Composite

Part:BBa_K5276033

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


Selection plasmid

A118-rha_Promoter-PhiX174E-GFP

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Fig1. Expression plasmid of traditional mutagenesis
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Fig2. Selection plasmid of traditional mutagenesis
We designed expression plasmid(Fig.1, BBa_K5276034) and selection plasmid(Fig.2, BBa_K5276033). The expression plasmid induces recombinant enzyme expression with IPTG, while the selection plasmid uses a phage lysis gene φX174 E (Din et al., 2016). We incorporated mixed NNK mutation sequence primers into the PCR system to generate a library of various mutants. The mutation and screening plasmids were then introduced into E. coli, and the screening plan was designed as follows: Initial screening: The growth of E. coli on the medium indicates whether the φX174 E gene has been successfully inverted. If the recombinant enzyme mutant retains biological function, the φX174 E gene will not express, allowing for colony growth on the plate. Conversely, if the mutant loses biological function, the φX174 E gene will express, lysing E. coli, resulting in no observable colonies. Secondary screening: The brightness of GFP was measured using a microplate reader. The higher the recombinant enzyme's recombination efficiency, the greater the proportion of RNA transcribed from the inverted GFP gene to the total RNA, visually represented by increased unit brightness of GFP (assuming transcription levels before and after gene inversion remain constant).

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 87
    Illegal EcoRI site found at 843
    Illegal EcoRI site found at 2380
    Illegal EcoRI site found at 2695
    Illegal XbaI site found at 83
    Illegal XbaI site found at 2699
    Illegal PstI site found at 849
    Illegal PstI site found at 1002
    Illegal PstI site found at 2221
    Illegal PstI site found at 2374
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 87
    Illegal EcoRI site found at 843
    Illegal EcoRI site found at 2380
    Illegal EcoRI site found at 2695
    Illegal PstI site found at 849
    Illegal PstI site found at 1002
    Illegal PstI site found at 2221
    Illegal PstI site found at 2374
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 87
    Illegal EcoRI site found at 843
    Illegal EcoRI site found at 2380
    Illegal EcoRI site found at 2695
    Illegal BamHI site found at 861
    Illegal BamHI site found at 2362
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 87
    Illegal EcoRI site found at 843
    Illegal EcoRI site found at 2380
    Illegal EcoRI site found at 2695
    Illegal XbaI site found at 83
    Illegal XbaI site found at 2699
    Illegal PstI site found at 849
    Illegal PstI site found at 1002
    Illegal PstI site found at 2221
    Illegal PstI site found at 2374
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 87
    Illegal EcoRI site found at 843
    Illegal EcoRI site found at 2380
    Illegal EcoRI site found at 2695
    Illegal XbaI site found at 83
    Illegal XbaI site found at 2699
    Illegal PstI site found at 849
    Illegal PstI site found at 1002
    Illegal PstI site found at 2221
    Illegal PstI site found at 2374
    Illegal AgeI site found at 138
    Illegal AgeI site found at 261
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 1376
    Illegal BsaI.rc site found at 3048


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