Part:BBa_K2243012

Bxb1 gp35

Bxb1 gp35 integrase comes from Mycobacterium Phage Bxb1 and can bind to specific attB/P sites to catalyze DNA recombination. It helps the phage to integrate its genome into bacterial genome naturally. By constructing the attB/P sites in different directions, Bxb1 gp35 can catalyze the recombination of DNA between their sites, leading to inversion when attB/P are in opposite directions and excision when attB/P are in the same directions. Bxb1 gp35 is widely used to construct combinational logic gate and performs well in changing DNA sequence

Usages

Many researchers have paid attention to recombinases because of their ability of changing genetic circuits. Integrase Bxb1-gp35 is one of the recombinases with outstanding performance. In existence of recombinase Bxb1 gp35, different orientation of attB and attP allows the sequence to be flipped, excised, or inserted between recognition sites, which makes it useful for gene editing. In our project, we selected Bxb1-gp35 to flip the sequence flanked by attB and attP site for Bio-Flip-Flop construction.

Biology

Integrase Bxb1-gp35 comes from Mycobacteriophage, which allows the phage to insert its DNA into the host’s genome. We got the sequence by commercial synthesis.

Design Notes

We got the coding sequence by de novo synthesis and introduced three point mutations to remove the BsmB1 restriction enzyme cutting sites.

Characterization

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.

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 1. Gating of the RFP and GFP subsets and change of fluorescence after induction. Left: no induc-er. Right: 10 mM arabinose for 15h.

Fig 2. Bxb1 gp35 recombination efficiency with a variety of calculated RBS and RBS sequences from iGEM (B0030~B0035). T.I.R = Translation Initiation Rate

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.

< img src="https://static.igem.org/mediawiki/2017/9/93/Peking_flipflop_fig17.svg" height="500" width="500"/>

Fig 3. Bxb1 gp35 transfer curve

Source

Mycobacterium Phage Bxb1

References

Roquet, N., Soleimany, A.P., Ferris, A.C., Aaronson, S. & Lu, T.K. Synthetic recombinase-based state machines in living cells. Science 353, aad8559 (2016).

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 192
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 466
  Illegal XhoI site found at 553
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 1105
  Illegal NgoMIV site found at 1192
  Illegal AgeI site found at 242
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 1300