Part:BBa_K3350859
yqjF1st (promoter)
Our yqjF (BBa_K1316002) promoter 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 conducted semi-rational mutagenesis in its -35 region and generated its mutated version, the yqjF1st promoter.
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 landmine 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 conducted semi-rational mutagenesis in its -35 region and generated its mutated version, the yqjF1st promoter. 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.
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
Previously reported detection threshold of the yqjF promoter is as high as 25 mg/L of DNT [1]. To improve the strength of the yqjF promoter in response to DNT induction, we carried out mutagenesis studies of its essential elements and screened for the mutants with increased transcription activity in the reporter system (Fig. 1). 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.
First, we conducted the semi-rational mutagenesis in the -35 region of the yqjF promoter. We designed the primer yqjF-35-F containing random DNA sequences covering the 9 nucleotides of this region (Fig. 2), which was used together with a downstream primer in PCR amplification. We tested about 500 clones with the yqjF promoter randomly mutated at the 9 nucleotides and tested their response to 30 mg/L of DNT(Fig. 3).
As shown in Fig. 3, among screened clones, we found that many of them showed reduced EGFP induction by 30 mg/L DNT, but one mutant reporter, marked as a red bar. We sequenced this clone and found that it contained three nucleotide mutations around the -35 region (Fig. 4). We named this mutant as the yqjF1st promoter, whith enhanced responsiveness to DNT induction.
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. 5. 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.
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