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Team:UCL/Tests
From 2014.igem.org
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<div id="view7"><div class="textTitle"><h4>Stage 07: Assembling azo-reductase BioBrick Device(s)</h4></div><br> | <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> | <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 17:04, 16 October 2014
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 | Source | Size | |
|---|---|---|---|---|
| |
BBa_K314103 | IPTG-inducible LacI Expression Cassette | Spring 2014 BioBrick Distribution. Plate 1, Well 4D. | 1638 bp |
| |
BBa_J04450 | RFP Coding Device | Spring 2014 BioBrick Distribution. Plate 4, Well 4B. | 1069 bp |
| |
BBa_R0010 | IPTG-inducible LacI Promoter | Spring 2014 BioBrick Distribution. Plate 3, Well 4G. | 200 bp |
| |
BBa_B0034 | Ribosomal Binding Site (RBS) | Spring 2014 BioBrick Distribution. Plate 4, Well 1N. | 12 bp |
| |
BBa_K518012 | RBS + RFP + double Terminator | Spring 2014 BioBrick Distribution. Plate 1, Well 18C. | 828 bp |
| |
BBa_K206000 | pBAD Strong Promoter | Spring 2014 BioBrick Distribution. Plate 3, Well 14A. | 130 bp |
| |
BBa_R0011 | LacI-Regulated, Lambda pL Hybrid Promoter | Spring 2014 BioBrick Distribution. Plate 2, Well 6D. | 55 bp |
| |
BBa_B0012 | Transcription Terminator for E. coli RNA Polymerase | 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
Extraction of Bacillus subtilis genomic DNA
Protocols
DNA extraction
Our literature search identified a number of bacterial species that have been proven to degrade azo dye compounds including B. subtilis and P. aeruginosa. We were able to obtain a B. subtilis strain for use in our project from ?. We extracted the genomic DNA from this strain using a Promega Wizard Genomic DNA extraction kit so that we could subsequently amplify the azo-reducatase gene (AzoR1) and create our first azo-reductase 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-reductase plasmids
We were gratefully provided with a set of five plasmids from the Microbial & Enzyme Technology Lab led by Dr Lígia O. Martins at the Universidade Nova de Lisboa. They are currently researching how azo-dye degrading enzymes function and are keen to collaborate with us. These plasmids contained a number of genes encoding azo-dye degrading enzymes from both B. subtilis and P. putida including mutated forms found to exhibit enhanced degradation activity. As the DNA concentration of the plasmids we were sent was insufficient to perform PCR amplification on we transformed each of these plasmids into our E. coli NEB5alpha derivative competent cells. After growing the cells overnight we then mini-prepped each of them to obtain plasmids at sufficient concentrations for future experimental work.
Stage 04: Diagnostic digest of azo-reductase plasmids
...
Stage 05: Creation of azo-reductase BioBrick parts from plasmids
...
Stage 06: Diagnostic digest of azo-reductase BioBrick parts
...
Stage 07: Assembling azo-reductase BioBrick Device(s)
...
Stage 08: Characterisation of azo-reductase BioBrick devices
...
