Team:Manchester/Enzymetest

Week 1 in the Manchester iGEM house... Exams are over and we’ve officially started full-time work on the project! At the start of the week we had a big group meeting, complete with instructors and advisors, and were presented with our very own pins and stickers set. The pins quickly vanished, but we held on to the stickers for an Outreach activity we have in the pipeline (more on that on the Outreach page!). We took advantage of the rare sunshine and had our first (almost) group photo taken, to go in an article in the uni newsletter (and to also reveal what Team Manchester looks like to the rest of the world). We were introduced to our beautiful lab space, and are all really eager to get our hands/gloves dirty!

The wiki designers have begun tweaking the page to make it a little more presentable for the masses and, apart from a little accident where Ali’s face was pasted over everyone else’s on the Team page, it’s going quite swimmingly!

After quickly realising that biology is SO COMPLEX, the main bulk of the team began an intensive literature search, looking for anything and everything that may be of use to us in the following weeks. We’ve located a BioBrick that we would really love to use and attempt to improve, but are having doubts about whether or not it is available for us to order. Hope so, or it’s back to square one! Fingers crossed.

Preliminary modelling research has also begun. None of the team has firsthand experience of modelling biology, so this should be interesting. We’re currently scouring the internet and some 1970s handbooks in search of kinetic information for the enzymes involved in prokaryotic fatty acid biosynthesis. One small step for man and all that. Wish us luck!

Week 2 in the Manchester iGEM house... (that’s old now, I won’t use it again.) This has been a busy week for the less sciencey aspects of the project. We finalised our team logo, made a great poster for a Community open day we will be attending in July, and made the final changes to the sponsor packet we will be sending out soon. The team attempted to split into two subteams: core experimental team and core modelling team. Despite working on these two separate things, the group still managed to come together regularly to update the others on any progress made. Both experimental and modelling research started in Week 1 was built upon, and we feel that we are at least going in the right direction!

We put a request in for 3 BioBricks from the Repository, and are eagerly awaiting a response. Lab work will start soon. To begin with it will just be building up a stock of media and plates etc, but it will make the lab feel more like home. Which is good, because we plan on living there for the next 10 weeks.

We also met with Dr. Andy Balmer to have a chat about Human Practices. There was so much we hadn’t thought about! He definitely gave us a few things to consider, and we set a date for another meeting in Week 3. Following the meeting we thought it would be a good idea to appoint a Head of Human Practices and a Head of Ethics on the team, which fell to Rob and Tan respectively (congrats on your new job, here is a cake).

5 of the team will be visiting London in July for the first ever Young Synthetic Biologists meet-up! Accommodation and travel has already been paid, so now it’s just a matter of waiting for the day to come. We’ll be there with bells on (and also with a soil sample, at Norwich iGEM’s request!)

This week we started thinking about our FadD knockout. After a meeting about primer design with our new supervisor, Jay; we found the gene sequence, worked out our primers and put them to order! Later in the week we had another meeting with Andy Balmer, which once again gave us a lot to think about. We’ve had a few lightbulb moments and we’re pretty excited to get stuck in! Back in the lab we found out that we didn’t have any supplies, but after a quick shopping trip we ended up with plenty of supplies...ok a LOT of supplies (Not sure if we’re going to get through 3000 1.5ml Eppendorf tubes).

Unfortunately, our first attempt at transformation failed, but to raise our spirits we had our first iGEM social! Divita cooked the team a fantastic curry, and we had a great time. The food adventures continued the next day when Elsa brought in some Icelandic dried fish: certainly something different!

Meanwhile all through the week, the modelling team have been doing a great job making the experimental team look bad. They’ve been learning how to use Copasi (made here in Manchester!) and are cross-referencing the data ready to start simulation.

Week 4 started off slowly, but by the end things were really starting to come together. We realised that the pkd46 plasmid we were trying to transform is heat sensitive, so heat-shocking and incubating at 37C really wasn’t the best idea! Instead we tried electroporation, which we managed to do first time, with some successful colonies! Hopefully the rest of the fadD knockout will now run smoothly.

After realising just how expensive biology can be, we’ve started to make a big push on the sponsorship front. So far this has led to some free kits from QIAGEN, so we're off to a good start.

