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Team:UCL/Science/Experiment
From 2014.igem.org
Experiments
Laboratory Team
List of Experiments
- Stage 01: Extraction of useful BioBrick plasmids from iGEM 2014 Distribution Kit
- Stage 02: Identification of useful genes for making new BioBricks
- Stage 03: Transforming E. coli with azo-reductase plasmids
- 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
Experiments
Stage 01: Extraction of useful BioBrick plasmids from iGEM 2014 Distribution Kit
...
Stage 02: Extraction of useful BioBrick plasmids from iGEM 2014 Distribution Kit
...
Stage 03: Transforming E. coli with azo-reductase plasmids
...
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
...
Random Stuff will go here based on what you want to say. This text is just filler text so all I can do is chuck it in here for mucking around
Extraction of Bacillus subtilis genomic DNA
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.
Transforming E. coli with Azo-reductase plasmids
We were gratefully provided with a set of five plasmids from a group of researchers working at the University of Lisbon, Portugal who are researching how azo-dye degrading enzymes function and who were 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 competent cells. After growing the cells overnight we then mini-prepped each of them to obtain plasmids at sufficient concentrations for future experimental work.
| Name | Function | Source | Concentration | Sequence | Initial Plasmid / Vector | Comments |
|---|---|---|---|---|---|---|
| pAzoR | FMN-dependent NADH-azoreductase 1 | Pseudomonas putida | Miniprep,
48 ng/uL, |
597 bp [Check! Not 612 bp?] | Expression vector pET-21a (+) (ampicillin resistant (ampR)), initially cloned between NdeI and BamHI. | Plasmid provided by Lisbon |
| p1B6 (AzoR 1B6) | Mutant: Heat-stable; FMN-dependent NADH-azoreductase 1 | Pseudomonas putida | Miniprep,
68 ng/uL, |
597 bp [Check! Not 612 bp?] | Expression vector pET-21a (+) (ampR), initially cloned between NdeI and BamHI. | Plasmid provided by Lisbon. |
| pCotA | Spore Coat Protein Laccase | Bacillus subtilis | Miniprep,
103 ng/uL |
1733 bp [Check! Not 1539 bp?] | Expression vector pET-21a (+) (ampR), initially cloned between NheI and BamHI. | Plasmid provided by Lisbon. |
| pBsDyP | Dye Decolourising Peroxidase BSU38260 | Bacillus subtilis | Miniprep,
51 ng/uL, |
1251 bp | Expression vector pET-21a (+) (ampR), initially cloned between NdeI and BamHI. | Plasmid provided by Lisbon. |
| pPpDyP | Dye Decolourising Peroxidase PP_3248 | Pseudomonas putida | Miniprep,
55 ng/uL |
861 bp [Check! Not 864 bp?] | Expression vector pET-21a (+) (ampR), initially cloned between NdeI and BamHI. | Plasmid provided by Lisbon. |
Diagnostic digest of azo-reductase plasmids
After successfully transforming these plasmids into competent E. coli NEB5alpha cells we then performed a diagnostic digest and gel electrophoresis experiment to ascertain that these plasmids contained the gene we expected. Each plasmid was digested using two restriction enzymes chosen to digest DNA as specific points on the plasmids and create fragments of known length which we could then confirm using gel electrophoresis.
Creation of azo-reductase BioBrick parts from plasmids
senectus et netus et malesuada
Diagnostic digest of azo-reductase BioBrick parts
senectus et netus et malesuada
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 expresing 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.
Assembling azo-reductase BioBrick Device(s)
senectus et netus et malesuada
Characterisation of azo-reductase BioBrick devices
senectus et netus et malesuada
