| Types | DnaRegion
|
| Roles | CDS
Coding
|
| Sequences | BBa_K1716001_sequence (Version 1)
|
Description
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. subtitles (please see Results).
Notes
CDS was obtained from NC_004166.2. The stop codon (TAG) was replaced by two TAA stop codons.
Source
Bacillus subtilis phage SPP1
