Team:Cardiff Wales/Notebook
Log
13/6/16 - 17/6/16
Week 1 - Bootcamp
- Training week for all team members
- Basic Wet lab planning
Not 3 days after the end of exam season we began, with a crash course in synthetic biology (from a plant biologist). Both student and instructor were a bit nervous - as the first team from Cardiff this would all be learning experience for all. We spent the first 3 days going through all the procedures, the bread and butter that would be essential for all labwork moving on. Having been trained in GMO safety, we could dive straight in and get practice. Progress-wise, all members got lab experience and we transformed some reference samples for later comparison. We also had an Human practices planning session to work out how to best approach our problem.
20/6/16 - 24/6/16
Week 2 - First Steps
- Rivalry between Chemists and Biologists
- Outreach work gets started
The training wheels were off, and we were free to put our planning into practice. Unfortunately delivery issues hampered our progress, so it was less of a leap and more of a small step, but progress is progress. Miniprep yields were improving, the Chemists finally stopped calling the incubator 'the oven', and we successfully did our first solo transformation (pet16b into C3019H competent cells). Not only this, but we managed to get in touch with the Cardiff University PR officer to discuss ideas about how to promote ourselves. We organised a piece in our University paper, Ghairr Ryhdd (link?), to stir up interest in our project.
27/6/16 - 1/7/16
Week 3 - Gantt Charts
- Lab organisation
- PARIS :D
- First Outreach event
"If we follow the plan we can get all this done in 10 days" David McMaster
With all our g-blocks in the freezer we could finally arrange all our pieces and see how we could move forward, hence the start of the lab management phase - as we grew more comfortable in the labwork we looked for ways to increase our efficiency. This included distributing lab work (e.g. Asal and Rob focusing on making lux plates while others worked on Cas-Find, and (later on) Rob following up on FLIM work), creating Gantt charts, Asal taking on Human Practices solo, and another delivery delay which evened it all out. In H.P., we hosted our first outreach event, with a stall at a STEM event at our university, allowing us to reach out to prospective students and introduce them to iGEM and international science. Finally, a stroke of luck ended up with Cardiff being invited to EE16, a European meet-up of iGEM teams hosted by iGEM IONIS, so Chris and David got to spend a weekend away in Paris while the rest of the team stayed in sunny exotic Wales to present a stall at a second STEM open day, this time with an interactive display with glow in the dark plates.
4/7/16 - 8/7/16
Week 4 - Lots of Ligations
- Laura returns from Tobago
- Team members who went to Paris wont stop talking about it
After a hectic weekend in international outreach at EE16 Paris, it was back to the grind - running the guide RNA parts from MP to transformation into pColaDuet & SB1C£ in one week, a coordinated effort made possible through the training and planning so far. Lab work was less of a team effort for each stage - each man working as much as each pair was in the initial weeks. A welcome addition to the team, Laura came back from a marine biology project in Tobago, and promptly showed everyone up by immediately integrating into the wet lab team and proved a quick study.
11/7/16 - 15/7/16
Week 5 - Progress evaluation
- Stocktake
- Mid-Lab Crisis
As we were getting into our stride and our transformations started stacking up, we were soon drowning in eppendorfs, and the freezer shelf looked more like a school locker than a lab shelf. Rob spent an entire day fixing this and creating a new system, whilst David and Andrew created new protocols for labelling. The rest of us carried on with our work - Asal, now focused almost entirely on Human Practices, was starting to get replies from professionals on the viability & necessity of our diagnostic tool, while Chris and Nik worked through the wet lab load. At this point as well we reflected on our progress so far, and moved to align ourselves to achieving our aims. This led to the start of FUEL planning, an idea touched upon in our planning briefings as one of many possible side-projects.
18/7/16 - 22/7/16
Week 6 - PCR problems
- Pain, Primers & PCRs
With our guides and spilt luciferase cloned into pET16b (or so we thought), we set about on troubleshooting our PCRs - we were skeptical of the official guides, and tested a range of temperatures and combinations (appropriate to each construct) to achieve the strongest signal. Outside of the lab, we searched for collaboration opportunities - CRSIPR-CAS9 was a common enough system that there must be someone else using it in a similar way to us?
25/7/16 - 29/7/16
Week 7 - PCR PCR PCR
- Dreaming of PCRs
We found the golden ratio, and we went into overdrive to make up for lost time, running hundreds at a time, (record = 105 in an afternoon). Despite a low success rate, we found SB1C3 - R1 almost immediately and Andrew sent it off for sequencing. Concurrently Chris and Nik were busy re-doing all previous stages for the constructs, and troubleshooting along the way to work out what had failed and why. In a few cases it was human error, but we discovered more problems with our protocols, and a the week after we found out a full third of our constructs were doomed from the start
1/8/16 - 5/8/16
Week 8 - Success?
- First Biobrick - worthy construct
- A New Hope
Huzzah! Our first bonafide success, even if it was just a guide RNA for a CRISPR-dCAS9 system it counts as our first biobrick-able construct. No time to celebrate though. With over 20 gels to run at times it felt like a Sisyphean task, but as always, what needs to get done gets done. With multiples samples sent off for sequencing, the end seemed near. Alongside all this Rob continued to toil on his FUEL project on the side.
/8/16 - 12/8/16
Week 9 - FUELed up
The sequences came back, and they were a surprise to say the least - only the guides were viable, and those were the ones which have been biobricked (link). Neither LacZ/a or LucC/N was correctly cloned into pET16b or pColaDuet. Upon further investigation, we discovered the reason for some of it - our sourced pET16b vector had an insert with the wrong restriction sites. This means a third of our potential clones (or half of what we tested) never stood a chance. This lead to an emergency meeting whereupon it was decided that, with 1 week left in the main lab we should divert efforts into FUEL. On the bright side, we had a whole week to finish our FUEL improvement, and with the whole wet lab team on the job (instead of just Rob) we made rapid headway and were ready to PCR over the weekend.
15/8/16 - 18/8/16
Week 10 - Bottom of the Ninth
- Closure of main wet lab team
- Westminster presentation
The team were once again invited to a meetup, this time Westminster. To gain experience for Boston, Andrew went with Chris this time whilst David stayed behind to help the wetlab team finish up. It was the final week of labs and things couldnt have been more tense. It was hard to work on the Westminster presentation when you knew how close to the wire the wet lab was cutting it - if the chimera wasnt cloned this week then we were buggered. On the thursday, the day of the presentation, we fortunately had a breakthrough - a successful clone of mK into lux operon in SB1c3. However, this relief was mulled somewhat - our main lab space was to shut the next day, and the team split up, some to Toronto and Marseille, others to Warwick for placements. The rest would work in the PIs lab in restriced space. On the last day, there was a relaxed feel to it - knowing that as a teamyou have accomplished all you can is a sastisfying moment to say the least. The next time we would all be together was Boston in 3 months time, and whilst that seems an age, when you have a wiki to write it passes in an instant -_-
