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 aeruginosa). Finally, rpfC, rpfG, clp and PmanA were selected to deal with DSF; BCAM0227, BCAM0228 and Pcbld for BDSF; and pqsR and PpqsA 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 pv. 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 PpqsA 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 clone 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 concentrations 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, 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 removing 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.

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Week 5

It turned out that our very first transformations of YdeH into E. coli JM110 didn’t seem to be working. Nothing we did was different from any other transformations so we wondered if YdeH is toxic to the cells - maybe too much c-di-GMP is a bad thing! We decided to try cloning it under an inducible promoter…later! The DSF model was slightly harder to work with than PQS as it contained a lot of components and our first model started with about 12 equations! We were able to reduce these down and simplify the model, which enabled us to input them into MATLAB and produce some graphs. We had a few problems determining exactly what our GFP production was reliant on, but after a lot of work on our blackboard, we came to a conclusion. Again, our deterministic model allowed us to change our initial conditions allowing us to see what would happen.
PqsR and PpqsA were successfully BioBricked!
A lot of stochastic modelling going on this week, PQS wild type and PQS E.coli being our priority. Adding in an external signal is proving a little difficult as we’re not getting our expected results! A lot of trial and error in trying to match up parameters from MatLab and Maple, but we think we’re there! Netlogo is also coming along nicely, with PQS wild type and PQS E. coli finished! BDSF E. coli is almost finished too, so after that only DSF to tackle!

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Week 6

We successfully removed the first illegal site from RpfC after analysing sequencing results. Meanwhile clp BioBricking was underway and work had begun on the BDSF system…halfway through the 10 weeks and we were feeling productive (if showing the first signs of frazzling)! The dry lab were certainly ahead of the game as far as BDSF was concerned; after working through the mammoth DSF system, the closely related two-component BCAM0227/BCAM0228 system was a breeze. In fact, with such a head-start it was decided that there would be a presentation given by the maths boffins of the team entitled: “Modelling for the Perplexed Biologists and Dave”. This was an interactive lesson on how to apply equations to biological systems which helped the lab-rats get to grips with how the figures being generated by the dry team could inform the final design of the biological systems.
Around this time, the “Device Committee” was formed; a small group of wet and dry members who would begin the design of a physical unit to house and interpret the output from our engineered bacteria. This began with background reading on creating a GFP-fluorescence reading device. Order initial parts to build a light sensing device based on LED light source so we could modulate the light levels. We considered that we would have to use MatLab to carry out calculations. RpfG, RpfC and Clp cloned into pUniprom!

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Week 7

Initial experiments with a photodiode, Arduino and a green LED for the physical device. First designs showed us that we would need an excitation source to induce fluorescence, this would necessitate the inclusion of filters and amplifiers…extremely complicated! Discussions were had with the entire team about eventually using firefly luciferase as a reporter in the place of fluorescent proteins.
BCAM0227 successfully cloned into pUniprom and clp BioBricked! However, sequencing results for RpfG QuikChange showed that it was actually RpfC…oops!
All promoters had now been successfully cloned and sequenced, so were cloned along with their fluorescent reporters into pUniprom.
Repeated the RpfG quickchange.

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Week 8

Good catch by a studious member of the dry lab – it turned out that B.cenocepacia produces a signaling molecule called HHQ which is so similar to PQS that it can bind to PqsR and activate the system! After some modelling, the decision was made to re-design the P. aeruginosa system to detect Pseudomonas autoinducer 1 (PAI-1). Lots of work for the wet team!
BioBricking of RpfG commences upon receiving good sequencing results.
DSF system looked like it was ready for testing with our newly acquired synthetic DSF (minus YdeH – this is still killing our cells). A culture of E. coli MC1061 transformed with the DSF plasmid is set up for blotting – alas, RpfC-HA is not being expressed! Back to the cloning board…

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Week 9

The Device Committee met up with Dr. Stephen Reynolds from the University’s physics department who helped us to optimize our light-detecting circuit. Constructed a crude (but effective) Faraday cage from aluminium foil and a cardboard box to eliminate electrical interference from the buzzing computers in the dry lab…
RpfC was, at great length, cloned into pUniprom along with the rest of the DSF system, time for some tests!
BCAM0227 was cloned from X. campestris DNA and sent for sequencing with good results first time! It was then swiftly cloned into pUniprom (already containing the P_cbld-Fgap fusion).
BCAM0228 quick-changes were completed and sent for sequencing.

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Week 10

We obtained a culture of Vibrio fischeri from Dr Sarah Coulthurst’s lab and began streaking plates to test out our physical device for detection of the bioluminescence from V. fischeri luciferase – it worked! Although, at this point our device was essentially half of a water bottle covered in insulating tape, an Arduino and a voltage meter. However, we had come up with a product name: the Light Amplifying Signal Sensing Object, because every good cowboy needs a L.A.S.S.O., right? Time for a more robust design, maybe 3D printed… iGEM alumnus and polymath Nasir Ahmad showed us how to draw up plans using Google Sketch. BCAM0228 sequence came back looking good, into pUniprom and pSB1C3 it went!

