Team:UCL/Science/Experiment

From 2014.igem.org

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<div id="view1"><div class="textTitle"><h4>Stage 01: Extraction of useful BioBrick plasmids from iGEM 2014 Distribution Kit</h4></div><br>
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<div id="view1"><div class="textTitle"><h4>Stage 01: Extraction of Useful BioBrick Plasmids from iGEM 2014 Distribution Kit</h4></div><br>
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<div><strong>Protocols&nbsp;&nbsp;</strong>
<div><strong>Protocols&nbsp;&nbsp;</strong>
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                 <td> <center>T</center> </td>
                 <td> <center>T</center> </td>
                 <td> &nbsp;<a href="http://parts.igem.org/Part:BBa_K518012">BBa_K518012</a> </td>
                 <td> &nbsp;<a href="http://parts.igem.org/Part:BBa_K518012">BBa_K518012</a> </td>
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                 <td> &nbsp;RBS + RFP + double Terminator</td>
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                 <td> &nbsp;RBS + RFP + Double Terminator</td>
                 <td> &nbsp;Chloramphenicol</td>
                 <td> &nbsp;Chloramphenicol</td>
                 <td> &nbsp;Spring 2014 BioBrick Distribution. Plate 1, Well 18C.</td>
                 <td> &nbsp;Spring 2014 BioBrick Distribution. Plate 1, Well 18C.</td>
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<div id="view2"><div class="textTitle"><h4>Stage 02: Identification of useful genes for making new BioBricks</h4></div><br>
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<div id="view2"><div class="textTitle"><h4>Stage 02: Identification of Useful Genes for Making New BioBricks</h4></div><br>
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     <strong>Identifying azo dye degrading enzymes</strong>
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     <strong>Identifying Azo-Dye Degrading Enzymes</strong>
     <p>Searching through the literature, we identified a number of bacterial species (including <em>Bacillus subtilis</em> and <em>Pseudomonas sp.</em>) that have proven to degrade azo dye compounds <a href="http://www.ncbi.nlm.nih.gov/pubmed/21655981">[1]</a><a href="http://www.ncbi.nlm.nih.gov/pubmed/24475252">[2]</a><a href="http://www.itqb.unl.pt/martins/index_files/JBC2002.pdf">[3]</a><a href="http://www.ncbi.nlm.nih.gov/pubmed/23820555">[4]</a>. <br>
     <p>Searching through the literature, we identified a number of bacterial species (including <em>Bacillus subtilis</em> and <em>Pseudomonas sp.</em>) that have proven to degrade azo dye compounds <a href="http://www.ncbi.nlm.nih.gov/pubmed/21655981">[1]</a><a href="http://www.ncbi.nlm.nih.gov/pubmed/24475252">[2]</a><a href="http://www.itqb.unl.pt/martins/index_files/JBC2002.pdf">[3]</a><a href="http://www.ncbi.nlm.nih.gov/pubmed/23820555">[4]</a>. <br>
     We contacted the <a href="http://www.itqb.unl.pt/martins">Microbial & Enzyme Technology Lab</a> led by Dr Lígia O. Martins at the Universidade Nova de Lisboa, who are currently researching how azo dye degrading enzymes function, and they were keen to collaborate with us on our project. They agreed to send us a set of five plasmids, each containing different genes encoding azo dye degrading enzymes from both <em>B. subtilis</em> and <em>P. putida</em> (including mutated forms found to exhibit enhanced degradation activity), for us to use in our investigations (see Table below). <br>
     We contacted the <a href="http://www.itqb.unl.pt/martins">Microbial & Enzyme Technology Lab</a> led by Dr Lígia O. Martins at the Universidade Nova de Lisboa, who are currently researching how azo dye degrading enzymes function, and they were keen to collaborate with us on our project. They agreed to send us a set of five plasmids, each containing different genes encoding azo dye degrading enzymes from both <em>B. subtilis</em> and <em>P. putida</em> (including mutated forms found to exhibit enhanced degradation activity), for us to use in our investigations (see Table below). <br>
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             <tr>
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                 <td> &nbsp;<a href="http://www.ncbi.nlm.nih.gov/pubmed/24475252">p1B6</a> </td>
                 <td> &nbsp;<a href="http://www.ncbi.nlm.nih.gov/pubmed/24475252">p1B6</a> </td>
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                 <td> &nbsp;AzoR heat-stable mutant</td>
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                 <td> &nbsp;AzoR Heat-stable Mutant</td>
                 <td> &nbsp;<em>Pseudomonas putida</em> </td>
                 <td> &nbsp;<em>Pseudomonas putida</em> </td>
                 <td> &nbsp;612 bp </td>
                 <td> &nbsp;612 bp </td>
