Part:BBa_K1716000

Lambda Beta 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 an optimised coding sequence for lambda phage derived beta recombinase. When expressed in Bacillus subtilis, oligo recombineering efficiencies were increased. [http://2015.igem.org/Team:DTU-Denmark/Project/MAGE Please see our project page].

How to use

1. Express lambda beta in B. subtilis (or another organisms) by insertion into must (mismatch repair) gene. Read more about how we did that using standard B. subtilis BioBricks available from the registry under our project [http://2015.igem.org/Team:DTU-Denmark/Project/MAGE here]. 2. Make electro/MAGE-competent B. subtilis cells. Find our protocol [http://dtuwiki-drewt.rhcloud.com/files/Electro_competent_protocol.pdf here]. 3. Design your 90-mer oligo. If you want to design oligos targeting NRPS use our NRPS Oligo Designer (NOD). Otherwise we suggest using [http://modest.biosustain.dtu.dk MODEST]. You can also read more about how to to multiplex engineering to design oligo chips [http://2015.igem.org/Team:DTU-Denmark/Project/Background here] for low-cost library generation. 4. Electroporate the cells. Read more about our optimisation of MAGE in Bacillus subtilis and cos-MAGE for higher efficiencies [http://dtuwiki-drewt.rhcloud.com/Team:DTU-Denmark/Project/MAGE here].

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 Bonde, M. T., Klausen, M. S., Anderson, M. V., Wallin, A. I. N., Wang, H. H., & Sommer, M. O. A. (2014). MODEST: a web-based design tool for oligonucleotide-mediated genome engineering and recombineering. Nucleic Acids Research, 42(W1), W408–W415. doi:10.1093/nar/gku428 Photo credit go MAGE cycle: Michael Schantz Klausen

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
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 245