Source:
Generated By: https://synbiohub.org/public/igem/igem2sbol/1
Created by: Pernille Neve Myers
Date created: 2015-09-15 11:00:00
Date modified: 2015-09-17 02:32:10
NahR Biosensor for detection of acetylsalicylic acid/aspirin with blue chromoprotein reporter
| Types | DnaRegion |
| Roles | Composite engineered_region |
| Sequences | BBa_K1716002_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). 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.Notes
The construct was originally assembled into pDG268neo using Gibson assembly. pDG268neo contains amyE sites for integration into Bacillus subtilis. The expression cassette with promoter, RBS, CDS, and terminator was amplified by PCR. The PCR product was inserted into pSB1C3 using Gibson assembly. The plasmid was transformed into E. coli and subsequently purified and sequenced. The plasmid was linearised before transformation into B. subtilis.Source
Bacillus subtilis phage SSP1 gene product 35Homoloogus regions upstream and downstream from must (genomic DNA template)
Promoter:
Terminator:
Neomycin resistance marker from Bacillus subtilis plasmid pDG268neo
| Access | Instance | Definition |
| public public | BBa_J61051 BBa_K592009 | BBa_J61051 amilCP |
| Sequence Annotation | Location | Component / Role(s) |
| BBa_J61051 BBa_K592009 | 1,1268 1275,1943 | BBa_J61051 amilCP |