Difference between revisions of "Part:BBa K5226090"

 
 
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<partinfo>BBa_K5226090 short</partinfo>
 
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<h2>Sequence and Features</h2>
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<partinfo>BBa_K5226090 SequenceAndFeatures</partinfo>
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<body>
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<h2>Introduction</h2>
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<p>
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<br>
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One of the goals of iGEM SCUT-China-A is to use synthetic biology tools to build <b>thermosensitive bio-switch</b> for <i>Halomonas</i> TD. We successfully <b>built one using ci857 regulatory protein and superfolder green fluorescence protein(sfGFP)</b>. And for better performance and broader range of usage, we <b>replaced the promoter of ci857, characterized together with PR-sfgfp and a thermalsensitive bio-switch with lower leakage and higher fold change was successfully obtained.</b>
  
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<h2>Usage and Biology</h2>
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<br>
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This is a composite part acting as a part of the T-switch we’ve created for Halomonas TD. CI857 is a widely used mutant of cI from bacteriophage λ as a thermo-genetic tool, exhibits strong repression on PR promoter at 30 °C by forming dimmer CI857 complex to achieve active and inactive transcriptional control of repressor and reporter under 37 °C and 30 °C, respectively. The PRM promoter can be regulated by the native bacteriophage lambda proteins CI and CRO. PR promoter is a special promoter regulated by CI857. When CI857 formed dimer, it was inhibited, so that the following genes could not be normally expressed; The inhibition is released when the dimer is depolymerized, and then the gene is normally expressed. J23100 through J23119 are a family of constitutive promoter parts isolated from a small combinatorial library by iGEM06_Berkeley. Its expression intensity is higher than that of the corresponding promoter of CI857 in our original thermosensitive bio-switch. Therefore, we planned to replace the PRM promoter with the J23 series promoter, but unfortunately we were only able to construct a plasmid containing the J23100 promoter and the J23110 promoter. Among them, the plasmid replaced with the J23110 promoter shows great potential. When PR-sfgfp was expressed together, we obtained a new version of T-switch with lower leakage and higher fold change.
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<h2>Experimental characterisation</h2>
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<h3>Protocol</h3>
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<b>Strain construction</b>
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Because <i>Halomonas</i> TD is easy to lose plasmids, we converted plasmid into <i>E. coli</i> s17-1 and transferred plasmids to it by junction and then characterisd it.
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<html> <img src="https://static.igem.wiki/teams/5226/parts/pr-prm-sfgfp-protocol.png" width="700px">
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<b>Fluorescence Characterisation</b>
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<br>
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-Add 1ml 60LB, 0.1% chloramphenicol and spectinomycin to the well, pick up the strain and add it to the hole, and then put the deepwell plate in the shaker at the corresponding temperature for 12-14 hours. Usually, for each strain we would prepare two wells in case of emergency.
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<br>
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-Add 1ml 60LB, 10μl bacterial fluid of last step,0.1% chloramphenicol and spectinomycin to the well, then put the deepwell plate in the shaker at the corresponding temperature for 12 hours.
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<br>
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-Take 100μl bacterial fluid of last step, add 400μl deionised water to the bacterial fluid, then centrifuge for 5 minutes, discard the supernatant and add 500μl 1X PBS, take 200μl into the enzyme standard plate to measure fluorescence using microplate reader（absorbance 600, excitation/emission wavelength 488/520）
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<h3>Result</h3>
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The result showed that the fluorescence leakage of J23110-ci857 was <b>reduced by 44%</b> and the leakage of J23100-ci857 was <b>reduced by 25%.</b> The fluorescence intensity of them were controlled <b>at around 100 and 80</b>, which means this version of T-switch could be used to control genes with a desired gene expression intensity of around 100 and 80.
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<html> <img src="https://static.igem.wiki/teams/5226/parts/result-of-j23110-ci857.png" width="700px">
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===Usage and Biology===
 
  
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<span class='h3bb'>Sequence and Features</span>
 
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<partinfo>BBa_K5226090 SequenceAndFeatures</partinfo>
 
  
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<h2>References</h2>
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[1]Wang, X., Han, JN., Zhang, X. et al. Reversible thermal regulation for bifunctional dynamic control of gene expression in Escherichia coli. Nat Commun 12, 1411 (2021). https://doi.org/10.1038/s41467-021-21654-x
  
 
<!-- Uncomment this to enable Functional Parameter display  
 
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Latest revision as of 07:02, 30 September 2024

J23110-ci857

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]

Introduction


One of the goals of iGEM SCUT-China-A is to use synthetic biology tools to build thermosensitive bio-switch for Halomonas TD. We successfully built one using ci857 regulatory protein and superfolder green fluorescence protein(sfGFP). And for better performance and broader range of usage, we replaced the promoter of ci857, characterized together with PR-sfgfp and a thermalsensitive bio-switch with lower leakage and higher fold change was successfully obtained.

Usage and Biology


This is a composite part acting as a part of the T-switch we’ve created for Halomonas TD. CI857 is a widely used mutant of cI from bacteriophage λ as a thermo-genetic tool, exhibits strong repression on PR promoter at 30 °C by forming dimmer CI857 complex to achieve active and inactive transcriptional control of repressor and reporter under 37 °C and 30 °C, respectively. The PRM promoter can be regulated by the native bacteriophage lambda proteins CI and CRO. PR promoter is a special promoter regulated by CI857. When CI857 formed dimer, it was inhibited, so that the following genes could not be normally expressed; The inhibition is released when the dimer is depolymerized, and then the gene is normally expressed. J23100 through J23119 are a family of constitutive promoter parts isolated from a small combinatorial library by iGEM06_Berkeley. Its expression intensity is higher than that of the corresponding promoter of CI857 in our original thermosensitive bio-switch. Therefore, we planned to replace the PRM promoter with the J23 series promoter, but unfortunately we were only able to construct a plasmid containing the J23100 promoter and the J23110 promoter. Among them, the plasmid replaced with the J23110 promoter shows great potential. When PR-sfgfp was expressed together, we obtained a new version of T-switch with lower leakage and higher fold change.

Experimental characterisation

Protocol

Strain construction

Because Halomonas TD is easy to lose plasmids, we converted plasmid into E. coli s17-1 and transferred plasmids to it by junction and then characterisd it.

Fluorescence Characterisation
-Add 1ml 60LB, 0.1% chloramphenicol and spectinomycin to the well, pick up the strain and add it to the hole, and then put the deepwell plate in the shaker at the corresponding temperature for 12-14 hours. Usually, for each strain we would prepare two wells in case of emergency.
-Add 1ml 60LB, 10μl bacterial fluid of last step,0.1% chloramphenicol and spectinomycin to the well, then put the deepwell plate in the shaker at the corresponding temperature for 12 hours.
-Take 100μl bacterial fluid of last step, add 400μl deionised water to the bacterial fluid, then centrifuge for 5 minutes, discard the supernatant and add 500μl 1X PBS, take 200μl into the enzyme standard plate to measure fluorescence using microplate reader（absorbance 600, excitation/emission wavelength 488/520）

Result

The result showed that the fluorescence leakage of J23110-ci857 was reduced by 44% and the leakage of J23100-ci857 was reduced by 25%. The fluorescence intensity of them were controlled at around 100 and 80, which means this version of T-switch could be used to control genes with a desired gene expression intensity of around 100 and 80.



References

[1]Wang, X., Han, JN., Zhang, X. et al. Reversible thermal regulation for bifunctional dynamic control of gene expression in Escherichia coli. Nat Commun 12, 1411 (2021). https://doi.org/10.1038/s41467-021-21654-x