Difference between revisions of "Part:BBa K5226089"

 
 
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<h2>Sequence and Features</h2>
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<partinfo>BBa_K5226089 SequenceAndFeatures</partinfo>
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<h2>Introduction</h2>
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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 sfgfp, characterized together with PRM-ci857, and a thermalsensitive bio-switch with lower leakage was successfully obtained.</b>
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<h2>Usage and Biology</h2>
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This is a composite part acting as a part of the T-switch we’ve created for <i>Halomonas</i> TD. CI857 is a widely used mutant of cI from <i>bacteriophage</i> 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 reporter genes sfgfp could not be normally expressed; The inhibition is released when the dimer is depolymerized, and then the following genes could normally expressed. By using both PRM and PR promoters simultaneously, the OR binding site is increased. When PRM-ci857(<a href="https://parts.igem.org/Part:BBa_K5226063">BBa_K5226063</a>)  was expressed together, we obtained a combination of thermosensitive bio-switch with lower leakage.
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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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-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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-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 leakage was <b>reduced by 58%</b> and the fluorescence intensity of sfgfp was controlled <b>at around 40</b>, which means this version of T-switch could be used to control genes with a desired gene expression intensity of around 40.
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<html> <img src="https://static.igem.wiki/teams/5226/parts/result-of-pr-prm-sfgfp.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_K5226089 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  
 
<!-- Uncomment this to enable Functional Parameter display  

Latest revision as of 07:05, 30 September 2024

PR/PRM-sfgfp

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
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 141

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 sfgfp, characterized together with PRM-ci857, and a thermalsensitive bio-switch with lower leakage 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 reporter genes sfgfp could not be normally expressed; The inhibition is released when the dimer is depolymerized, and then the following genes could normally expressed. By using both PRM and PR promoters simultaneously, the OR binding site is increased. When PRM-ci857(BBa_K5226063) was expressed together, we obtained a combination of thermosensitive bio-switch with lower leakage.

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 leakage was reduced by 58% and the fluorescence intensity of sfgfp was controlled at around 40, which means this version of T-switch could be used to control genes with a desired gene expression intensity of around 40.



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