Difference between revisions of "Part:BBa K3350858"

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Several transcriptional regulatory protein families are involved in the metabolism of aromatic compounds in bacteria, and have difference in both their structures and in the activation mechanisms. The LysR-type transcriptional regulators (LTTRs) are characterized by an N-terminal DNA-binding helix-turn-helix motif and a C-terminal co-inducer-binding domain. According to Tropel and V an Der Meer, all LTTRs involved in aromatic compounds degradation pathways can act as transcriptional activators with the co-inducers, which are either degraded compounds or intermediates in the degradation pathways. The YhaJ protein, a member of the LysR type family, acts as a transcriptional regulator to activate the the<i> yqjF</i> promoter (<a href="https://parts.igem.org/Part:BBa_K1316002">BBa_K1316002</a>).[1]
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Several transcriptional regulatory protein families are involved in the metabolism of aromatic compounds in bacteria, and have difference in both their structures and in the activation mechanisms. The LysR-type transcriptional regulators (LTTRs) are characterized by an N-terminal DNA-binding helix-turn-helix motif and a C-terminal co-inducer-binding domain. According to Tropel and V an Der Meer, all LTTRs involved in aromatic compounds degradation pathways can act as transcriptional activators with the co-inducers, which are either degraded compounds or intermediates in the degradation pathways &nbsp;<sup>[1]</sup>. The YhaJ protein, a member of the LysR type family, acts as a transcriptional regulator to activate the the<i> yqjF</i> (<a href="https://parts.igem.org/Part:BBa_K1316002">BBa_K1316002</a>) promoter.
 
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The YhaJ protein, a member of the LysR type family, acts as a transcriptional regulator to activate the the<i> yqjF</i> promoter (<a href="https://parts.igem.org/Part:BBa_K1316002">BBa_K1316002</a>).We generated the <i>yqjF3rd </i>(<a href="https://parts.igem.org/Part:BBa_BBa_K3350862">BBa_K3350862</a>)promoter that show much reduced DNT detection threshold of 5 mg/L versus 25 mg/L of the wild-type<i> yqjF</i> promoter,and we overexpressed the <i>yhaJ1st </i>gene together with an <i>yqjF3rd</i>-containing reporter construct in the engineered bacteria, we could reduce the DNT detection threshold of the <i>yqjF </i>promoter from 25 mg/L to 0.1 mg/L, which is a 250-fold increase of the sensitivity.
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The YhaJ protein, a member of the LysR type family, acts as a transcriptional regulator to activate the the<i> yqjF</i> promoter (<a href="https://parts.igem.org/Part:BBa_K1316002">BBa_K1316002</a>).We generated the <i>yqjF3rd </i> (<a href="https://parts.igem.org/Part:BBa_K3350862">BBa_K3350862</a>) promoter that show much reduced DNT detection threshold of 5 mg/L versus 25 mg/L of the wild-type<i> yqjF</i> promoter,and we overexpressed the <i>yhaJ1st </i>gene together with an <i>yqjF3rd</i>-containing reporter construct in the engineered bacteria, we could reduce the DNT detection threshold of the <i>yqjF </i>promoter from 25 mg/L to 0.1 mg/L, which is a 250-fold increase of the sensitivity.
 
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We had optimized the <i>yqjF </i>promoter to obtain its new version<i> yqjF3rd </i>(<a href="https://parts.igem.org/Part:BBa_BBa_K3350862">BBa_K3350862</a>). Thus, when we overexpressed the <i>yhaJ </i>gene together with an <i>yqjF3rd</i>-containing reporter construct in the engineered bacteria, we could reduce the DNT detection threshold of the <i>yqjF </i>promoter from 25 mg/L to 0.25 mg/L, which is a 100-fold increase of the sensitivity.
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We had optimized the <i>yqjF </i>promoter to obtain its new version<i> yqjF3rd </i>(<a href="https://parts.igem.org/Part:BBa_K3350862">BBa_K3350862</a>). Thus, when we overexpressed the <i>yhaJ </i>gene together with an <i>yqjF3rd</i>-containing reporter construct in the engineered bacteria, we could reduce the DNT detection threshold of the <i>yqjF </i>promoter from 25 mg/L to 0.25 mg/L, which is a 100-fold increase of the sensitivity（Fig. 1).
  
 
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<img src="https://static.igem.org/mediawiki/parts/e/e9/T--NEFU_China--yhaJ.png
 
<img src="https://static.igem.org/mediawiki/parts/e/e9/T--NEFU_China--yhaJ.png
 
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Fig. 1. Effects of YhaJ overexpression on DNT detection sensitivity of engineered bacteria.
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Given the importance of the YhaJ transcription factor, we used random error prone PCR mutagenesis to mutated the <i>yhaJ </i>coding sequence. Based on the experimental results, we obtained an improved version of the transcription factor, <i>yhaJ1st</i>.
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Given the importance of the YhaJ transcription factor, we further investigated whether we could optimize the transcription activator YhaJ to further improve the detection sensitivity. Employing random mutagenesis approach, we generate the YhaJ mutant library and screen over 500 clones to evaluate their effects on <i>yqjF</i> promoter-mediated EGFP expression in response to 10 mg/L DNT. Based on the data of Fig. 2, we discovered that one YhaJ mutant showed markedly increased EGFP production compared to the wild-type YhaJ. Based on DNA sequencing analysis, this mutant, designed as <i>yhaJ1st</i>.  
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<img src="https://static.igem.org/mediawiki/parts/a/a5/T--NEFU_China--yhaJst1%E7%AD%9B%E9%80%89.png
 