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<div id="view3"><div class="textTitle"><h4>Stage 03: Transforming E. coli with azo-dye degrading plasmids from Lisbon</h4></div><br>
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<div id="view3"><div class="textTitle"><h4>Stage 03: Transforming E. coli with Azo-Dye Degrading Plasmids from Lisbon</h4></div><br>
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     <strong>Transforming <em>E. coli</em> with azo-dye degrading plasmids</strong>
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     <strong>Transforming <em>E. coli</em> with Azo-Dye Degrading Plasmids</strong>
     <div><strong>Protocols&nbsp;&nbsp;</strong>
     <div><strong>Protocols&nbsp;&nbsp;</strong>
         <a href="/Team:UCL/Science/Proto"><span class="label label-warning">competent cells</span></a>
         <a href="/Team:UCL/Science/Proto"><span class="label label-warning">competent cells</span></a>
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     <a data-tip="true" class="top large" data-tip-content="Here's the gel visualisation showing an analytical digest of the plasmids we received from Lisbon!" href="javascript:void(0)" style="width: 40%;float: right;margin-left:1%"><img src="http://2014.igem.org/wiki/images/0/08/UCL_23-07-2014_Analytical_Digest_Visualisation.pptx.png" style="max-width: 100%;"></a>
     <a data-tip="true" class="top large" data-tip-content="Here's the gel visualisation showing an analytical digest of the plasmids we received from Lisbon!" href="javascript:void(0)" style="width: 40%;float: right;margin-left:1%"><img src="http://2014.igem.org/wiki/images/0/08/UCL_23-07-2014_Analytical_Digest_Visualisation.pptx.png" style="max-width: 100%;"></a>
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     <strong>Diagnostic digest of azo-dye degrading plasmids</strong>
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     <strong>Diagnostic Digest of Azo-Dye Degrading Plasmids</strong>
     <div><strong>Protocols&nbsp;&nbsp;</strong>
     <div><strong>Protocols&nbsp;&nbsp;</strong>
         <a href="/Team:UCL/Science/Proto"><span class="label label-warning">digest</span></a>
         <a href="/Team:UCL/Science/Proto"><span class="label label-warning">digest</span></a>
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<div id="view4"><div class="textTitle"><h4>Stage 04: Creation of azo-reductase BioBrick parts from plasmids</h4></div><br>
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<div id="view4"><div class="textTitle"><h4>Stage 04: Creation of Azo-Reductase BioBrick Parts from Plasmids</h4></div><br>
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<div><strong>Protocols&nbsp;&nbsp;</strong>
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     <strong>Diagnostic digest of azo-dye degrading plasmids</strong>
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     <strong>Diagnostic Digest of Azo-Dye Degrading Plasmids</strong>
     <div><strong>Protocols&nbsp;&nbsp;</strong></div>
     <div><strong>Protocols&nbsp;&nbsp;</strong></div>
     <p>We confirmed the success of the PCR through gel visualisation, comparing PCR products with and without prefix and suffix.</p>
     <p>We confirmed the success of the PCR through gel visualisation, comparing PCR products with and without prefix and suffix.</p>
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<div id="view5"><div class="textTitle"><h4>Stage 05: Creation of azo-reductase BioBrick parts from plasmids</h4></div><br>
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<div id="view5"><div class="textTitle"><h4>Stage 05: Creation of Azo-Reductase BioBrick Parts from Plasmids</h4></div><br>
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<p>We decided to assemble both BsDyP and ispB in a LacI cassette, inducible by IPTG.  
<p>We decided to assemble both BsDyP and ispB in a LacI cassette, inducible by IPTG.  
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<div id="view6"><div class="textTitle"><h4>Stage 06: Diagnostic digest of azo-reductase BioBrick parts</h4></div><br>
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<div id="view6"><div class="textTitle"><h4>Stage 06: Diagnostic Digest of Azo-Reductase BioBrick Parts</h4></div><br>
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<div id="view7"><div class="textTitle"><h4>Stage 07: Assembling azo-reductase BioBrick Device(s)</h4></div><br>
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<div id="view7"><div class="textTitle"><h4>Stage 07: Assembling Azo-Reductase BioBrick Device(s)</h4></div><br>
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<div id="view8"><div class="textTitle"><h4>Stage 08: Characterisation of azo-reductase BioBrick devices</h4></div><br>
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<div id="view8"><div class="textTitle"><h4>Stage 08: Characterisation of Azo-Reductase BioBrick Devices</h4></div><br>
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Revision as of 01:18, 18 October 2014

