Team:UCL/Science/Experiment

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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


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.

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Stage 03: Transforming E. coli with azo-dye degrading plasmids from Lisbon


Transforming E. coli with azo-dye degrading 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.


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.

Diagnostic digest of azo-dye degrading plasmids
Protocols   digest gel

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.







Stage 04: Diagnostic digest of azo-reductase plasmids


Protocols   digest gel

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.

Stage 05: Creation of azo-reductase BioBrick parts from plasmids


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Stage 06: Diagnostic digest of azo-reductase BioBrick parts


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Stage 07: Assembling azo-reductase BioBrick Device(s)


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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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Biochemical Engineering Department
Phone: +44 (0)20 7679 2000
Email: ucligem2014@gmail.com

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