Team:Edinburgh/Notebook

Wednesday 28th

The team assembled in the Waddington building, ready to pit our five ideas against each other. Some had not survived the research process (the glowsticks), some did not seem to constitute a full project (the switch), and the others were not without their problems either. No clear consensus emerged, and team stuck around until late at night, trying to make them work.

Thursday 29th

Both L-glucose and dye manufacture were more or less ruled out on this day, which left us with no actual project to speak of. This was widely considered an undesirable state of affairs, and great efforts were gone to in the search for something new. Another late night, another intensive search through the track lists – still no success.

Friday 30th

Two fresh ideas came out of this day’s efforts. One – synthetic honey – went pretty much nowhere since bacteria would just be killed by honey, but the other seemed to have more life to it. This was metabolic wiring, and arose from a recent paper on the subject. This occupied most of the day.

Saturday 31st

Work continued into metabolic wiring, and a second project idea arose – could simple biological logic gates enable bacteria to play a game of Iterated Prisoner’s Dilemma against each other? The two ideas were developed in parallel, and it seemed we were getting somewhere at last.

Sunday 1st

The two project ideas continued their development, with diagrams and schematics being produced. Population control was looked at as a possible use for the metabolic wiring.

Monday 2nd

We presented the two ideas to one of the supervisors, Jon Marles-Wright, and it was agreed to combine the two into one project – develop metabolic wiring and BioBrick it, and use IPD as a showcase for how it can be used. More detailed research also began.

Tuesday 3rd – Friday 6th

Much more intensive research could now be done, now that the idea was ‘official.’ Actual genes and pathways were found which could be implemented, and circuit diagrams for different IPD strategies were devised.

Saturday 7th – 8th

It was time to get ready for the SULSA conference next week, where we would be presenting our idea for the first time, and making our first poster. This redefined what we had previously considered a ‘late night’ for iGEM.

Monday 9th – Tuesday 10th

SULSA! The week kicked off with the two day SULSA conference held here in Edinburgh. This was a really great experience. We got to meet the other three Scottish iGEM teams [LINKS], Kim de Mora from iGEM HQ, and lots of professionals in the field of Synthetic Biology who presented their work on the second day. We also got to present our idea to the world for the first time.

Wednesday 11th

Today we focused mainly on filling out the kind of overdue safety form, and also started handing out official roles within the group. The wiki was begun today, though nothing was uploaded yet, and we got to see our lab for the very first time.

Thursday 12th

Our first meeting of any real organisation was today, thanks to Chiara. The main outcome of this meeting was the role allocation. Philip is the ‘Trello man,’ Sam and Rikki are responsible for the wiki, Carrie and Charlotte are safety officers, Sam and Charlotte are the tweetsters, Chiara is the secretary, Philip and Elize are in charge of organising collaborations, Yuma is the Google Drive organiser.

We also started looking at organising the scientific aspects of the project. The Biologists broke into three teams to look at finding metabolic wires, creating IPD circuits, and looking at the population control aspect.

Finally, Philip also worked on trying to get funding for the iGEM meetup in Oxford later this month – we may be able to get SULSA funding (is there anything they can’t do?).

Friday 13th

We were given a tour of our lab by Jon today, who wisely advised us not to injure ourselves whilst in the lab. We also continued with experiment plans and started looking into wiki design.

We had two excellent pieces of input from actual professionals today also. First of all, Maryia Trubitsyna came to explain how her now paperclip method of parts assembly works and left us really excellent notes. And secondly, Yuma and Cesar attended an Informatics forum and gave a surprise presentation of the project. The feedback we got from some of the guys over there was to think about using metabolic wiring as a digilog signal as well as a digitial signal (so as to provide information on the metabolic load of the sender strain) and, again, to look at introducing elements of selection pressure to the IPD system. We decided to think about these suggestions but take them with a grain of salt from people who might be less familiar with iGEM than other advisers we have had.

Weekend 14th-15th

After a long sunny week where we were trapped inside for all of it, the weekend was mostly cloudy with occasional rain. Welcome to Scotland.

The team mostly relaxed over the weekend, but Philip and Yuma’s dedication recognises no weekend. Philip read some reviews on aromatics degradation, among other things, and Yuma looked some more into an idea brought up by advisors Chris French and Jon – sugar signalling. He and Philip decided it was promising but would require more research.

