Difference between revisions of "Part:BBa K3257101"

 
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<partinfo>BBa_K3257101 short</partinfo>
 
<partinfo>BBa_K3257101 short</partinfo>
  
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According to our modeling, the number of Cre recombinase in one E.coli cell should be a relatively small one, more specifically 5 per cell, so that mutations will accumulate after rounds of R-Evolution process.  
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For the best composite part, we would like to present you with our Cre recombinase together with degradation tag (https://2019.igem.org/Team:Fudan-TSI/Part_Collection). Cre recombinase (BBa_K3257044 https://parts.igem.org/Part:BBa_K3257044) is responsible for the recombination process between the reverse-transcribed cDNA and the target Gene of Interest (GOI), which is rather one of the most important processes in our system.
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Based on this instruction, we have spent much effort in lowering the steady-state expression level of Cre. An important approach is to attach 5 different types of degradation tags after the CDS of Cre. Using EGFP as a reporter, we can see that the degradation rate of degradation tag with the amino acid residue sequence of AANDENWVLAA is moderate for Cre recombinase to reach a steady but low level of expression.
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Transformed into BL21 (DE3) with another plasmid containing lox sites and analyzed by agarose gel, we have seen that Cre is slightly restricted to knockout DNA fragment between two loxP sites.
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Because of the leakage of uninduced Cre expression, we have spent much effort in lowering the steady-state expression level of Cre. An important approach is to attach 5 different types of degradation tags after the CDS of Cre. Using EGFP (BBa_E0040 https://parts.igem.org/Part:BBa_E0040) as a reporter, we can see that the degradation rate of degradation tag with the amino acid residue sequence of AANDENWVLAA (BBa_K3257073 https://parts.igem.org/Part:BBa_K3257073) is moderate for Cre recombinase to reach a steady but relatively low level of expression.
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Transformed into BL21 (DE3) with another plasmid containing lox sites and analyzed by colony PCR, we can see that homologous recombination happens between the same lox sites instead of gene knockout (Figure 1).
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<span class='h3bb'>Sequence and Features</span>
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<span class='h3bb'>'''Sequence and Features'''</span>
 
<partinfo>BBa_K3257101 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K3257101 SequenceAndFeatures</partinfo>
  

Revision as of 03:34, 10 October 2019

Cre Recombinase with SsrA(WVLAA)-aTc Inducible

For the best composite part, we would like to present you with our Cre recombinase together with degradation tag (https://2019.igem.org/Team:Fudan-TSI/Part_Collection). Cre recombinase (BBa_K3257044 https://parts.igem.org/Part:BBa_K3257044) is responsible for the recombination process between the reverse-transcribed cDNA and the target Gene of Interest (GOI), which is rather one of the most important processes in our system.

Because of the leakage of uninduced Cre expression, we have spent much effort in lowering the steady-state expression level of Cre. An important approach is to attach 5 different types of degradation tags after the CDS of Cre. Using EGFP (BBa_E0040 https://parts.igem.org/Part:BBa_E0040) as a reporter, we can see that the degradation rate of degradation tag with the amino acid residue sequence of AANDENWVLAA (BBa_K3257073 https://parts.igem.org/Part:BBa_K3257073) is moderate for Cre recombinase to reach a steady but relatively low level of expression.

Transformed into BL21 (DE3) with another plasmid containing lox sites and analyzed by colony PCR, we can see that homologous recombination happens between the same lox sites instead of gene knockout (Figure 1).


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 436
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 122
  • 1000
    COMPATIBLE WITH RFC[1000]