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

GluR0

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 BamH1 sites and medium strength RBS J61117 were also included in the design.

InFusion

InFusion primers were designed for both OsmY-RBS and KDP 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 product. The cells were then grown on kanamycin plates at 37°C and single colonies were then PCRed using standard BioBrick protocol.