Goodbye Azodye UCL iGEM 2014

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Experiments

Stage 01: Extraction of Useful BioBrick Plasmids from iGEM 2014 Distribution Kit


We began our project by identifying a range of BioBrick parts present in the iGEM 2014 distribution kit which we required as part of our cloning strategy. These parts primarily consisted of both constituitive and inducible promoter systems with ribosome binding sites which we could then use in conjunction with our azo-reductase BioBricks to assemble a functional azo dye degrading gene. We also decided that we would use the Red Florescent Protein expressing BioBrick as a control for any further transformation experiments. As the level of DNA present within each plate of the distribution kit is insufficient to perform digest and ligation reactions on it was necessary to transform each of these plasmids into our NEB5alpha competent cells. After growing our transformed cells overnight we then mini-prepped each of them to obtain BioBrick plasmids at suitable concentrations for future experiments.


Registry ID Name / Function Antibiotic Resistance Source Size
U
 BBa_K314103  IPTG-inducible LacI Expression Cassette  Chloramphenicol  Spring 2014 BioBrick Distribution. Plate 1, Well 4D.  1638 bp
T
 BBa_J04450  RFP Coding Device  Chloramphenicol  Spring 2014 BioBrick Distribution. Plate 4, Well 4B.  1069 bp
T
 BBa_R0010  IPTG-inducible LacI Promoter  Chloramphenicol  Spring 2014 BioBrick Distribution. Plate 3, Well 4G.  200 bp
T
 BBa_B0034  Ribosomal Binding Site (RBS)  Chloramphenicol  Spring 2014 BioBrick Distribution. Plate 4, Well 1N.  12 bp
T
 BBa_K518012  RBS + RFP + Double Terminator  Chloramphenicol  Spring 2014 BioBrick Distribution. Plate 1, Well 18C.  828 bp
N
 BBa_K206000  pBAD Strong Promoter  Chloramphenicol  Spring 2014 BioBrick Distribution. Plate 3, Well 14A.  130 bp
! N
 BBa_R0011  LacI-Regulated, Lambda pL Hybrid Promoter  Chloramphenicol  Spring 2014 BioBrick Distribution. Plate 2, Well 6D.  55 bp
! N
 BBa_B0012  Transcription Terminator for E. coli RNA Polymerase  Chloramphenicol  Spring 2014 BioBrick Distribution. Plate 2, Well 2B.  41 bp
Note: U = Used in experiments; T = Used for testing purposes but not for making BioBrick Devices; N = Transformed from Distribution Kits, but not used in experiments; ! = Problematic parts (see Parts Registry), were not used.

Stage 02: Identification of Useful Genes for Making New BioBricks


Identifying Azo-Dye Degrading Enzymes

Searching through the literature, we identified a number of bacterial species (including Bacillus subtilis and Pseudomonas sp.) that have proven to degrade azo dye compounds [1][2][3][4].
We contacted the Microbial & Enzyme Technology Lab led by Dr Lígia O. Martins at the Universidade Nova de Lisboa, who are currently researching how azo dye degrading enzymes function, and they were keen to collaborate with us on our project. They agreed to send us a set of five plasmids, each containing different genes encoding azo dye degrading enzymes from both B. subtilis and P. putida (including mutated forms found to exhibit enhanced degradation activity), for us to use in our investigations (see Table below).


Gene ID Name / Function Source Size Plasmid
 pAzoR  FMN-dependent NADH-azoreductase 1  Pseudomonas putida  612 bp  In expression vector: pET-21a (+) (ampicillin resistant) [2] [3] ,
initially cloned between NdeI and BamHI restriction sites.
 p1B6  AzoR Heat-stable Mutant  Pseudomonas putida  612 bp  In expression vector: pET-21a (+) (ampicillin resistant) [2] [3] ,
initially cloned between NdeI and BamHI restriction sites.
 pCotA  Spore Coat Protein Laccase  Bacillus subtilis  1542 bp  In expression vector: pET-21a (+) (ampicillin resistant (ampR)) [2] [3] ,
initially cloned between NheI and BamHI restriction sites.
 pBsDyP  Dye Decolourising Peroxidase BSU38260  Bacillus subtilis  1251 bp  In expression vector: pET-21a (+) (ampicillin resistant) [2] [3] ,
initially cloned between NdeI and BamHI restriction sites.
 pPpDyP  Dye Decolourising Peroxidase PP_3248  Pseudomonas putida  864 bp  In expression vector: pET-21a (+) (ampicillin resistant) [2] [3] ,
initially cloned between NdeI and BamHI restriction sites.

Extraction of B. subtilis genomic DNA
Protocols   DNA extraction

In the meantime, Helina (in our team), was able to obtain B. subtilis and P. aeruginosa strains for us to test whether we could retrieve azo dye degrading enzymes from their genomes, specifically, the azo-reductase gene (AzoR). This would be the first step for our first azoreductase BioBrick.
We extracted the genomic DNA from B. subtilis strain using a Promega Wizard Genomic DNA extraction kit so that we could subsequently amplify the azoreducatase gene (AzoR1) and create our first azoreductase BioBrick. After completing the genomic DNA extracton we ran a gel to show that we had successfully extracted the B. subtilis genomic DNA.



