IGEM:Cambridge/2008/Notebook/Voltage/GluR0 Manipulation

=GluR0 - K090001= GluR0 has been well characterised in Synechocystis and the amino acid sequence fully documented. The sequence was reverse translated using Gene Designer software from DNA2.0. It was specified to have both E.coli and B.subtilis compatible codon bias, and also all restriction sites were designed out of it. A medium strength promoter J23116 flanked by BamHI sites and medium strength RBS J61117 were also included in the design. The BamHI sites allow the promoter to be changed if necessary. This whole construct was then synthesised de novo by DNA2.0 as K090001

=InFusion= The plasmid received from DNA2.0 was not biobrick compatible, and did not contain a terminator. To rectify this the whole construct was cloned into part B1002, behind the terminator. This was done by infusion. InFusion primers were designed for both GluR0 construct as well as the terminator containing plasmid B1002. These primers were designed to give the backbone with the terminator still attached. 10μL of a 1:1 mass ratio mixture was put into the InFusion reaction and TOP10 cells were then transformed with 5μL of the diluted product. The cells were then grown on kanamycin plates at 37°C and single colonies were then PCRed using standard BioBrick protocol. Successful colonies were grown up and plasmid harvested

Primers
GluR0 InFusion Forward: CGG CCG CTT CTA GAG GGA TCC TTG ACA GCT AGC TCA G

GluR0 InFusion Reverse: GCG GGG TTT TTT GCG TTA GGA CGG GGA TTC GCC AA

B1002 Plasmid InFusion Forward: CGC AAA AAA CCC CGC TTC GGC GGG G

B1002 Plasmid InFusion Reverse: CTC TAG AAG CGG CCG CGA ATT CCA GAA ATC ATC CTT AGC G