Difference between revisions of "Part:BBa K1316002"
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<p>And we used semi-rational mutagenesis and error-prone PCR mutagenesis to randomly mutate the <i>yqjF</i>(<a href="https://parts.igem.org/Part:BBa_K1316002">BBa_K1316002</a>) promoter and obtained its mutant versions <i>yqjF1st </i>and <i>yqjF2nd</i> (<a href="https://parts.igem.org/Part:BBa_K3350859">BBa_K3350859 </a>and <a href="https://parts.igem.org/Part:BBa_K3350860">BBa_K3350860</a>) with reduced DNT detection thresholds. After combining the favorable mutation sites of the <i>yqjF1st</i> and <i>yqjF2nd</i>, we generated the <i>yqjF3rd</i> promoter that show much reduced DNT detection threshold of 5 mg/L versus 25 mg/L of the wild-type <i>yqjF</i> promoter.</p> | <p>And we used semi-rational mutagenesis and error-prone PCR mutagenesis to randomly mutate the <i>yqjF</i>(<a href="https://parts.igem.org/Part:BBa_K1316002">BBa_K1316002</a>) promoter and obtained its mutant versions <i>yqjF1st </i>and <i>yqjF2nd</i> (<a href="https://parts.igem.org/Part:BBa_K3350859">BBa_K3350859 </a>and <a href="https://parts.igem.org/Part:BBa_K3350860">BBa_K3350860</a>) with reduced DNT detection thresholds. After combining the favorable mutation sites of the <i>yqjF1st</i> and <i>yqjF2nd</i>, we generated the <i>yqjF3rd</i> promoter that show much reduced DNT detection threshold of 5 mg/L versus 25 mg/L of the wild-type <i>yqjF</i> promoter.</p> | ||
Revision as of 10:22, 26 October 2020
yqjF promoter
Promoter of the yqjF gene, which is activated by the presence of several nitrogen-based compounds such as 2,4,6-TNT, 2,4-DNT and 1,3-DNB.[1]
Characterisation
The functionality of the yqjF promoter is proven by the production of mKate2 in response to 2,4-DNT on the characterisation of BioBricks BBa_K1316007 (plate reader experiment) and BBa_K1316003 (FACS experiment).
Experience
The iGEM20 NEFU_China uses optical biosensors in the Bio-optical Landmine Detection, with the yqjF promoter as the core component. The yqjF promoter has been documented to correspond to the volatile substance DNT released by landmines. The original yqjF has a detection threshold of 25 mg/L for DNT, as we presented in the figure below.
And 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.
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.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 128
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]