Part:BBa_K5005002
The BFP protein is located downstream of the T7 promoter.
BFP is located downstream of the T7 promoter (derived from site-directed mutagenesis of EGFP), expressing the target gene BFP (blue fluorescence), which could potentially be evolved back into EGFP by Editor(BBa_K5005003).
The directed evolution system TRACE was first reported by the Chen Fei research group at MIT. The key plasmid, pEditor (BBa_K5005003), used in our experiments, was provided by Professor Yu Yang from the Institute of Biophysics, Chinese Academy of Sciences. Firstly, we verified the feasibility of our directed evolution system (TRACE) using a customized EGFP plasmid. In principle, we mutated the 199th base of the EGFP gene (BBa_K5005001) from T-A to C-G, converting tyrosine to histidine and changing the fluorescence color from green to blue (BBa_K5005000). Therefore, if the TRACE system is feasible, we can mutate the BFP gene obtained from site-directed mutagenesis back to the EGFP gene, achieving a change in cell fluorescence from blue to green. In specific steps, (1) we first constructed an empty plasmid, pTarget (BBa_K5005014): the target plasmid contains a T7 promoter downstream of the CMV promoter from the pCDH vector; (2) Then, we mutated the reporter gene from EGFP (BBa_K5005001) to BFP (BBa_K5005000); (3) Through Biobricks provided by iGEM and homologous recombination, we got pTarget-BFP (BBa_K5005002). Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Plasmid lacks a prefix.
Plasmid lacks a suffix.
Illegal EcoRI site found at 8002
Illegal NheI site found at 315
Illegal SpeI site found at 2
Illegal PstI site found at 16
Illegal NotI site found at 9
Illegal NotI site found at 1053
Illegal NotI site found at 8008 - 21INCOMPATIBLE WITH RFC[21]Plasmid lacks a prefix.
Plasmid lacks a suffix.
Illegal EcoRI site found at 8002
Illegal BglII site found at 2719
Illegal BglII site found at 2785
Illegal BglII site found at 2826
Illegal BglII site found at 6250
Illegal BamHI site found at 1047 - 23INCOMPATIBLE WITH RFC[23]Illegal prefix found at 8002
Illegal suffix found at 2 - 25INCOMPATIBLE WITH RFC[25]Illegal prefix found at 8002
Plasmid lacks a suffix.
Illegal XbaI site found at 8017
Illegal SpeI site found at 2
Illegal PstI site found at 16
Illegal NgoMIV site found at 1571
Illegal NgoMIV site found at 2604 - 1000INCOMPATIBLE WITH RFC[1000]Plasmid lacks a prefix.
Plasmid lacks a suffix.
Illegal BsaI site found at 2808
Illegal BsaI site found at 6232
Illegal BsaI.rc site found at 1524
Illegal BsaI.rc site found at 4673
Illegal SapI site found at 3590
Illegal SapI.rc site found at 6881
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