Team:XHD-Wuhan-A-China/Improve
Improvement
Improved part design
We improved part: BBa_K216005 (PyeaR promoter), which is the promoter of the Escherichia coli yeaR/yoaG operon. The most remarkable feature of this promoter is its ability to sense nitrate and nitrite. In order to better regulate the response of the promoter to nitrate, we use machine learning models to predict and design new PyeaR sequences. Compared to the original sequence, five or six bases have been changed.
Construction of improved PyeaR
We obtained the wild-type PyeaR promoter from HZAU-China team and constructed it into the amilCP-MCS2 plasmid. Plasmid map is in Figure 1. Based on the original sequence, we designed and predicted three mutation sequences that can increase the intensity of the promoter by using our machine learning model. By modifying the PCR primers, we successfully obtained the three mutated PyeaR promoters. Through homologous recombination, we replaced the wild-type promoter with the improved promoter.
Figure 1. Design of PyeaR-amilCP-pBBR1MCS2
Results
Figure 2. Gel electrophoresis of PyeaR-B0034-amilCP-B0015 fragments
We have successfully constructed the pyear-amilCP-MCS2 plasmid through molecular biology technology. And the results are shown in figure 2. We obtained the correct fluorescent band with the right size. We used the machine learning model to predict the strength of the promoter before and after the mutation, and the results are shown in Figure 3. The data presented in Figure 3 shows that all mutant PyeaR have stronger strength than wild-type.
Figure 3. The predicted strength of the wild-type PyeaR and mutant PyeaR
In order to verify the true strength of our redesigned promoter, we replaced the wild-type promoter with a mutant promoter. After culturing the engineered bacteria overnight at 220 rpm, it was reactivated at a ratio of 1:100 in LB liquid medium for 4 hours. And then we tested OD588 of the samples every half hour for 6 hours. The results are shown in Figure 4. The results show that mutant PyeaR (BBa_K3926002) has a stronger promoter strength than the wild-type.
Figure 4. Verified the strength of the wild-type PyeaR and mutant PyeaR
Figure 5. The expression levels of amilCP genes are different between wild-type PyeaR and mutant PyeaR
Conclusion
Compare to the unmodified PyeaR promoter (WT-PyeaR in Figure 3), the histogram of amilCP (OD588) data demonstrated that the higher strength in mutant PyeaR. In short, we have successfully improved the pyear promoter to make it have a higher promoter strength.
Reference
Lin, H.-Y., Bledsoe, P.J., and Stewart, V. 2007. Activation of yeaR-yoaG operon transcription by the nitrate-responsive regulator NarL is independent of oxygen-responsive regulator Fnr in Escherichia coli K-12. J. Bacteriol. 189, 7539-7548
