Week 0

A few members of the team, fresh from their exams, began the initial literature reviews necessary to identify the genes and promoters we would need to clone in order to detect and respond to the signaling molecules DSF (from Stenotrophomonas maltophilia), BDSF (from Burkholderia cenocepacia) and PQS (from Pseudomonas aeriginosa). Finally, RpfC, RpfG, Clp and PmanA were selected to deal with DSF; BCAM0227, BCAM0228 and Pcbld for BDSF; and PqsR and PpqsABCD for PQS. Primers (including restriction sites for cloning and tags to detect protein expression) were designed for the smallest system – PQS - and ordered from Sigma Aldrich.
All sequences were analyzed with Webcutter 2.0 to detect restriction sites that conflict with the BioBrick assembly standard RFC [10] and primers were designed for use with QuikChange™ Site-Directed Mutagenesis Kit to remove illegal sites. It was decided at this stage that for the DSF system, homologues of all sequences would be taken from Xanthomonas campestris cv. campestris would be used as they have identical functions but contain fewer illegal restriction sites.

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Week 1

The team are all together! Work begins… We began organizing our raw materials, setting up overnight cultures of our vectors (pSB1C3 for BioBricks, and our supervisor’s own pUniprom for experiments) and obtaining gDNA from P.aeruginosa, X.campestris and B.cenocepacia from generous lab members. By the end of the week we had cloned PqsR and PpqsABCD into pSB1C3, phew! As for the dry-lab, the first week consisted mostly of reading papers and harassing Gillian, our mathematical biologist, for definitions of all the bio-jargon. In fact, most of our early discussions about modelling our systems consisted of furrowed brows from both sides and questions such as “a differential what?” and “Uhhh, transcriptional whatnow?”

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Week 2

In order to test our systems we decided to cone them into pUniprom, a high copy-number plasmid with a large and varied multiple cloning site downstream of a strong, constitutive promoter: Ptat. In order to arrange our sequences in such a way that there would be no leakage of our reporter (see above), we had to employ a bit of cloning trickery by inserting our sensing and responding fragments in opposite orientations. This involved cloning them into separate fragments and then flipping them round into pUniprom. To the bench! Now that the plan for the plasmid was in place, we began to enter all the information onto Netlogo to model how the system would behave in the presence of different amounts of PQS.

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Week 3

Time to start on the DSF system. We decided to use GFP rather than mCherry from this point onwards as we already have antibodies against it for blotting later. Because the DSF system acts via a reduction in cellular levels of the 2nd messenger c-di-GMP (SEE BIOLOGY PAGE), we thought it best to include a diguanylate cyclase, YdeH, in the system to replenish this important molecule afterwards. So now the DSF system has four sensory components! This posed a bit of a problem for cloning as we thought we would need to use more restriction enzymes than we had access to. The solution? Order primers that include a 5’ BglII site and 3’ BamHI-XbaI sites. This way, fragments can be cloned in sequentially with each new insert cut with BglII-XbaI and the ever-growing vector cut with BamHI-XbaI as the BglII and BamHI overhangs are complementary but upon ligation leave a scar that is not recognized by either enzyme (see below). The 3’ BamHI-XbaI site allows this process to be repeated indefinitely. Woo, idempotency!

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Week 4

A week of sequencing… We have a sequencing service downstairs from the lab who kindly gave us 100 free sequencing runs (which we quickly exceeded!) so after every cloning step we sent a sample to be sequenced – the results were a source of merriment and heartbreak in equal measure…it turned out that our 1st clone (PQS system from week 1) wasn’t quite right. So began Dimitrios’ epic journey into the world of BioBricks! We also began the lengthy task of quick-changing out all the illegal restriction sites from the fragments bound for BioBricking. So it turned out that, after a bit more sifting through papers, the Netlogo model for PQS was not quite right. While we figured that out we began to calculate the ordinary differential equations for the same system.