Part:BBa_K3350860
yqjF2nd (promoter)
Based on the sequences provided in the literature, we synthesized the original yqjF (BBa_K1316002) promoter. To improve the sensitivity of the yqjF (BBa_K1316002) promoter to DNT for its practical application, we made error-prone PCR mutagenesis to randomly mutate the yqjF promoter and generated its mutated version, the yqjF2nd.
Usage and Biology
Landmines pose a great threat to human lives and health. In our project, we designed a Bio-optical Landmine Detection device to achieve landmines detection with high sensitivity.
Our yqjF promoter (BBa_K1316002) was originally involved in the metabolism of aromatic compounds in bacteria and was later found to respond to chemicals, such as 2,4-dinitrotoluene (DNT) constantly released from landmines.
To improve the sensitivity of the yqjF (BBa_K1316002) promoter to DNT for its practical application,we made error-prone PCR mutagenesis to randomly mutate the yqjF promoter and generated its mutated version, the yqjF2nd. This mutagenesis decreased the DNT detection threshold from 25 mg/L of the original yqjF promoter to 15 mg/L of the yqjF1st promoter, which is a double fold increase of the sensitivity approximately.
Finally,aromatic compounds are the main components of water pollutants, and we hope that other iGEM teams can use this basic part to achieve highly sensitive detection of different water pollutants.
Characterization and Measurement
Previously reported detection threshold of the yqjF promoter is as high as 25 mg/L of DNT [1] (Fig. 1), thus, we must improve the sensitivity of the yqjF promoter for its practical application. We wanted to construct a system to evaluate the responsiveness of the wild-type yqjF promoter to DNT and its detection threshold. We amplified the wild-type yqjF promoter from the genomic DNA of bacteria, inserted the EGFP, as a reporter, at its downstream, and thus generated the reporter plasmid pYB1a-yqjF-EGFP. This reporter was transformed into Escherichia Coli DH5α for the following experiments.
We employed a random mutagenesis kit to introduce random mutations in the yqjF promoter through PCR amplification. We first optimized the amounts of the mutagenesis enhancer to evaluate the correlation of its concentration with the mutation rate, and determined its appropriate amount to be used in our study. Using this optimized condition, we carried out the error-prone PCR and screened about 300 clones for their responsiveness to the induction of 30 mg/L DNT. As shown in Fig. 2, we discovered one mutated yqjF promoter with higher induction by DNT than that of the wild-type promoter. This yqjF promoter mutant was named as yqjF2nd and we sequenced this clone and found that it contained 3 mutation sites (Fig. 3)
Finally, we tested the induction of the reporter system by different concentrations of DNT (10, 15, 20, 25, 30, 40 and 50 mg/L). EGFP intensity in response to DNT changes was shown in Fig. 4. The results indicated that the DNT detection threshold decreased from 25 mg/L of the original yqjF promoter to 15 mg/L of the yqjF1st promoter, which is a double fold increase of the sensitivity approximately.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
References
[1] Yagur-Kroll, S., Lalush, C., Rosen, R., Bachar, N., Moskovitz, Y., & Belkin, S. (2014). Escherichia coli bioreporters for the detection of 2,4-dinitrotoluene and 2,4,6-trinitrotoluene. Applied microbiology and biotechnology, 98(2), 885–895.
//dna
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