Difference between revisions of "Part:BBa K1716001"

 
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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 &#955; 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 &#955; 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. subtitles (please see Results).
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<html><div style="float:right;"><img src="https://static.igem.org/mediawiki/2015/9/91/DTU-Denmark_Magecycle.png" style="width:400px" /></div></html>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 &#955; 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 &#955; 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].
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'''Conclusion: '''We suggest that you use Lambda Beta ([https://parts.igem.org/Part:BBa_K1716000 BBa_K1716000]) for recombineering in ''E. coli'' and ''B. subtilis''.
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===References===
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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
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Photo credit go MAGE cycle: Michael Schantz Klausen
  
 
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Revision as of 01:35, 19 September 2015

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.

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

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]