Monday 16th

Today we got to do actual lab work for the first time. We might have done little more than plate out some bacterial cells to make them competent, but it felt very encouraging. We learned some of the practical stuff you never learn about in the course, like how to prepare agar plates, and who to ask for various pieces of equipment.

We met with Dr. Garry Blakely to discuss a mechanism for slowing growth in our proposed population control demonstration of metabolic wiring, and he suggested a ribosome bases approach, such as targeting rpsL. We also further discussed the sugar logic idea as a way of getting started, though it was agreed a better metabolic pathway should be sought out. The main problem we are having at the moment is that all goof pathways produce toxic chemicals that cause horrific nerve diseases or cancer – not ideal.

Presentation skills were also worked on – rather fun – and looked into multicellular IPD, and alternative aromatic pathways.

Tuesday 17th

In the lab we just plated the cells from yesterday onto LB media – apparently the streaking went well. Small victories are heroic victories this early on.

Today was pretty research-heavy, with more metabolic pathways being sought out with limited success. We have a database of possible molecules we can use but they’re all so toxic, presumably because they are membrane-diffusible which is what we need. The sugar logic system would provide an alternative but it is not without its own problems.

Some of the groundwork was laid for the modelling too – mainly skill learning today – and primers were designed.

Many people seem stressed about the amount of time left - probably more because we have only a vague idea of how long things will take rather than any actual shortage of time. Time will tell.

Wednesday 18th

We finished making our cells competent today, and learnt how to make up the TSS buffer necessary to do so. Transformations should begin tomorrow. We also really need to source more materials and pieces of equipment for our lab – it has a real ‘abandoned’ feel.

Meanwhile a number of problems with the theoretical aspects were worked on. Four of us met with Dr. French to see if he had a workaround for a problem with our sugar logic system – the problem that each strain is too sensitive to false positives because it doesn’t rely on the prior strain for precursor. He didn’t have a solution, but did offer a possible solution to the secondary problem of signal quenching – make the enzymes unstable rather than use a degradation tag (which wouldn’t work outside the cell). We also looked for a way to source E. coli cells with the tetR gene as the strain we are working with is missing it, and we sort of need it for all our IPD circuits.

In addition, more organised circuit diagrams were made alongside matching plasmid diagrams, which will hopefully make the transition to multi-cell IPD easier, and a more in depth investigation of the Hbp operon (required for the metabolic wiring) was done. Philip and Cesar left us today to go and attend the Oxford iGEM meetup as our representatives. Good luck guys!

Finally, it was far too hot today. We almost denatured.

Thursday 19th

The first transformations were done today, which was exciting. They’re being incubated overnight, ready for a miniprep tomorrow. We were also very kindly given some TetR+ cells by Dr. Garry Blakely (once we’d prepared the tetracycline plate for him).

Meanwhile there was a discussion about what our ultimate aims should be, and some questions from the dry team about the specifics of metabolic wiring were answered. It was felt we need to make sure we are all on the same page, so to speak, lest we drift apart into different, unrelated subprojects.

Elize is looking into a Synthetic Biology Film Festival which previous iGEM teams have entered into with some success. The deadline for entries is in 11 days however, so this might not be feasible. Looks fun though.

Philip and Cesar attended the Oxford iGEM meetup but since they didn’t write up their logs they have been lost to history.

Friday 20th

The famous transformation efficiency kit was a complete failure – our first failed experiment! The first biobrick transformations seemed to have transformed quite well however, so the cells are at least competent.

Not a lot else happened today. Elize continued with the film, some more wiki research was done, and otherwise the day generally consisted of reading and research. The first minipreps will be done on Monday.

Weekend 21st-22nd

Recovery, mostly. First day of Summer, and the approach of July, all starting to get a bit stressful.

Monday 23rd

Lab work is now a major component of every day – as it should be. Today we performed our first minipreps of the three biobrick plasmids, ran them on a gel, and prepared for our first PCR. With then weeks to go until the end of August, it’s good that . We also have our first lab book!

The sugar project lumbered onward. We had a meeting with a resident expert on the subject who gave us a list of potential sugars we might use. The ongoing quest for potential metabolic pathways seems to be making some headway too, with some good potential operons found. Getting them on the other hand...

The film submission idea seems to be looking more doable – we churned out the first draft of a script and we have a vague idea of what we might do. The deadline is in seven days though...

