Difference between revisions of "Part:BBa K3081003"
 
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<partinfo>BBa_K3081003 short</partinfo>
 
<partinfo>BBa_K3081003 short</partinfo>
  
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A fluorescent protein sfGFP is fused to the C-terminal of dCas9 for quantitative characterization.
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In this part, a fluorescent protein sfGFP is fused to the C-terminal of dCas9 for quantitative characterization.
 
A degradation tag ssrA is added to the end of sfGFP to alleviate the burden caused by dCas9.
 
A degradation tag ssrA is added to the end of sfGFP to alleviate the burden caused by dCas9.
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The expression of this fusion protein is driven by pBAD promoter, which can be triggered by arabinose.This part is an improvement of dCas9-linker-sfGFP(<partinfo>BBa_K3081119</partinfo>), in which we add a degradation signal peptide ssrA to sfGFP.
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The expression of this fusion protein is driven by pBAD promoter, which can be triggered by arabinose.This part is an improvement of pBAD dCas9-linker-sfGFP(<partinfo>BBa_K3081002</partinfo>).Then we found that the protein with ssrA was expressed at a lower level than the control protein, with the same concentration of inducer.
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<center>
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https://2019.igem.org/wiki/images/7/78/T--Peking--dls_ssrA.png
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</center>
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<center>
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Figure 1: Comparison between dCas9 and dCas9-ssrA system by expression level (on a low copy number plasmid pSB4A5)
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</center>
  
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Latest revision as of 20:23, 21 October 2019


pBAD-dCas9-linker-sfGFP-ssrA

In this part, a fluorescent protein sfGFP is fused to the C-terminal of dCas9 for quantitative characterization. A degradation tag ssrA is added to the end of sfGFP to alleviate the burden caused by dCas9. The expression of this fusion protein is driven by pBAD promoter, which can be triggered by arabinose.This part is an improvement of pBAD dCas9-linker-sfGFP(BBa_K3081002).Then we found that the protein with ssrA was expressed at a lower level than the control protein, with the same concentration of inducer.

T--Peking--dls_ssrA.png

Figure 1: Comparison between dCas9 and dCas9-ssrA system by expression level (on a low copy number plasmid pSB4A5)

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 1205
    Illegal NheI site found at 2321
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 1144
    Illegal BamHI site found at 4600
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 979
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI site found at 961
    Illegal SapI.rc site found at 5369