Composite

Part:BBa_K1716002

Designed by: Pernille Neve Myers   Group: iGEM15_DTU-Denmark   (2015-09-16)
Revision as of 20:40, 16 September 2015 by Registry (Talk | contribs)

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NahR Biosensor for detection of acetylsalicylic acid/aspirin with blue chromoprotein reporter

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). This BioBrick contains a construct for expression of GP35 in Bacillus subtilis using neomycin as resistance marker. The construct is designed to be delete must (mismatch repairing protein) in Bacillus subtilis. Deletion of mismatch repair systems improve oligo recombineering.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 786
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 77
    Illegal NgoMIV site found at 618
  • 1000
    COMPATIBLE WITH RFC[1000]


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