Stage 03: Transforming E. coli with Azo-Dye Degrading Plasmids from Lisbon


Transforming E. coli with Azo-Dye Degrading Plasmids

The five azo dye degrading enzymes from Lisbon arrived as the respective genes in pET-21a (+) ampicillin resistant (ampR) expression vectors/plasmids (size: 5443 bp)[1][2]. The DNA concentrations of these plasmids, however, were insufficient to perform PCR amplification, therefore we transformed each into our own E. coli competent cells (grown from NEB DH5α derivatives). After growing the cells overnight, we made bacterial glycerol stocks and miniprepped the cells to obtain plasmids at sufficient concentrations for further work.


Diagnostic Digest of Azo-Dye Degrading Plasmids
Protocols   digest gel

A diagnostic digest was performed to ascertain that these pET-21a (+) plasmids contained the gene we expected. As each plasmid possessed EcoRI and XbaI restriction sites close to the genes of interest, we performed double-digests using these recognition enzymes and predicted the digest fragments. The digestion products were visualised using gel electrophoresis (see image right).







Stage 04: Creation of Azo-Reductase BioBrick Parts from Plasmids


Protocols   digest gel

After isolating our genes of interest we attempted to use PCR as a method of prefix and suffix generation to fit the BioBrick standard assembly.


Achieving a successful PCR proved difficult, this may have been due to poor reagent quality. We repeated the PCR using Taq, phusion and Pfu polymerases. We took an alternative route successfully used directionless ligation to generate the prefix and suffix for 1B6. Taq polymerase eventually gave us a successful generation of prefix and suffix for BsDyP, AzoR and ispB. Unfortunately, due to time constraints we were unable to implement site directed mutagenesis on 1B6 required to remove two illegal PstI and therefore did not submitt the part to the registry.


Diagnostic Digest of Azo-Dye Degrading Plasmids
Protocols  

We confirmed the success of the PCR through gel visualisation, comparing PCR products with and without prefix and suffix.

Stage 05: Creation of Azo-Reductase BioBrick Parts from Plasmids


We decided to assemble both BsDyP and ispB in a LacI cassette, inducible by IPTG. Issues with inconclusive antibiotic effectivity led to major delays in construction of these composite parts. We first had to prove our antibiotics were functioning properly before making progress on our project.


We confirmed the construction of our BsDyP and ispB cassettes using analytical gel digest cutting at sites E and P.

Stage 06: Diagnostic Digest of Azo-Reductase BioBrick Parts


...


Stage 07: Assembling Azo-Reductase BioBrick Device(s)


...


Registry ID Gene ID Name / Function Source Size Status
 BBa_K1336000  AzoR  FMN-dependent NADH-azoreductase 1  Pseudomonas putida  612 bp  [In Progress]: primers designed
 BBa_K1336001  1B6  AzoR heat-stable mutant  Pseudomonas putida  612 bp  [In Progress]: to remove 2 illegal PstI sites
 BBa_K1336002  CotA  Spore Coat Protein Laccase  Bacillus subtilis  1542 bp  [In Progress]: primers designed
 BBa_K1336003  BsDyP  Dye Decolourising Peroxidase BSU38260  Bacillus subtilis  1251 bp  [New BioBrick Part]: submitted
 BBa_K1336004  PpDyP  Dye Decolourising Peroxidase PP_3248  Pseudomonas putida  864 bp  [In Progress]: primers designed
 BBa_K1336005  ispB RNAi  RNAi of Octaprenyl Diphosphate
Synthase fragment
 Escherichia coli, K12 strain  562 bp  [New BioBrick Part]: submitted
 BBa_K1336006  LacIEC+ispB  IPTG inducible ispB RNAi  Escherichia coli, K12 strain  2208 bp  [New BioBrick Device]: submitted
 BBa_K1336007  LacIEC+BsDyP  IPTG inducible BsDyP  Bacillus subtilis  2895 bp  [New BioBrick Device]: submitted
 BBa_K729006  CueO  Laccase  Escherichia coli  1612 bp  [In Progress]: ascertaining identity
()
 BBa_K500000  LiP  Lignin Peroxidase  Phanerochaete chrysosporium  1116 bp  [Improved Characterisation]: toxicity issues in gene synthesis.
 [In Progress]: to subclone into pSB1C3/pSB3C5.
 BBa_K729004  nucB  Extracellular nuclease  Staphylococcus aureus  561 bp  [Improved Function]

Stage 08: Characterisation of Azo-Reductase BioBrick Devices


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Protocols   PCR analytical digest gel (digest ligation competent cells transformation miniprep)

[Insert table of Our Genes]


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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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