Difference between revisions of "Part:BBa K1316002"
 
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<h3>Experience </h3>
 
<h3>Experience </h3>
 
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<p>The iGEM20 NEFU_China uses optical biosensors in the Bio-optical Landmine Detection, with the <i>yqjF</i> promoter as the core component. The <i>yqjF</i> promoter has been documented to correspond to the volatile substance DNT released by landmines. The original <i>yqjF</i> has a detection threshold of 25 mg/L for DNT, as we presented in the figure below.</p>
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The iGEM20 NEFU_China uses optical biosensors in the Bio-optical Landmine Detection, with the <i>yqjF</i> promoter as the core component. The <i>yqjF</i> promoter has been documented to respond to the volatile DNT released from landmines. The original <i>yqjF</i> promoter has a detection threshold of 25 mg/L of DNT, as we presented in the figure below.
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<img src="https://static.igem.org/mediawiki/parts/d/d2/T--NEFU_China--yqjF%E9%98%88%E5%80%BC.png
 
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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>
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To improve the strength of the <i>yqjF </i> promoter in response to DNT induction, we used semi-rational mutagenesis and error-prone PCR mutagenesis to randomly mutate the <i>yqjF</i>  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>) , respectively, with markedly reduced DNT detection thresholds. After combining the mutations of the<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>) , we generated the <i>yqjF3rd</i> and its transcription factor <i>yhaJ1st</i> (<a href="https://parts.igem.org/Part:BBa_K3350858">BBa_K3350858</a>) promoter. When overexpressing the <i>yqjF3rd-EGFP </i>and its transcription activator  <i>yhaJ1st</i> (<a href="https://parts.igem.org/Part:BBa_K3350858">BBa_K3350858</a>)  in the engineered bacteria, we could significantly reduce the DNT detection threshold to 0.1 mg/L. Compared to 25 mg/L of the wild-type yqjF promoter without <i>yhaJ</i>(<a href="https://parts.igem.org/Part:BBa_K3350857">BBa_K3350857</a>) overexpression, we have increased the DNT detection sensitivity of the engineered bacteria by 250-fold.
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Latest revision as of 12:34, 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 respond to the volatile DNT released from landmines. The original yqjF promoter has a detection threshold of 25 mg/L of DNT, as we presented in the figure below.

To improve the strength of the yqjF promoter in response to DNT induction, 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) , respectively, with markedly reduced DNT detection thresholds. After combining the mutations of theyqjF1st and yqjF2nd (BBa_K3350859 and BBa_K3350860) , we generated the yqjF3rd and its transcription factor yhaJ1st (BBa_K3350858) promoter. When overexpressing the yqjF3rd-EGFP and its transcription activator yhaJ1st (BBa_K3350858) in the engineered bacteria, we could significantly reduce the DNT detection threshold to 0.1 mg/L. Compared to 25 mg/L of the wild-type yqjF promoter without yhaJ(BBa_K3350857) overexpression, we have increased the DNT detection sensitivity of the engineered bacteria by 250-fold.

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


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 128
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]