Team:UCL/Lab AzoReducateCloning Portal

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BioBrick and Primer Design
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<html><h2>Overview</h2></html>
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Two Azoreductase enzymes will be cloned from Bacilus subtilis (strain 168) and Pseudomonas Aeruginosa (PA01) using DNA PCR amplification.
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A BioBrick part containing the azoreductase (AzoR) gene will created 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.
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Suitable forward and reverse primers were designed for each enzymes using the following strategy.
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<html><h2>AzoR BioBrick Design</h2></html>
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The protein coding sequences for each enzyme were obtained from ?.
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Azoreductase genes will be cloned from two species, B. subtilis (strain 168) and P. Aeruginosa (PA01) using DNA PCR amplification.  Suitable forward and reverse primers were designed for each enzyme that facilitated the creation of two AzoR BioBrick parts.
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Protein coding sequences
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B.Subtilis 168
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<html>
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<h4>B. subtilis 168 AzoR</h4>
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<code>
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ATG TCT ACA GTT TTA TTT GTA AAA TCA AGC GAC CGT ACA GCT GAA GAA GGC GTT TCA ACT AAA CTT TAC GAA</br>
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GCT TTC TTA GCT GCT TAT AAA GAA AAC AAC CCT AAT GAT GAA GTG GTT GAA TTA GAT CTT CAT AAG GAA AAC</br>
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CTT CCT TAC CTT GGC AGA GAT ATG ATT AAC GGA ACA TTT AAA GCA GGT CAA GGA ATG GAA ATG ACA GAA GAT</br>
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GAG AAA AAA CAA GCA GCA ATT GCT GAC AAA TAT CTG AAC CAG TTT GTA AAA GCT GAC AAA GTT GTT TTC GCA</br>
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TTC CCG CTT TGG AAC TTC ACA GTG CCA GCA GTG CTT CAT ACT TAT GTT GAT TAT CTG TCT CGC GCA GGC GTT</br>
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ACA TTC AAA TAC ACA CAA GAA GGA CCA GTC GGT TTA ATG GGC GGC AAA AAA GTT GCG CTT CTT AAC GCT CGC</br>
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GGC GGT GTC TAC TCA GAA GGA CCA ATG GCT GCA CTT GAA ATG TCA TTA AAC TTC ATG AAA ACA GTT CTT GGT</br>
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TTC TGG GGT GTT CAA GAC TTG CAC ACA GTT GTC ATC GAA GGA CAT AAC GCA GCA CCT GAT CAA GCG CAA GAA</br>
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ATC GTT GAA AAA GGT TTA CAA GAA GCA AAA GAT CTT GCT GCA AAA TTC TAA
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</code></html>
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IGM-Bsubt-F 5’-3’
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<html>
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            ATG TCT ACA GTT TTA TTT G
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<h3>P. aeruginosa PA01 AzoR</h3>
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    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
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<code>
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    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
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ATG AGT AGA ATT CTT GCA GTG CAT GCC AGT CCG CGA GGC GAG CGC TCG CAG TCC CGG CGT CTC GCC GAG GTT</br>
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TTC CTG GCG GCC TAT CGC GAA GCC CAT CCG CAG GCC CGC GTG GCT CGC CGC GAA GTC GGC CGG GTA CCG CTT</br>
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CCG GCG GTC ACC GAG GCC TTC GTC GCC GCC GCC TTC CAT CCC CAG CCG GAA CAG CGT TCG CTG GCG ATG CAG</br>
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GCC GAC CTG GCG CTG AGC GAC CAA CTG GTC GGC GAA CTG TTC GAC AGC GAC CTG CTG GTG ATC TCC ACG CCG</br>
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ATG TAC AAC TTC AGC GTG CCC AGC GGC CTG AAG GCC TGG ATC GAC CAG ATC GTG CGC CTC GGG GTG ACC TTC</br>
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GAT TTC GTC CTC GAC AAT GGC GTC GCC CAG TAC CGG CCG CTG CTG CGT GGC AAG CGT GCG CTG ATC GTC ACC</br>
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AGT CGC GGT GGC CAT GGC TTC GGC CCG GGC GGC GAG AAC CAG GCG ATG AAC CAC GCC GAT CCC TGG TTG CGC</br>
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ACC GCG CTG GGT TTC ATC GGC ATC GAC GAG GTC ACG GTG GTC GCG GCG GAA GGC GAG GAA TCC GGC GGC AGG</br>
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TCC TTC GAG GAC TCC TGC GAC GAG GCG GAA CAG CGC CTG CTG GCG CTG GCG CGG TCG GCC TGA
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</code></html>
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The forward primer for the B. subtilis AzoR gene consists of the first 19 nucleotides of the protein coding sequence.
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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
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iGM SUBF4 : 5'-<html><code>ATGTCTACAGTTTTATTTG</code></html>-3'
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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
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The reverse primer for the B. subtilis AzoR gene consists of the last 19 nucleotides of the protein coding sequence.
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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
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iGM REVSUB: 5'-<html><code>TTAGAATTTTGCAGCAAGA</code></html>-3'
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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
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The forward primer for the P. aeruginosa AzoR gene consists of the first 17 nucleotides of the protein coding sequence.
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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
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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
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iGM AER4 : 5'-<html><code>ATGAGTAGAATTCTTGC</code></html>-3'
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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
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The reverse primer for the P. aeruginosa AzoR gene consists of the last 18 nucleotides of the protein coding sequence.
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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
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iGM REV AER :5'-<html><code>TCAGGCCGACCGCGCCAG</code></html>-3'
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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
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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.
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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
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BioBrick CDS Prefix  5'-<html><code>GAATTCGCGGCCGCTTCTAG</code></html>-3'
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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
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BioBrick Suffix  5'-<html><code>TACTAGTAGCGGCCGCTGCAG</code></html>-3'
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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
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    A GAA CGA CGT TTT AAG ATT
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The full primer sequences for B. subtilis AzoR and P. aeruginosa AzoR are therefore
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IGM-Bsubt-R, 3’-5’
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iGM PRESUB4 : 5'-<html><code>GAATTCGCGGCCGCTTCTAGATGTCTACAGTTTTATTTG</code></html>-3'
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iGM SUF REV SUB:5'-<html><code>TACTAGTAGCGGCCGCTGCAGTTAGAATTTTGCAGCAAGA</code></html>-3'
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IGM-Bsubt-F : 5’-ATG TCT ACA GTT TTA TTT G-3’
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iGM PRE AER4: 5'-<html><code>GAATTCGCGGCCGCTTCTAGATGAGTAGAATTCTTGC</code></html>-3'
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IGM-Bsubt-F + Prefix: 5’-GTT TCT TCG AAT TCG CGG CCG CTT CTA GAG ATG TCT ACA GTT TTA TTT G-3’
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iGM SUF REV AER: 5'-<html><code>TACTAGTAGCGGCCGCTGCAGTCAGGCCGACCGCGCCAG</code></html>-3'
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IGM-Bsubt-R:5’-TTA GAA TTT TGC AGC AAG A-3’
 
