Part:BBa_K3926002

An improved PyeaR with higher expression strength

We use machine learning model to design this promoter.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Usage and Biology

Introduction

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

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.

Characterization

We used machine learning methods to predict the promoter strength after mutation, and the results are shown in Figure 1.

Figure 1 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 2 and Figure 3. The results show that mutant PyeaR (BBa_K3926002) has a stronger promoter strength than the wild-type.

Figure 2 Verified the strength of the wild-type PyeaR and mutant PyeaR


Figure 3 The expression levels of amilCP genes are different between wild-type PyeaR and mutant PyeaR

Conclusion

Compare to the unmodified PyeaR promoter (BBa_K216005, 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.