Composite

Part:BBa_K5276032

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


T7_Promoter-lacO-tetR-A118-lacI-Gp44

Recombination direction factors (RDF) can control the activity of integrases to promote specific recombination reaction directions. Research indicates that the Gp44 protein(Mandali et al.,2017), as a recombination direction factor, can enhance the excision or inversion reaction of A118 between attL and attR sites. We designed a bistable switch system focused on regulating the expression of the A118 integrase and the Gp44 protein. The "Turn over" switch is controlled by the lactose operon, while the "Turn back" switch is regulated by the tetR protein.

img
Fig.1 Target plasmid of continuous directed evolution system


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 2593
    Illegal EcoRI site found at 3703
    Illegal XbaI site found at 1492
    Illegal XbaI site found at 2159
    Illegal SpeI site found at 1127
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 2593
    Illegal EcoRI site found at 3703
    Illegal NheI site found at 3751
    Illegal SpeI site found at 1127
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 2593
    Illegal EcoRI site found at 3703
    Illegal BglII site found at 2225
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 2593
    Illegal EcoRI site found at 3703
    Illegal XbaI site found at 1492
    Illegal XbaI site found at 2159
    Illegal SpeI site found at 1127
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 2593
    Illegal EcoRI site found at 3703
    Illegal XbaI site found at 1492
    Illegal XbaI site found at 2159
    Illegal SpeI site found at 1127
    Illegal NgoMIV site found at 2257
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI site found at 730


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