Unfortunately this week we also had to say goodbye to Ali and Johanna, who need to spend time on other projects. Luckily we can say hello to Marco (who will be helping in the lab) and our new advisor Denis (who’s will be helping us out with implementing our wiki ideas).

As you can see it’s been a fairly quiet week, but with our upcoming public outreach events and YSB 1.0, we’ve got a busy fortnight ahead of us!

This week has certainly been a mix of ups and downs! On Tuesday and Wednesday the outreach team ran 12 workshops for children aged 11-13 at the university’s Science Stars event. They taught the children about the structure of DNA by getting them to create a DNA molecule from Mario sweets (Mario pairs to Yoshi, Donkey Kong to Diddy Kong) and strawberry pencils. The activity was received amazingly well by the children and teachers alike. We also taught them about what synthetic biology is, and asked them to think what ideas they’d have as well as the ethical implications associated with them. Some of the ideas were phenomenal, and some were let’s say...a little more abstract. The team is doing the workshop again at the Community Open Day on Saturday, where they’ll be bringing SynBio to the wider public!

Unfortunately, experimental progress has been plagued by admin issues, delaying us from getting the FAS module and our primers. On the plus side our electrocompetent bacterial colonies were successful, meaning our Court-Lamba recombinase knockout of the fadD gene is ready for the next step! Now we just need our primers. Modelling have had a bit more success, but both subteams have had issues with finding the source for delta-9-desaturase. Fortunately, this was more or less resolved by the end of the week!

We also had another social! Elsa cooked the team a delicious vegetarian lasagna, we found out more about Jess’ love of rodents, and Rob tried (and failed) to do Bhangra dancing.

Next week most of the team is off to YSB 1.0 in London. We’re really looking forward to meeting the other UK iGEM teams!

This has been a good week of collaborations for us! At the start of the week we had a Google hangout with Perdue to discuss their idea to standardise entry of parts into the registry. It emerged that there are many issues raised when it comes to standardising modelling. We agreed to help them with this. By the end of the week, this had somewhat grown...

On Thursday we headed off to London for the very first Young Synthetic Biologists (YSB) conference. This was a big meeting of all the UK iGEM teams. On the first day we listened to presentations from each team in the morning. The afternoon featured workshops covering topics such as public engagement, business startups and bioart.

The second day brought exciting news to the team. After our presentation, we had a lot of interest in our model. This led to a proposed collaboration with several other UK teams. Our aim is to each make a video tutorial on how to use various different software commonly used to model in iGEM, and to then make these videos accessible to future teams. Hopefully having introductions like this will encourage and inspire more people to embrace modelling in their projects!

This week’s been a quiet one, but it’s given some fairly substantial progress for the experimental and human practice teams.

Experimentally, the FAS module arrived from Prof Mattheos Koffas of the Rensselaer Polytechnic Institute, Troy, New York. We successfully extracted lots of it (78ng/µl!). This FAS module will help increase fatty acid synthesis in our E. coli so we get a greater yield of product. We are incredibly grateful for Prof Koffas for his generosity in donating this module, as this will be a big help to our project.

In human practice news, the team first had a meeting with James Leigh, a chemistry PhD student who has worked with the Oxbridge Biotech Roundtable. He gave the team some advice about how to organise a roundtable for a possible talk with industry we have in mind. It sounds like a lot of work, so we’re not sure if this is going to be a viable idea in the timescale we have left. There was also a meeting with Dr Catherine Rhodes, an expert in science ethics. She made us consider the rights needed to keep the Malaysian and Indonesian economies stable, and possible patenting issues with our project.

Even more economic progress was made (having Matt on the team was definitely a good decision!). We got concrete figures showing there is a link between palm oil growth and deforestation, that there is going to be a sharp rise in the price of naturally grown palm oil in the future.

This week finally brought good news in sponsorship! We would like to thank the Hain Daniels Group and Eccelso for their very generous donations to our team! They are very much appreciated and will no doubt help us travel to the European jamboree.

