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 and the RBS sequences upon the integrase, we measure the recombination efficiency under different conditions.

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