<img src="https://static.igem.org/mediawiki/parts/a/a5/T--NEFU_China--yhaJst1%E7%AD%9B%E9%80%89.png
 
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Fig. 2. The effects of the yhaJ mutations on the yqjF promoter-driven EGFP expression in response to 10 mg/L DNT.
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When we overexpressed the <i>yhaJ1st</i>, we observed the reduced DNT detection threshold  of the <i>yqjF</i> promoter from 25 mg/L to 0.1mg/L, which is a 250-fold increase of the sensitivity.
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When we overexpressed <i>yqjF3rd</i> and <i>yhaJ1st</i>, we observed the reduced DNT detection threshold  of the <i>yqjF</i> promoter from 25 mg/L to 0.1 mg/L, which is a 250-fold increase of the sensitivity (Fig. 3).
 
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<img src="https://static.igem.org/mediawiki/parts/c/c6/T--NEFU_China--yhaJ1st%E9%98%88%E5%80%BC.png
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<img src="https://static.igem.org/mediawiki/parts/8/86/T--NEFU_China--%E9%98%88%E5%80%BC%E9%99%8D%E4%BD%8E%E5%88%B00.1%281%29.png
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Fig. 3. Evaluation of overexpression of <i>yqjF3rd</i> and <i>YhaJ1st</i>.
  
 
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===References===
 
===References===
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[1] Palevsky N, Shemer B, Connolly JP, Belkin S. The Highly Conserved<i> Escherichia coli</i> Transcription Factor YhaJ Regulates Aromatic Compound Degradation. Front Microbiol. 2016 Sep 22;7:1490.
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[1] Palevsky, N., Shemer, B., Connolly, J. P., & Belkin, S. (2016). The Highly Conserved Escherichia coli Transcription Factor YhaJ Regulates Aromatic Compound Degradation. Frontiers in microbiology, 7, 1490.
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_K3350858 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K3350858 SequenceAndFeatures</partinfo>

Latest revision as of 10:04, 27 October 2020


yhaJ1st (DNA-binding transcriptional activator)
Several transcriptional regulatory protein families are involved in the metabolism of aromatic compounds in bacteria, and have difference in both their structures and in the activation mechanisms. The LysR-type transcriptional regulators (LTTRs) are characterized by an N-terminal DNA-binding helix-turn-helix motif and a C-terminal co-inducer-binding domain. According to Tropel and V an Der Meer, all LTTRs involved in aromatic compounds degradation pathways can act as transcriptional activators with the co-inducers, which are either degraded compounds or intermediates in the degradation pathways  [1]. The YhaJ protein, a member of the LysR type family, acts as a transcriptional regulator to activate the the yqjF (BBa_K1316002) promoter.


Usage and Biology


The YhaJ protein, a member of the LysR type family, acts as a transcriptional regulator to activate the the yqjF promoter (BBa_K1316002).We generated the yqjF3rd (BBa_K3350862) promoter that show much reduced DNT detection threshold of 5 mg/L versus 25 mg/L of the wild-type yqjF promoter,and we overexpressed the yhaJ1st gene together with an yqjF3rd-containing reporter construct in the engineered bacteria, we could reduce the DNT detection threshold of the yqjF promoter from 25 mg/L to 0.1 mg/L, which is a 250-fold increase of the sensitivity.

Characterization and Measurement


We had optimized the yqjF promoter to obtain its new version yqjF3rd (BBa_K3350862). Thus, when we overexpressed the yhaJ gene together with an yqjF3rd-containing reporter construct in the engineered bacteria, we could reduce the DNT detection threshold of the yqjF promoter from 25 mg/L to 0.25 mg/L, which is a 100-fold increase of the sensitivity（Fig. 1).

Fig. 1. Effects of YhaJ overexpression on DNT detection sensitivity of engineered bacteria.

Given the importance of the YhaJ transcription factor, we further investigated whether we could optimize the transcription activator YhaJ to further improve the detection sensitivity. Employing random mutagenesis approach, we generate the YhaJ mutant library and screen over 500 clones to evaluate their effects on yqjF promoter-mediated EGFP expression in response to 10 mg/L DNT. Based on the data of Fig. 2, we discovered that one YhaJ mutant showed markedly increased EGFP production compared to the wild-type YhaJ. Based on DNA sequencing analysis, this mutant, designed as yhaJ1st.

Fig. 2. The effects of the yhaJ mutations on the yqjF promoter-driven EGFP expression in response to 10 mg/L DNT.

When we overexpressed yqjF3rd and yhaJ1st, we observed the reduced DNT detection threshold of the yqjF promoter from 25 mg/L to 0.1 mg/L, which is a 250-fold increase of the sensitivity (Fig. 3).

Fig. 3. Evaluation of overexpression of yqjF3rd and YhaJ1st.

References

[1] Palevsky, N., Shemer, B., Connolly, J. P., & Belkin, S. (2016). The Highly Conserved Escherichia coli Transcription Factor YhaJ Regulates Aromatic Compound Degradation. Frontiers in microbiology, 7, 1490.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]