Tuesday 1st July

A slow day today. Some primers were designed for the metabolic wire stuff, human practices strategies were discussed (the festivals are coming up...), and we received word that the deadline for submission of movies to the film festival has been extended, so we can redo ours if need be. In the lab, we made some more cells competent, and ran yesterday’s minipreps on a gel, with mixed results.

Wednesday 2nd July

Today we had a meeting with some artists who are keen to be involved with the project. After a pizza and beer fuelled discussion, we met with Fraiser, who is keen to produce a piece of art that would tie in with our project. The idea of an actual exhibit event sounds fantastic, and everyone seemed enthusiastic. This sounds promising!

Otherwise things continued in a similar vein as previous days – transformations were carried out and paperclip primers were designed.

Thursday 3rd July

A new aspect of the project entered the lab stage today, as Mg1665 cells were made competent ready for the actual metabolic wire mining to begin – very exciting times.

Friday 4th July

The population control system moved closer to reality today, with the discovery of the Rcd short RNA mediated division checkpoint, which looks like it can induce exactly the kind of quiescence we want. A minor setback appeared in that it’s pretty heavily patented, but a very recently discovered possible mechanism of action for it involving DNA gyrase is not (yet) patented and so might provide a potential avenue. Still looks like very hard work however.

Some genomic extraction was done in the lab today also, ready for gene harvesting – soon our metabolic wires will be forged from the resultant gloop.

Weekend 5th-6th

A sunny weekend this time, which Anna nonetheless spent reading up on degron testing and part characterisation entries. What a hero.

Monday 7th

Another new week, another week closer to the end. Today was spent largely on paperclip primer design, circuit diagram creation for population control, and degron characterisation.

Tuesday 8th

We started to try and get some Human Practices balls rolling today, with the ‘Big List’ of ideas transformed into a rudimentary action plan, and more research into previous projects’ endeavours.

Meanwhile various lab tasks were got on with – the sugar logic components were miniprep’d out, primers were designed for the aromatic wires, and some sequencing was ordered.

Wednesday 9th

We finally got some plasmids designed for the population control circuits – all being well we will inactivate certain host genes using antisense RNA genes, to halt growth on demand. All being well.

Hopefully we’ll be meeting with our artist tomorrow to see what’s been happening in his world.

Thursday 10th

Today was that most magnificent of days – Master Plan creation day. With seven weeks to go we decided to formally set out what remains to be done in each aspect of the project, and a rough outline of what will happen when.

The meeting with Fraizer went well – he seems to have lots of good (and usable) ideas, and we’re hoping to meet with him again next week, hopefully at his studio.

Friday 11th

A very important day today, as we had the big meeting with the supervisors to assess how we were getting along with the gold criteria. It also served as the halfway point meeting to see how far along we are in general. It seemed to be quite productive. The bronze criteria are more or less in the bag, the silver criteria look equally sorted providing we can get the human practices stuff off the ground, and in terms of gold – we just need one of three done. We’ve sort of made progress on all three, though we decided we would make a much bigger effort to reach out to other teams for collaboration purposes.

Weekend 12th-13th

Cesar did some prep work for the modelling, and started the first BSim implementation. Otherwise it was a week of relaxation.

Monday 14th

Another day of slow but steady lab progress – primers were ordered (a batch of 80), kanamycin plasmids were transformed, and asRNA genes were dsigned – sort of.

Tuesday 15th

Primers are flooding in now, those genomes won’t know what hit them when we start PCR-ing out genes left right and centre.

In the evening we attended a discussion at the ongoing Edinburgh Biotech conference on the coming antibiotic apocalypse, in a bid to cheer ourselves up. It was very interesting.

Wednesday 16th

New targets for the population control system were sought out today, as an antisense system began to take shape. We will create a complimentary RNA gene with flanking inverted repeats to stabilise the gene, as the literature suggests.

Thursday 17th

AntisenseBot 5000 was made to automatically generate antisense genes from coding sequences, in Python. Meanwhile, paperclip half clips were made for the PCR assembly, and more glycerol stocks were made up.

Friday 18th

In the lab, we are now nearing the end of our degron endeavours – should be in a position to0 characterise soon.

Upstairs, the bot was characterised, and more human practices research done ahead of a tentative meeting next week with some HP people.

Weekend 19th-20th

Cesar finished work on a nifty little 3D model for the population control – it looks good!

