Team:NEFU China/Engineering
Overview
As a high-level competition based on synthetic biology, the iGEM encourages problem solving on engineering success. This year, This year, we focused on improving the relatively low efficiency and high detection threshold of the yqjF promoter that is used in the landmine detection.
Based on our review of previous literature, the DNT detection threshold of the wide-type yqjF promoter is much higher than the actual DNT amounts released by landmines. Due to this reason, the yqjF promoter cannot be directly used in landmine detection. Therefore, we put efforts to improve the yqjF promoter to reduce its DNT detection threshold and make it possible for our application. In related literature, we found that YhaJ is a positive transcriptional regulator of the yqjF promoter. We predicted that the overexpression of the yhaJ gene in bacteria could potentially reduce the DNT detection threshold. Therefore, we planned to make engineered bacteria with overexpressed yhaJ to enhance sensitivity of DNT detection.
Methods
First, we stably integrated the YhaJ expression cassette into the engineered bacteria that already harbored the yqjF promoter reporter system. When using the engineered bacteria to detect different concentrations of DNT, we remarkably reduced DNT detection threshold, which was lower than the detection levels of our expectation. Nevertheless, we still explored to further improve the detection sensitivity. We utilized error-prone PCR to introduce random mutations in different elements of the transcription activator YhaJ to generate an array of the transcription activator YhaJ mutants. After screening these mutants, we obtained a mutated transcription activator YhaJ with reduced DNT detection threshold. We then tested this transcription activator YhaJ mutant, we observed that the DNT detection threshold dropped to 0.1 mg/L versus 25 mg/L of the original transcription activator. Thus, our mutagenesis study of the transcription activator YhaJ led to 250-fold increase of DNT detection sensitivity.
Fig. 1. DNT detection by the reporter containing the yqjF3rd promoter.
Fig. 2. Effects of YhaJ overexpression on DNT detection sensitivity of engineered bacteria.
Fig. 3. Assay to evaluate the DNT detection threshold of the yhaJ mutations.
Conclusion
In our attempt to reduce the DNT detection threshold by introducing YhaJ, we found that the detection sensitivity of wild-type YhaJ was still not ideal. Thus, we employed an error-prone PCR random mutagenesis to generate different mutants of YhaJ. Finally, we obtained a YhaJ mutant that can significantly improve the DNT detection sensitivity of the engineered bacteria. Overall, our attempt to generate ideal engineered bacteria for landmines detection has been successfully conducted.
