Part:BBa_K2243005
C12_Bxb1 gp35
Through the random mutation from iGEM registry RBS, one well-behaved RBS C12 is constructed upstream Bxb1 gp35 coding sequence. The Bxb1 gp35 integrase performed high efficiency of DNA inversion using this construct.
Usage:
F8_Bxb1 gp35 is one of the best expression system. We added RBS C12 upstream the Bxb1 gp35 coding sequence (BBa_K22430012).
Biology:
The Bxb1 coding sequence is the part BBa_K22430012.
Design note:
We construct this structure by Gibson Assembly. The C12 RBS is generate by random mutation.
Characterize:
Since the viability of a bio-flip-flop relies on the performance of two integrases and their corresponding excisionases. To select integrases for the bio-flip-flop, we constructed expression vectors for different recombinases and tested their performance individually.
To make sure that Bxb1 have an optimal performance. We used the standard testing system, consisting of the integrase expression plasmid and the recombination reporter plasmid (BBa_K2243006). By changing the vector with different replication origins(a p15A origin with a pTac promoter, and a ColE1 origin with a pBAD promoter) and the RBS sequences upon the integrase, we measure the recombination efficiency under different conditions. We picked out our optimal RBS for ColE1 origin ,pBAD promoter backbone, which displayed low leakage and high efficiency.
Fig 1. The standard genetic structure used to characterize the recombinases.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 212
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 486
Illegal XhoI site found at 573 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 1125
Illegal NgoMIV site found at 1212
Illegal AgeI site found at 262 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 1320
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