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Week 11

At this stage, we had all of our systems (barring PAI-1) cloned into pUniprom and ready for blotting. So with a little (actually, rather a lot) of help from master-blotter Prof Frank Sargent, we set to the task with all the elation of a team that can see the end of cloning in sight.
The Device Committee were having a great time designing the L.A.S.S.O.; a cavity here, a plunger there, maybe some springs…
Some of the wet team set to the noble task of cloning our replacement Pseudomonas system. Luckily, all the components we needed were included in the 2014 iGEM distribution pack so we stabbed, rehydrated and got to work.
We decided to use the Ptet BioBrick as a vector and cloned in lasR downstream.
YdeH was successfully BioBricked – another one bites the dust!

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Week 12

The BDSF system, after a successful western blot to check for protein expression, was set up for an inducible GFP production assay by spiking overnight cultures of transformed E. coli MC1061 with a range of concentrations of synthetic BDSF. Unfortunately, GFP appeared to be expressed at all concentrations, including 0µM! This experiment clearly needed to be repeated. The L.A.S.S.O. design was almost complete, with only some means of safe disposal of our engineered bacteria to be factored in. We settled on a design that would involve sealed petri-dishes containing freeze-dried E. coli with a one-way valve on the lid through which a sample of sputum and water would be passed. The plate would then be inserted into the L.A.S.S.O. and all sources of external light eliminated by sliding flaps. The photodiode would pick up luminescence from the luciferase, expressed in response to the bacterial signaling molecule of interest, and send a voltage to the Arduino which would pass this signal via a µUSB cable to a computer where an application would interpret the signal into an email containing information about the bacterial species present which would be sent to a clinician. The plate would then be transferred mechanically into an isolated waste compartment which can be removed and emptied into a biological waste container when full.

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Week 13

The dry team set about modelling what potential savings could be made from the implementation of The Lung Ranger/L.A.S.S.O. by investigating the cost to the NHS of keeping patients in hospital for treatment that could be given domestically if the need was identified earlier. Also under consideration was the amount of days taken off work by the patients when ill or visiting the clinic – a concern mentioned by some patients we met at the clinic.
BCAM0227 and P_cblD BioBricked.
The growing PAI-1 system is complemented with the addition of the nanoluc BioBrick.

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Week 14

More L.A.S.S.O. testing with V. fischeri – this time with plates containing different amounts of bacteria, obtained by serial dilution of an overnight culture. This resulted in our first attempt to map the sensitivity of our device vs. c.f.u. of bacteria. PmanA BioBricked, one to go!
The BDSF GFP blot was repeated with similar results, the decision is made to begin removing genes from the system until we find out where the problem is.
Western blots of the DSF system show good protein expression, GFP assay to follow.
Sequencing of the PAI-1 system shows that the nanoluc sequence is missing, hmm…..

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Week 15

Repeated ligations of PAI-1 system with nanoluc.
RpfC BioBricked, all BioBricks finished, woohoo!
Received our 3D printed L.A.S.S.O., it looked great but didn’t fit together as planned… out with the Stanley knife! No problem. GFP assay of DSF system showed similar results to that of the BDSF system, surely not! The experiment was repeated and the same results obtained. Oh, dear!
The Device Committee met with Dr. Mike MacDonald from the Institute for Medical Science and Technology (IMSAT) to discuss plans for building a device and learned from him the importance of following international standards of operation (ISOs). From this point on we endeavoured to follow ISOs 13485 (medical devices) & 9001(general quality management) so that, if the device were to be taken on by a company, our design and parts would be easily replicable.

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Week 16

Instead of painstakingly removing each gene, we decided (on the advice of the University’s top microbiologist Prof. Tracy Palmer) to blot the DSF and BDSF systems for GFP when only the promoter/reporter fusions were present. This was a breakthrough as the PmanA-gap fusion still showed GFP expression while the PcblD-gap fusion did not. This lead us to perform a BLAST search of the Clp sequence from the DSF system against the E. coli genome to look for similar transcription factors in the wild type. CRP from E. coli showed significant similarity so we obtained a CRP-deletion mutant (BW25113 ∆CRP) and transformed it with our promoter/reporter fusion in preparation for blotting.
BCAM0228-P_cblD-GFP was set up in E. coli MC1061 for blotting.

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Week 17

PAI-1 system sequencing looked good so was transformed into E. coli MC1061 in preparation for blotting.
Western blot of BCAM0228-P_cblD-gfp showed GFP expression. Contrasted with the negative results from the P_cblD-gfp blot, this suggests that something native to E. coli is activating BCAM0228.
Western blot of P_manA-gfp in the CRP deletion strain of E. coli still shows expression of GFP.

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Week 18

PAI-1 system blot works! What a relief. After much head-scratching and brow-furrowing over the other two systems it was great to get a positive result. We have learned a lot about the potential cross-talk between signal transduction pathways when they are expressed in different species. We believe that research into untangling these interactions will significantly speed-up the process of applying synthetic biology solutions to real-world problems.

Time to pack up the lab and hand over our equipment to the new PhD students for training, it’s been a rollercoaster of a project that every one of us has benefited from in ways we will likely still be discovering years from now.

See you in Boston!