Team:UCL/Science/Experiment

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.

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).

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.

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.

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).

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 parts format.

Achieving a successful PCR proved difficult; this may have been due to poor PCR reagent quality. We repeated the PCR using various polymerases (Taq, Phusion and Pfu) and also different dNTP mixes. Eventually, we succeeded in amplifying AzoR 1B6, BsDyP, and ispB asDNA with the required BioBrick Prefix and Suffix. Given the time-constraints, we did not succeed in also amplifying AzoR, CotA, and PpDyP with the Prefix and Suffix.

Our next step was to ligate these into the required pSB1C3 backbone. For BsDyP and ispB asDNA, this proved to be fairly straightforward, and quickly resulted in the production of our first new BioBricks: BBa_K1336003 for BsDyP+pSB1C3, and BBa_K1336005 for ispB+pSB1C3.
This was trickier for 1B6, as this gene possessed 2 illegal PstI restriction sites. Site-directed mutagenesis primers were designed to remove these sites, however, we could not completed this in time for submission. We did, however, succeed in performing a directionless ligation into pSB1C3. From here, we screened for plasmids with the correct orientation, and started our characterisation assays with this pseudo-BioBrick part.

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.

Ideally, we wanted a Azo-Remediation Chassis (ARC), our BioBrick System, to be assembled as follows:

A further development on this prototype would be to have Bba_K1336000, the AzoR gene, to be inducibly transcribed by one promoter (say BBa_K314103, the LacI Expression Cassette) such that it is expressed in the reductive step of our azo dye remediation process. This would form a distinct BioBrick Device of "promotor A + AzoR + double terminator".
A secondary BioBrick Device (of "promoter B + gene 2 + double terminator") would follow this, where gene 2 would be one of our enzymes that function in the oxidative step of the azo dye remediation process, e.g. laccase or one of the dye decolourising peroxidases. A further tertiary BioBrick Device, with another oxidative enzyme would also be ideal. At least 2 oxidative enzymes are proposed as the enzymes are specialised for different substrates (as described in our BioBrick page.
As always, time works against us, and we succeeded only in constructing 2 composite parts (BioBrick Devices): BBa_K1336007 for LacIEC+BsDyP+pSB1C3, and BBa_K1336006 for LacIEC+ispB+pSB1C3. This latter ispB asDNA device has functions in the biosafety aspects of our ARC.

	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]

Results and protocols for the characterisation pages can be found here