Part:BBa_K2243013

Bxb1 gp35-gp47 fusion protein

catalyze recombination between attL and attR site to reset the DNA sequence.

Usage

In the last few years, researchers also investigate into integrases work with the help of their Recombination Directionality Factors (RDFs). Bxb1 gp47 is an Bxb1-encoded RDF, the product of gene 47. With gp47, Bxb1 gp35 allows the sequence to be flipped, excised, or inserted between attL and attR sites, which makes it useful for gene editing. We design a fusion protein for the integrase and its RDF. In our project, Bxb1 gp35-gp47 fusion protein can flip the sequence flanked by attL and attR site to reset the sequence flipped by Bxb1 gp35.

Biology

Integrase Bxb1 gp35 comes from Mycobacteriophage, which allows the phage to insert its DNA into the host’s genome at host’s attP site using viral attB site. With RDF Bxb1 gp47, the recombined attL and attR can be recognized and undergo recombination, which enables the excision of phage genome. We got the sequence by de novo synthesis.

excisionases are able to recognize and bind attL and attR sequences. With the help of excisionases, the state transitions become reversible. Recombination Directionality Factors (RDF) are a kind of protein that achieves reverse flipping to recover the sequence flipped by recombinases. By co-expression of the integrase with the corresponding RDF, or expression of the integrase-RDF fusion protein, recombination between the attL and attR sites can be induced. Thus, the flip-flop can restore to the previous state.

Fig 1. Schematic drawing of RDF mechanism.

Experience

Excisionase characterization To make sure that the sequence reversing process is efficient and complete, we use two strategies: one is constructing the integrase and its corresponding RDF into polycistronic structure, and the other one is to build the fusion protein of integrases and RDFs. We have been trying to improve the recombination efficiency by replacing the expression vector with different replication origins or inducible promoters and changing the RBS sequence before the coding sequence.

Fig 2. Two strategies we used. Upper: The polycistronic structure composed the integrase and its corresponding RDF. Lower: The fusion protein structure of integrase with its corresponding RDF.

High flipping efficiency and leakage were seen for Bxb1 gp35-gp47 fusion proteins. Still, the fusion proteins are able to flip the sequence between attL and attR site (indicated by observed GFP subset), although tuning work remains to be done to construct a controllable, functional bio-flip-flop device.

Fig 3.Flipping efficiency for pTac integrase-RDF fusion protein of phiC31 and Bxb1. Proportion of GFP subset indicates flipping efficiency.

Design Notes

We assembled the Bxb1 gp35 and gp47 with a linker adapted from a recent research[1] by Gibson Assembly.

Source

Mycobacterium Phage Bxb1

References

[1]Olorunniji, F. J., McPherson, A. L., Rosser, S. J., Smith, M. C., Colloms, S. D., & Stark, W. M. (2017). Control of serine integrase recombination directionality by fusion with the directionality factor. Nucleic acids research, 45(14), 8635-8645.

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 BglII site found at 1507
  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
  COMPATIBLE WITH RFC[1000]