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Team:UCL/Lab AzoReducateCloning Portal
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<html><h2>Overview</h2></html> | <html><h2>Overview</h2></html> | ||
| - | E. coli will be transformed with overexpression plasmids containing three genes expressing azoreductase enzymes in order to create an azo-dye decolourising and detoxifying organism with potential applications on an industrial scale. Further transformation of E. coli with laccase and lignin peroxidase to oxidize the aromatic products of azo-dye reduction by azoreductase will reduce the toxicity levels down to the point | + | E. coli will be transformed with overexpression plasmids containing three genes expressing azoreductase enzymes in order to create an azo-dye decolourising and detoxifying organism with potential applications on an industrial scale. Further transformation of E. coli with laccase and lignin peroxidase to oxidize the aromatic products of azo-dye reduction by azoreductase will reduce the toxicity levels down to the point below the initial toxicity of the intact dye. |
<html><h2>BioBrick and Primer Design</h2></html> | <html><h2>BioBrick and Primer Design</h2></html> | ||
Revision as of 13:26, 29 May 2014
Overview
E. coli will be transformed with overexpression plasmids containing three genes expressing azoreductase enzymes in order to create an azo-dye decolourising and detoxifying organism with potential applications on an industrial scale. Further transformation of E. coli with laccase and lignin peroxidase to oxidize the aromatic products of azo-dye reduction by azoreductase will reduce the toxicity levels down to the point below the initial toxicity of the intact dye.
BioBrick and Primer Design
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 AzoR
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 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 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 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 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 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 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
P. aeruginosa PA01 AzoR
ATG AGT AGA ATT CTT GCA GTG CAT GCC AGT CCG CGA GGC GAG CGC TCG CAG TCC CGG CGT CTC GCC GAG GTT
TTC CTG GCG GCC TAT CGC GAA GCC CAT CCG CAG GCC CGC GTG GCT CGC CGC GAA GTC GGC CGG GTA CCG CTT
CCG GCG GTC ACC GAG GCC TTC GTC GCC GCC GCC TTC CAT CCC CAG CCG GAA CAG CGT TCG CTG GCG ATG CAG
GCC GAC CTG GCG CTG AGC GAC CAA CTG GTC GGC GAA CTG TTC GAC AGC GAC CTG CTG GTG ATC TCC ACG CCG
ATG TAC AAC TTC AGC GTG CCC AGC GGC CTG AAG GCC TGG ATC GAC CAG ATC GTG CGC CTC GGG GTG ACC TTC
GAT TTC GTC CTC GAC AAT GGC GTC GCC CAG TAC CGG CCG CTG CTG CGT GGC AAG CGT GCG CTG ATC GTC ACC
AGT CGC GGT GGC CAT GGC TTC GGC CCG GGC GGC GAG AAC CAG GCG ATG AAC CAC GCC GAT CCC TGG TTG CGC
ACC GCG CTG GGT TTC ATC GGC ATC GAC GAG GTC ACG GTG GTC GCG GCG GAA GGC GAG GAA TCC GGC GGC AGG
TCC TTC GAG GAC TCC TGC GAC GAG GCG GAA CAG CGC CTG CTG GCG CTG GCG CGG TCG GCC TGA
The forward primer for the B. subtilis AzoR gene consists of the first 19 nucleotide of the protein coding sequence.
iGM SUBF4 : 5’-ATGTCTACAGTTTTATTTG-3’
The reverse primer for the B. subtilis AzoR gene consists of the last 19 nucleotide of the protein coding sequence.
iGM REVSUB:5’-TTAGAATTTTGCAGCAAGA-3’
The forward primer for the P. aeruginosa AzoR gene consists of the first 17 nucleotide of the protein coding sequence.
iGM AER4 : 5’ATGAGTAGAATTCTTGC-3’
The reverse primer for the P. aeruginosa AzoR gene consists of the last 18 nucleotide of the protein coding sequence.
iGM REV AER :5’-TCAGGCCGACCGCGCCAG-3’
The BioBrick prefix for protein coding sequences and suffix sequences used in Standard Assembly and belonging to BioBrick RFC 10 were appended to the forward and reverse primers respectively.
BioBrick CDS Prefix
5' GAATTCGCGGCCGCTTCTAG '3
BioBrick Suffix
5' TACTAGTAGCGGCCGCTGCAG '3
The full primer sequences for B. subtilis AzoR and P. aeruginosa AzoR are therefore
iGM PRESUB4 : 5’-GAATTCGCGGCCGCTTCTAGATGTCTACAGTTTTATTTG-3’ iGM SUF REV SUB:5’-TACTAGTAGCGGCCGCTGCAGTTAGAATTTTGCAGCAAGA-3’
iGM PRE AER4: 5’-GAATTCGCGGCCGCTTCTAGATGAGTAGAATTCTTGC-3’ iGM SUF REV AER: 5’-TACTAGTAGCGGCCGCTGCAGTCAGGCCGACCGCGCCAG-3’