The experimental team had some frustrations this week (it wouldn’t be real science without them!). After waiting a long time for our primers to arrive, they had some very confusing contamination after our PCR. The next challenge is to work out where it came from! The economics team drew up a list of things that need doing by time the project ends, and there’s lots of work to do! This week they found out that ‘sustainable’ palm oil isn’t as sustainable as you’d think. You can read much more about this on our ethics pages

At the end of the week we had another iGEM social where we tried the Icelandic delicacy of Hákarl (fermented shark). As you can imagine, it tasted bad but smelled even worse! The vegetable chilli that Jess made the team was much more appetising...

This week was once again plagued with failed PCR runs, which then stalled the FadD knockout. It seems that the parameters on the thermocycler had been adjusted, leading to inconsistent results. Whilst we waited around for PCR cycles to complete, we decided to use the time to make up more stock solutions and media that we will (hopefully!) need in the near-future. That and to make ourselves feel a little more productive...

The ethics research is still coming along nicely. This week the team did more research into what policies are currently in place to protect rainforests, and looked into several case studies detailing previous instances where a synthetic alternative to a naturally occurring product has been introduced, amongst other things. We also contacted a number of companies in the hopes that one or two of them will be interested in sharing their opinions on the palm oil industry with us.

We also of course had more socials! First up was a Thai meal that lead onto a Gypsy jazz gig at a small bar in town, which the team thought was great. Then on friday we spent the evening chatting on the roof terrace of Marco’s flat, and took some really blurry photos (as you can see below)!

It’s Week 10 already? Not sure how that happened. The pressure is on (...even more) now! The FadD knockout is still proving elusive, we’re sort of losing hope that it will ever work. This week will be our final attempt to complete it. Fingers crossed!

This week we had a meeting with Eriko and Rainer to discuss our progress with the project. They gave us some invaluable advice regarding the finer details of our experimental protocols, so we will put them into practice soon and hope to see some results! We received approval to order our 2 required genes: 𝛥9 and 𝛥12. This means that we should have them with us by the end of August. It will be a push to get them ready in time for the BioBrick deadline, but we’re hopeful that we will make it.
We have exciting news regarding our future fatty acid analyses. We found a new supervisor in Dr. Nik Rattray, a postdoc working with Prof. Roy Goodacre. Nik works with Orbitrap LC-MS, and will be helping us to characterise the fatty acid profiles of our constructs (once we make them!).
Next week we will be cloning the FabA gene out of the E. coli BL21 (DE3) genome, which we extracted from wild-type this week. The primers needed for the cloning of FabA were also ordered.

The economics and ethics research is feeling close to completion, and the write up has begun! Now it will be a race to get it all on the wiki looking pretty in time. No small feat, but the ethics guys can do it!.

The project is getting increasingly stressful, which is seeing a rise in our team socialising time! Coincidence? I think not. This week the team visited Tim’s place for food on Saturday, then went to a few bars in town, our favourite of which was The Alchemist. If they serve drinks in conical flasks it still counts as research, right?

Sorry, this may be a long one! We started out the week with a big team meeting, during which we came up with a list of everything that needs to be done this week. Busy would be an understatement. We even had to seek out permission to enter the building at 7am, a first for this project!
The work was split into 4 sections: 1. Cloning and inserting ribosomal binding site (RBS) gene into pUC18 vector, 2. Cloning of FabA from WT E. coli BL21 (DE3), 3. FadD knockout, 4. LC-MS characterisation.

1. RBS BioBricks from the kit were ligated into plasmid, transformed into E. coli DH5-alpha (?), and miniprepped. (this is probably wrong. Rob or Tim clarify please)

2. We really want to BioBrick FabA because it is an important part of the fatty acid biosynthesis pathway of E. coli that is yet to be submitted to the Registry, and also we would really like to attempt to measure the kinetic properties of the enzyme to improve our model. This week we cloned the gene from E. coli BL21 (DE3) using PCR and stored in the freezer. The team are now thinking on the best ways to characterise this gene.

3. Sadly, this week we had to say goodbye to our hopes of achieving knockout of the FadD gene. 10 long weeks and nothing to show for it (other than a significant improvement in our PCR and gel electrophoresis skills)!

4. We met with Nik who guided us through the quenching and metabolite extraction of prokaryotic cells. Initially we are running reference samples through Orbitrap LC-MS (WT E. coli BL21 (DE3) cells in different media, solid and liquid fractions of authentic palm oil), which we will then compare to the fatty acid profiles of our constructs. Nik also kept us entertained with stories about how the Orbitrap technology was developed in a cellar here in Manchester, and showed us a signed mass spectrometer by Alexander Makarov himself!

