Difference between revisions of "Part:BBa K3350862"
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<span class='h3bb'>Sequence and Features</span> | <span class='h3bb'>Sequence and Features</span> | ||
Revision as of 03:20, 26 October 2020
yqjF3rd (promoter)
We used semi-rational mutagenesis and error-prone PCR mutagenesis to randomly mutate the yqjF(BBa_K1316002) promoter and obtained its mutant versions yqjF1st and yqjF2nd (BBa_K3350859 and BBa_K3350860) with reduced DNT detection thresholds. After combining the favorable mutation sites of the yqjF1st and yqjF2nd, we generated the yqjF3rd promoter that show much reduced DNT detection threshold of 5 mg/L versus 25 mg/L of the wild-type yqjF promoter.
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]
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.
Model
Our modeling analyses of the yqjF promoter showed that DNT could induce the yqjF promoter, and there was a three-way relationship between the fluorescent protein expression and DNT concentration, with an R2 of 0.990, which was higher than 0.7. Thus, our proposed model could well fit the experimental data, suggesting that there was a strong correlation between fluorescent protein expression and DNT concentration , and the 95% confidence interval for the slope of the data was 1263.861-1716.902, with a slope of the p-value 0.000. Therefore, the difference between the slope value and 0 was statistically significant, and there was a strong linear relationship between fluorescent protein expression and DNT concentration.
Characterization and Measurement
Previously reported detection limit of the yqjF promoter is as high as 25 mg/L of DNT[1], thus, we must improve the sensitivity of the yqjF promoter for its practical application.
We optimized the response of the yqjF promoter to DNT in the following three aspects. We used semi-rational mutagenesis and error-prone PCR mutagenesis to randomly mutate the yqjF promoter and obtained its mutant versions yqjF1st and yqjF2nd (BBa_K3350859 and BBa_K3350860) with reduced DNT detection thresholds. After combining the favorable mutation sites of the yqjF1st and yqjF2nd , we generated the yqjF3rd promoter that show much reduced DNT detection threshold of 5 mg/L versus 25 mg/L of the wild-type yqjF promoter.
Based on previous literature, the yqjF promoter is likely activated by THT, which is a metabolite of DNT, and when binding to THT, the transcription factor yhaJ can activate the yqjF promoter. Based on these evidence, we generated three types of bacteria to further understand the connection between the metabolic processes of DNT and its regulatory mechanism. The nemA-nfsA-nfsB genes are responsible to convert DNT to THT. However, when we overexpressed the nemA-nfsA-nfsB (BBa_K1316006)genes and simultaneously introduced the yhaJ gene in the engineered bacteria, the fluorescence intensity was markedly reduced compared to the bacteria carrying the yhaJ gene alone. The results suggested that the simultaneous expression of all these genes could adversely affects bacterial metabolism.
Finally, we used error-prone mutagenesis to optimize the YhaJ transcription factor and obtained its mutated version yhaJ1st (BBa_K3350858). With overexpressed yhaJ1st in the engineered bacteria, we reduced the DNT detection threshold from 25 mg/L to 0.1 mg/L, which is a 250-fold increase of the sensitivity.
Aromatic compounds are the main components of water pollutants, and we hope that other iGEM teams can use this collection part to achieve highly sensitive detection of different water pollutants.
References
[1] S. Yagur-Kroll, S. Belkin et al., “Escherichia Coli bioreporters for the detection of 2,4-dinitrotoluene and 2,4,6-trinitrotoluene”, Appl. Microbiol. Biotechnol. 98, 885-895, 2014.