Team:UCL/Lab AzoReducateCloning Portal

BioBrick and Primer Design

Two Azoreductase enzymes will be cloned from Bacilus subtilis (strain 168) and Pseudomonas Aeruginosa (PA01) using DNA PCR amplification.

Suitable forward and reverse primers were designed for each enzymes using the following strategy.

The protein coding sequences for each enzyme were obtained from ?.

B.Subtilis 168

IGM-Bsubt-F 5’-3’

           ATG TCT ACA GTT TTA TTT G

ATG TCT ACA GTT TTA TTT GTA AAA TCA AGC GAC CGT ACA GCT GAA GAA GGC GTT TCA ACT AAA CTT TAC GAA GCT TTC TTA GCT TAC AGA TGT CAA AAT AAA CAT TTT AGT TCG CTG GCA TGT CGA CTT CTT CCG CAA AGT TGA TTT GAA ATG CTT CGA AAG AAT CGA

GCT TAT AAA GAA AAC AAC CCT AAT GAT GAA GTG GTT GAA TTA GAT CTT CAT AAG GAA AAC CTT CCT TAC CTT GGC AGA GAT ATG ATT AAC GGA CGA ATA TTT CTT TTG TTG GGA TTA CTA CTT CAC CAA CTT AAT CTA GAA GTA TTC CTT TTG GAA GGA ATG GAA CCG TCT CTA TAC TAA TTG CCT

ACA TTT AAA GCA GGT CAA GGA ATG GAA ATG ACA GAA GAT GAG AAA AAA CAA GCA GCA ATT GCT GAC AAA TAT CTG AAC CAG TTT GTA AAA GCT TGT AAA TTT CGT CCA GTT CCT TAC CTT TAC TGT CTT CTA CTC TTT TTT GTT CGT CGT TAA CGA CTG TTT ATA GAC TTG GTC AAA CAT TTT CGA

GAC AAA GTT GTT TTC GCA TTC CCG CTT TGG AAC TTC ACA GTG CCA GCA GTG CTT CAT ACT TAT GTT GAT TAT CTG TCT CGC GCA GGC GTT ACA CTG TTT CAA CAA AAG CGT AAG GGC GAA ACC TTG AAG TGT CAC GGT CGT CAC GAA GTA TGA ATA CAA CTA ATA GAC AGA GCG CGT CCG CAA TGT

TTC AAA TAC ACA CAA GAA GGA CCA GTC GGT TTA ATG GGC GGC AAA AAA GTT GCG CTT CTT AAC GCT CGC GGC GGT GTC TAC TCA GAA GGA CCA AAG TTT ATG TGT GTT CTT CCT GGT CAG CCA AAT TAC CCG CCG TTT TTT CAA CGC GAA GAA TTG CGA GCG CCG CCA CAG ATG AGT CTT CCT GGT

ATG GCT GCA CTT GAA ATG TCA TTA AAC TTC ATG AAA ACA GTT CTT GGT TTC TGG GGT GTT CAA GAC TTG CAC ACA GTT GTC ATC GAA GGA CAT TAC CGA CGT GAA CTT TAC AGT AAT TTG AAG TAC TTT TGT CAA GAA CCA AAG ACC CCA CAA GTT CTG AAC GTG TGT CAA CAG TAG CTT CCT GTA

AAC GCA GCA CCT GAT CAA GCG CAA GAA ATC GTT GAA AAA GGT TTA CAA GAA GCA AAA GAT CTT GCT GCA AAA TTC TAA TTG CGT CGT GGA CTA GTT CGC GTT CTT TAG CAA CTT TTT CCA AAT GTT CTT CGT TTT CTA GAA CGA CGT TTT AAG ATT A GAA CGA CGT TTT AAG ATT

IGM-Bsubt-R, 3’-5’

IGM-Bsubt-F : 5’-ATG TCT ACA GTT TTA TTT G-3’ IGM-Bsubt-F + Prefix: 5’-GTT TCT TCG AAT TCG CGG CCG CTT CTA GAG ATG TCT ACA GTT TTA TTT G-3’

IGM-Bsubt-R:5’-TTA GAA TTT TGC AGC AAG A-3’ IGM-Bsubt-R + Suffix:5’-GTT TCT TCC TGC AGC GGC CGC TAC TAG TAT TAG AAT TTT GCA GCA AGA-3’