This week’s social saw us at Jess’ house once more, this time for delicious Malaysian food and a film night after a particularly gruelling day in the lab. It goes without saying that everyone enjoyed letting their hair down for a bit.

Disaster struck this week! The stock of DH5-a cells from next door’s lab became contaminated with a mystery ampicillin resistant plasmid, meaning our fabA transformed cells are useless. After a week of success, it was very disappointing to find this out. But hey, these things can happen in science. Time to put this behind us, find some new cells and start again! A Skype conversation with our supervisor was very reassuring, and we’re confident we can easily pull this back.

Meanwhile, in the ethics department, the research is done and the write-up for the wiki has started! We’ve found so much we want to write about, our main issue is wondering how we’re going to do it! It’s a good job we didn’t leave this until a week before wikifreeze…

Monday also saw Divita leave to go on holiday, so Tan (well, her sister!) cooked the team a great meal to say goodbye.

This week has flown by! Partly because of how busy we were, but probably because it was a bank holiday on Monday so we’ve had a 4 day week (got to adhere to health and safety regulations!)

Tuesday finally saw the arrival of our synthesised delta 9 and delta 12 genes, so the main objective of our project can finally begin!. Without any hesitation we hydrated them, transformed them into E. coli , and miniprepped them to get a lovely stock of DNA ready for us to use for a biobrick! We also inserted fabA in to a blunt-end vector and transformed it to get more DNA to work with. Next, they were all digested ready to ligate into the iGEM submission vector on Friday. Everything was going well right up to our awful gel extraction yields, meaning biobricks didn’t happen this week! It was very disappointing, but our weekend will be spent finding ways to improve our techniques.

Once again this week we said goodbye to certain members of the team. Not one, not two, but THREE team members left us this week (obviously optimistic we’d have the project finished by now!). Now that Tim, Marco and Elsa are gone, we have 3 on the lab team and 2 on the modelling team. With not long to go until the biobrick submission deadline, here’s hoping we work well under pressure!

This week was filled with frustration, as our gel extractions refused to cooperate with us! Luckily however, Lorna soon became a gel extraction pro, and our DNA yield soon increased to workable levels.

This week we also ordered our polo shirts, and we got the (correct!) sequencing results back from our fabA gene.

It’s starting to feel like all of the effort we’ve been putting into the project will pay off! This week we managed to get our genes of interest (fabA, delta 9 and delta 12) into the iGEM submission plasmid! Sequencing showed that our genes are in fact ligated, and so we eagerly arranged a fedEX shipment to send our samples on their merry way to Boston. It’s not over though! We still needed to successfully ligate our genes into an expression plasmid. We chose pSB1C3 with a ribosomal binding site (RBS) and promoter (P) (Part BB1_ K608002). After a few seemingly failed attempts (our colonies were very small and took a while longer than expected to grow), we decided to test digest anyway because what did we have to lose (except all hope)? Excitingly however, the test digestions suggested that we’d put the genes into the expression plasmid! Time caught up with us and so we will be characterising next week. We think that the small colonies may be a result of the constitutive promoter used, and in an ideal world we’d check this theory. With one week to go though we will just have to leave it to any future iGEM teams out there!

The pressure is very much on now, the whole team is feeling it. We’re desperately trying to juggle a chaotic lab schedule, writing up the wiki and making the presentation/poster for the jamboree. However there's still human practices to be done! In addition to all our lab work, we met with a group of local environmentalists and spoke to large industries to aid us in gauging what the public and industry really think about synthetic biology.

This is it! The final stretch to wikifreeze and the pressure is hotting up. At the start of the week we inoculated our RBS/P and Delta9/Delta12/fabA constructs in FAS media containing the fatty acids we needed to feed the bacteria with. We left our samples in the very capable hands of Dr Rattray who ran our samples on the Orbitrap LC-MS so we could see if our fatty acid profile had changed. The results finally came through the day before wikifreeze, and we were thrilled to find out that our biobricks had worked!

There’s no time to celebrate though. It’s the day of wiki freeze and we’re furiously working to get our wiki finished. Talk about cutting it fine!