAD promoter can be controlled tightly by using arabinose. Then we constructed two plasmids containing these two circuits, and transformed the two plasmids into top10 respectively. After transformation, we measured the OD600 to draw the growth curve and observed the function of toxin protein. Besides the above two bacteria contained toxin genes, the control was the empty pSB1C3 vector. Add arabinose or not, there was another comparison. We add 10% arabinose 50 uL into 50 mL LB culture medium when the OD600 is 0.6 (the log phase). We measured the OD600 every hour until the bacteria reached the stationary phase.

Fig.1 Compared with the negative control (the empty pSB1C3 vector), OD600 of the two circuits containing toxin proteins are obviously lower, and the difference is evident as time going on. No obvious difference observed among the three groups with no induction. It showed that toxin proteins didn’t leak out.

Through growth curves, we concluded that toxin protein MazF and HokD have different lethal efficiency. Depending on different situations, they can be used.

Regulate the lethal efficiency of the toxin gene in an advanced way

To verify the toxin gene in an advanced way, we tested whether the toxin gene could be a well-regulated one. So we adjusted the translation efficiency of toxin proteins through replace ribosome binding site(RBS), thus to regulate the toxin gene. Through one-step mutation, we have separately replaced the B0032 (33.96%)in above circuits with B0031 (12.64%) and B0034 (100%). So we got： araC+PBAD+B0031+mazF BBa_K2120004， araC+PBAD+B0031+hokDBBa_K2120005， araC+PBAD+B0034+mazFBBa_K2120006， araC+PBAD+B0034+hokD，and here were the sequencing results.

Fig.2 The successful sequencing results of 4 mutations: B0031+mazF; B0034+mazF; B0031+hokD; B0034+hokD

By using the above methods that we used to verify the function of the toxin gene, we also tested OD600 to get the growth curve to show the lethal efficiency of MazF and HokD under different RBS. Compare with the negative control (pSB1C3 empty vector) and the positive control (B0032 circuits, the above two circuits), the experimental groups showed obvious difference. For MazF, the strong RBS B0034 was proved to have highest lethal efficiency. For HokD, the weak RBS B0031 was proved to have the lowest lethal efficiency.

Fig.3 For the toxin protein MazF, we concluded that the influence of strong RBS is more evident.

Fig.4 For the toxin protein HokD, the replacement of RBS will influence the expression of HokD. The weaker RBS B0031 leads to lower expression of toxin protein HokD.

All results showed both the toxin genes can be well-regulated by different RBS, thus the toxin proteins can be widely used in various situations according to different requirements.

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