Part:BBa_K1716001

GP35 recombinase optimized for expression in B. subtilis

Recombination-mediated genetic engineering (recombineering) utlises homologous recombination to facilitate genetic modifications at any desired target by flanking the mutated sequence with homologous regions. Multiplex Automated Genome Engineering (MAGE) is a method for rapid and efficient targeted programming and evolution of cells through cyclical recombineering using multiple single-stranded DNA oligonucleotides (oligos). The MAGE protocol utilises the λ Red recombination system in combination with an (temporary) inactivation of the mismatch repair system and consists of 7 steps that can be done with standard laboratory equipment (Wang, 2009). As MAGE utilises oligos, only the Beta protein of the λ Red system is required. This BioBrick encodes the coding sequence for a recombinase homologous to lambda beta. It originates from B. subtilis phage SPP1. It is based on Sun et. al. findings that GP35 had higher recombining frequencies than lambda beta in B. subtilis, when electroplated with a long (>1,000 nucleotide) ssDNA generated by PCR. We tested it with oligos (90-mers) and saw lower recombineering frequencies than lambda beta in B. subtilis. [http://2015.igem.org/Team:DTU-Denmark/Project/MAGE Please see our project page].

Conclusion: We suggest that you use Lambda Beta (BBa_K1716000) for recombineering in E. coli and B. subtilis.

Results

	Total number of CFUs	Number of colonies on antibiotic plates	Recombineering frequency
ΔmutS::beta-neoR	52	7	0.13
ΔmutS::GP35-neoR	100	1	0.01

Table 1. shows the data for the MAGE comparing efficiencies between GP35 and Lambda Beta.

Discussion and Conclusion

Recombineering efficiencies were higher, when Lambda Beta was used as recombinase than GP35. Previously, the opposite was concluded by Sun et al. that GP35. They used long ssDNA oligo of >1,000 nucleotides, which were generated by PCR. We used oligos with an length of around 90 nucleotides. This shows that the length of the optimal oligo depends on recombinase used. For our system, we wish to use Chip-based oligos. For that purpose long oligos are not suitable making lambda beta more applicable for our system. We do notice though that Sun et al. reach very high recombineering efficiencies which may be attributed to the long ssDNA used. B. subtilis has longer Okazaki fragments than E. coli and S. cerevisae.

References

Wang, H. H., Isaacs, F. J., Carr, P. A., Sun, Z. Z., Xu, G., Forest, C. R., & Church, G. M. (2009). Programming cells by multiplex genome engineering and accelerated evolution. Nature, 460(7257), 894–898. doi:10.1038/nature08187 Photo credit go MAGE cycle: Michael Schantz Klausen Sun, Z., Deng, A., Hu, T., Wu, J., Sun, Q., Bai, H., … Wen, T. (2015). A high-efficiency recombineering system with PCR-based ssDNA in Bacillus subtilis mediated by the native phage recombinase GP35. Applied Microbiology and Biotechnology, 99(12), 5151–5162. doi:10.1007/s00253-015-6485-5

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

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Categories

Parameters

None