Composite

Part:BBa_K5276030

Designed by: Yuming Luo   Group: iGEM24_UCAS-China   (2024-10-01)


J23119_Promoter-a118-GFP

img
Fig.1 Selection plasmid of continuous directed evolution system
A constitutive promoter, J23119, is used to express the flipped GFP gene.

We transferred three plasmids (BBa_K5276030, BBa_K5276031, BBa_K5276032) into E. coli. First, we added IPTG and arabinose to activate the "Turn over" switch, inducing the expression of eMutaT7 and mutating the A118 integrase sequence. If the expressed A118 mutant retains its biological function as a recombinase, the GFP gene will be flipped and normally expressed. After sampling the strain, we removed IPTG and arabinose and added ATc to activate the "Turn back" switch, relieving tetR repression on the PLtetRO promoter, which allows Gp44 protein expression to flip the GFP gene back to its original state. After multiple cycles, we can screen for mutant recombinant enzymes.

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 822
    Illegal PstI site found at 828
    Illegal PstI site found at 981
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 822
    Illegal NheI site found at 1128
    Illegal NheI site found at 1151
    Illegal PstI site found at 828
    Illegal PstI site found at 981
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 822
    Illegal BamHI site found at 840
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 822
    Illegal PstI site found at 828
    Illegal PstI site found at 981
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 822
    Illegal PstI site found at 828
    Illegal PstI site found at 981
    Illegal AgeI site found at 117
    Illegal AgeI site found at 240
  • 1000
    COMPATIBLE WITH RFC[1000]


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