Designed by: Yuming Luo Group: iGEM24_UCAS-China (2024-10-01)
Expression plasmid
Fig1. Expression plasmid of traditional mutagenesis
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 XbaI site found at 1506 Illegal SpeI site found at 1141
12
INCOMPATIBLE WITH RFC[12]
Illegal SpeI site found at 1141
21
INCOMPATIBLE WITH RFC[21]
Illegal BglII site found at 1572 Illegal BamHI site found at 107
23
INCOMPATIBLE WITH RFC[23]
Illegal XbaI site found at 1506 Illegal SpeI site found at 1141
25
INCOMPATIBLE WITH RFC[25]
Illegal XbaI site found at 1506 Illegal SpeI site found at 1141 Illegal NgoMIV site found at 1604