Monday 21st

We returned from the weekend very aware that there were now six weeks left of the project proper, an unpleasant thought. Today we performed more PCRs, adjusted the Antisense program to generate 2400 different antisense RNAs and Charlotte, in her own words, had to offer a prayer to Sanger, god of sequencing, that her mangled sequences might be fixable.

Tuesday 22nd

Sanger is good, Sanger is great, praise be to Sanger. It turned out the sequences were fine after all. Excellent stuff.

Meanwhile primers were finally designed and ordered for half of the population control genes, and the antisense optimiser moved ever closer to completion.

Wednesday 23rd

When they come to write the history of this era, they will call this day ‘the day in which they phosphorylated half-clips together.’

Thursday 24th

The population control model is now almost complete!

Friday 25th

This day will be known to history as ‘the day where no one did their logs except Cesar.’

Weekend 26th-27th

Not a lot happened, except that Sam's cat learned how to code and helped out with the antisense optimiser.

Monday 28th

With the population control subproject now at the lab stage too, that’s everything at the practical stage. Just the small matter of actually making our constructs now. Today we cloned out a PC gene, prepared our first PAGE gel for high resolution tests, and set up some degron ligations.

Tuesday 29th

So much PCR…

Wednesday 30th

Lots more PC genes were cloned out today, and the degron ligations seem to have worked!

Thursday 31st

Really need to start working on designing the wiki, but it’s so hard to get away from the lab at this stage. Today was more PCR, and some PCR purification ahead of sequencing.

Friday 1st

We had a meeting with today with some of the supervisors, who are keen to feel out aspects of the project we can ‘kill’ – now that there’s four weeks left. This caused some dismay, but there is some logic to it. There is to be a more official meeting next week where a final decision will be made.

We also had a meeting with Dr. Sam Brown, who advised us on the best way to go about modelling our populations and the mathematics of their growth patterns.

Weekend 2nd-3rd

It rained.

Monday 4th

Today we became acquainted with the mysterious ‘concentrator’ on the 8th floor, which we used to rescue some primers we diluted too much, like Superman if he was a Biologist.

We met with Frazer again today, who showed us the thing he’s made. It’s a boat with a tree in it, and it represents all kinds of stuff. Then we went to the pub.

Tuesday 5th

Had a Skype chat with the iGEM team from Colombia – seems they are an organising an interesting competition that they want us to be a part of – we shall investigate this.

The model is also becoming more sophisticated after a long discussion today.

Wednesday 6th

Elize and Chiara read all about ants and bees and viruses. They seem confident that this will further our human practices, but it also possible they have simply gone mad.

Thursday 7th

Another big, grand meeting with the supervisors, which ended up being mostly about branding. Some rash promises were made regarding wiki deadlines, and we talked about our art project by Frazer.

Friday 8th

The population control genes were transformed into our cells, and a PAGE gel was run for our degron ligations.

Weekend 9th-10th

Overnight incubations for some transformants were set up - nothing major.

Monday 11th

The population constructs were miniprepped out and examined on a gel, to mixed results. On a more positive note, we all had a branding meeting at the pub in the evening, where a sort of proto-logo was designed.

Tuesday 12th

We have tickets for London!

Wednesday 13th

The remake of Brian: The Movie began today – he’s never looked better. Meanwhile in the lab, data is (yes I know it should be ‘are’ – deal with it) starting to come in, but not fast enough.

Thursday 14th

We have tickets for Boston!

Friday 15th

The project description was finalised today. Infintiely more exciting was a visit from Eppendorf who gave us pens which look like pipettes. This changes everything.

A late night tonight though, as time points for a growth curve were needed until 9pm, and PCRs were still being done at 7pm.

Weekend 16th-17th

The pens aren’t that great actually – they ran out immediately.

Monday 18th

The start of the penultimate week now, and we might have a logo! The experiments continued apace, with the two week deadline being stolidly ignored by all.

Tuesday 19th

Another of our famed trips to the pub today, joined by one of our supervisors. This was the highlight of the day.

Wednesday 20th

We solved the mystery of the failed FullClips – our half clips were broken because the kinase is faulty because we… left the freezer open. Oh well!

Thursday 21st

More sequencing results today – they look… strange.

Friday 22nd

TAnother big meeting today with everyone – powerpoint slides and everything. Everyone recounted what stage they were up to and we started to look at exit strategies for getting thing to an acceptable end.

This was all fairly invigorating, and morale was generally restored after a hard week.

Weekend 23rd-24th

How can there be one week left??