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IGM-Bsubt-R + Suffix:5’-GTT TCT TCC TGC AGC GGC CGC TAC TAG TAT TAG AAT TTT GCA GCA AGA-3’
 
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{{:Team:UCL/Template:Footer}}
{{:Team:UCL/Template:Footer}}
[[Category:TeamUCL]]
[[Category:TeamUCL]]

Latest revision as of 14:08, 29 May 2014

Goodbye Azo Dye : iGEM 2014 - University College London

 

Overview

A BioBrick part containing the azoreductase (AzoR) gene will created 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.

AzoR BioBrick Design

Azoreductase genes will be cloned from two species, B. subtilis (strain 168) and P. Aeruginosa (PA01) using DNA PCR amplification. Suitable forward and reverse primers were designed for each enzyme that facilitated the creation of two AzoR BioBrick parts.

Protein coding sequences

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 nucleotides of the protein coding sequence.

iGM SUBF4 : 5'-ATGTCTACAGTTTTATTTG-3'

The reverse primer for the B. subtilis AzoR gene consists of the last 19 nucleotides of the protein coding sequence.

iGM REVSUB: 5'-TTAGAATTTTGCAGCAAGA-3'

The forward primer for the P. aeruginosa AzoR gene consists of the first 17 nucleotides of the protein coding sequence.

iGM AER4 : 5'-ATGAGTAGAATTCTTGC-3'

The reverse primer for the P. aeruginosa AzoR gene consists of the last 18 nucleotides 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'

Contact Us

University College London - Gower Street - London - WC1E 6BT - Biochemical Engineering Department
phone: +44 (0)20 7679 2000
email: ucligem2014@gmail.com

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