Part:BBa_K2243002
D4_Bxb1 gp35
Through the random mutation from iGEM registry RBS, one well-behaved RBS D4 is constructed upstream Bxb1 gp35 coding sequence. The Bxb1 gp35 integrase performed high efficiency of DNA inversion using this construct.
Usage:
D4_Bxb1 gp35 is one of the best expression system. We added RBS D4 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 D4 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.
Flow cytometry were used to evaluate the recombination efficiency, the expression vector and reporter of a recombinase were used to co-transform E. coli Top10 and samples were prepared for flow cytometry reading. Single colonies were picked and used to inoculate 1ml of LB media with antibiotics in a V-bottom 96-well plate. The cultures were grown at 37°C and 1000 RPM for 12h. Subsequently, an aliquot comprising 2 μL of the culture was transferred into 1ml of M9 glucose media with antibiotics and inducer (1mM IPTG or 10mM arabinose for RBS tuning, gradient concentration for transfer curve) in a V-bottom 96-well plate. The cultures were grown at 37°C and 1000 RPM for 15h. An aliquot comprising 2μL of the cul-ture was transferred into 198 μL of phosphate buffered saline (PBS) containing 2 mg/mL kanamycin in a 96-well plate. This mixture was incubated for one hour at room temperature before testing. Two lasers were used to excite GFP and RFP simultaneously. Single-cell fluorescence distribution at both emission wavelengths was recorded.
The counted cells were gated to eliminate the population which showed no fluorescence. The remaining cells were divided into two subsets by a diagonal: RFP sub-set and GFP subset. The recombination efficiency was estimated from the proportion of the RFP subset in the total fluorescent population.
Fig 2. Gating of the RFP and GFP subsets and change of fluorescence after induction. Left: no induc-er. Right: 10 mM arabinose for 15h.
Transfer curves
We evaluated the performance of Bxb1 gp35 on ColE1 expression vectors under a series of inducer concentration gradients. The results enabled us to determine the appropriate inducer concentration. And the curve also showed the performance of Bxb1 gp35 under the control of D4 RBS
Fig 3. Bxb1 gp35 transfer curve with selected RBS（including D4 RBS), ColE1 replication origin, pBAD promoter
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 192
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 466
Illegal XhoI site found at 553 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 1105
Illegal NgoMIV site found at 1192
Illegal AgeI site found at 242 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 1300
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