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

=OsmY RBS=

OsmY was PCRed out of the biobricks which contained it - BBa_J45996 (A), BBa_J45995 (B) and BBa_J45250 (C). The primers were designed to have the BioBrick prefix and suffix, but in addition to have RBSs of 3 different strengths (B0030, B0031 and B0032) included in the suffix. They were then cut with EcoRI and PstI and ligated into pSB4C5, which had been cut with the same enzymes. This was done at a 16:1 ratio of masses as OsmY is about 250bp and pSB4C5 is 4kbp. TOP10 cells were then transformed with the ligation product. The cells were then grown on chloramphenicol plates at 37°C and single colonies were then PCRed using standard BioBrick protocol.

Primers
OsmY Forward:       CTA TGA ATT CAT ATT CTA GAG CTG GCA CAG GAA CGT TAT CC     OsmY-B0030 Reverse:  CGC GCT GCA GCT CTA CTA GTT TTC TCC TCT TTA ATT TGT TAA ATA TAG A   OsmY-B0031 Reverse:  CTC TCT GCA GCT CTA CTA GTG GTT TCC TGT GTG ATT GTT AAA TAT AGA T    OsmY-B0032 Reverse:  CTC TCT GCA GCT CTA CTA GTC TTT CCT GTG TGA TTG TTA AAT ATA GAT CA

=Kdp= Kdp was PCRed out of the chromosome of MG1655. The primers were designed to have the BioBrick prefix and suffix. It was then cut with EcoRI and PstI and ligated into pSB4C5, which had been cut with the same enzyme. As Kdp is 4.4kb and the vector is 4kb approximatley the 1.1:1 ratio of mass was used (Kdp:pSB4C5). TOP10 cells were then transformed with the ligation product. The cells were then plated onto 35μg/mL chloramphenicol and grown at 37°C.

Primers
KDP Forward: ATA TGA ATT CAT ATT CTA GAT GAG TGC AGG CGT GAT AAC CGG CGT ATT KDP Reverse: CTC TCT GCA GCT CTA CTA GTT TAT TCA TCA AGT TTA TCC AGC GCC AGA T

=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 16:6:1 (KDP:vector:Osmy) 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.

Primers
OsmY InFusion Forward:       CGG CCG CTT CTA GAG CTG GCA CAG GAA OsmY-B0030 InFusion Reverse: TAT CAC GCC TGC ACT CGC GCG CGT TTC TCC TCT TTA ATT TGT TAA A

KDP InFusion Forward:        AGT GCA GGC GTG ATA ACC GGC GTA TTG CTG GTG

KDP InFusion Reverse:        GCG GGG TTT TTT GCG TTA TTC ATC AAG TTT ATC CAG CGC CAG A

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