Week 1 (15th June – 21st June)
Bootcamp
This is how it begins. The iGEM bootcamp at UCL is intended to prepare us for the roles we are going to play this summer. We get to talk to some of the founding members of iGEM and meet two other iGEM teams from London, from the Biohackspace in Hackney and Birkbeck College.
Monday 15th
We met at 09:30 in the lab. It is the first time we could all meet each other after our exams. The team members from the Biohackspace and Birkbeck were there as well. After the briefing on lab safety and other things we started with a SpeI and PstI digestion using the backbones from the InterlabStudy 2015:
J23101,
J23106,
J23117
.
As an insert we used an RFP containing vector from the iGEM 2014 distribution. We confirmed the digestions through an agarose gel electrophoresis and went for a break in the sun. Afterwards we performed the ligation of the insert and the backbone.
In the afternoon we had a skype meeting with Randy Rettberg from the iGEM foundation and learned something about the spirit and history of iGEM.
Tuesday 16th
We split up into two groups to transform bacteria with the ligation reaction from yesterday. One group was using electroporation and the other chemical transformation. We incubated the bacteria overnight.
For the afternoon we split up into different groups preparing us for the individual roles that we will take in the team.
DIYbio
We discussed about the principles of DIY biology and the barriers preventing people from participating in biology. We were inroduced to the concept of the microcontroller Arduino.
Software & Automation
We talked to Synthace founding member Rob Stanley. Automating lab work cannot start early enough! We later talked to computational biologists about software methods for modelling biological systems.
Extralab
Wednesday 17th
This morning we discovered that the RFP insert did not really fluoresce. At least not in red. We picked colonies to grow overnight in order to check whether something went wrong in the ligation or if the biobrick was not working. This could be done on Thursday through an agarose gel of the recombinant DNA.
In the afternoon we went back into our groups from yesterday.
DIYbio
We headed for the Biohackspace in Hackney and started doing manual. We 3D printed the parts and soldered everything together to make a spectrophotometer. Unfortunately, it didn't quite work. But being in the Hackspace was definitely worthwhile.
Software & Automation
We explored how the iGem wiki works which was more than this one sentence indicates.
Extra Lab
Thursday 18th
We made a mini prep of our bacterial cultures and digested the final plasmid again, this time with SpeI and XbaI to check the insert. We then did the anticipated gel-electrophoresis. The result was that the ligation worked as we got the expected bands on the gel. Our conclusion to why the fluorescence did not work is that something was wrong with the biobrick...
DIY bio
We went to the Hackspace again and finished the sensor for the photometer and measured
Interlab study meeting
We had a skype meeting with the head of InterLab Study, Jacob Beal, and had a chat about this year's interlab study.
Friday 19th
Mini Jamboree!
We have organised a mini jamboree in collaboration with Birkbeck iGEM and Biohackspace iGEM teams. We have invited people from the London synthetic biology community as well as the UCL Academy iGEM team to attend the jamboree. Each team presented what they did during the bootcamp.
Saturday 20th
Sunday 21st
Week 2 (22nd June – 28th June)
Monday 22nd
Tuesday 23rd
Wednesday 24th
Hackathon part 1
The beginning of our actual project. Before today we knew that our project would be about the mind gut axis which means trying to tackle mental health problems with engineered probiotics. Today we made an actual list of effectors and promoters that we want to use in our biobricks. It was brainstorming mayhem, but we cut down our list to about ten candidates from each category for which we are going to collect more information for tomorrow.
Thursday 25th
Hackathon part 2
We finished our list today and had a lengthy vote on the g Blocks to order for our biobricks. Our main criterium for now is the likeliness for the gene products to yield useful data. Here is our list for the genes and promoters that we are starting to work on next week:
- Effectors
- TPH1 gene with an adrenaline-sensitive promoter,
BBa_K554001.
TPH1 converts tryptophan to a precursor of serotonin.
- TPH1 gene with nitric oxide sensitive promoter, BBa_K381001
- naked sequence for GAD, which codes for the enzyme glutamate decarboxylase which produces GABA.
- naked sequence for KAT (kynurenine aminotransferase), an important enzyme in serotonin metabolism
- naked sequence for Cholin Acetyltransferase, which makes Acetylcholin, an important neurotransmiter
- Promoters
- biobrick of the adrenaline sensitive promoter BBa_K554001
- biobrick of an osmotic stress sensitive promoter BBa_R0082
- biobrick of the nitric oxide sensitive promoter BBa_K381001
- biobrick of a pH sensitive promoter BBa_K318512
- Constructs
- An estrogen induced construct that is not yet fully characterized.
We are going to transfect HeLa cells to model a pathway in mammalian
cells which is central to our project as it involves bacteria interfering with humans
- An anti-sense Tryptophanase construct. We want to use this to control the
tryptophanase expression which would allow us (or a cell) to regulate serotonin expression.
Friday 26th
Saturday 27th
Sunday 28th
Week 3 (29th June – 5th July)
Monday 29th
This week we finally managed to start the proper lab work! In the morning prepared agar plates with ampicillin/chloramphenicol. Then we carried out transformations of four promoters from the 2014 distribution that we plan to use for characterization of our parts:
- BBa_K554001 FlhDC promoter)
- BBa_R0082 (OmpR promoter)
- BBa_K144300 (blue light activated system) we got from our friends from Aalto-Helsinki team
- BBa_K864400 (PTac promoter)
+ DH5 alpha competent cells control
In the meantime some of us were cleaning and organizing our new lab to make it ours and pretty! We will post a picture once it's ready :)
We also tried to autoclave our tipette pits but they didn't quite like the autoclave!
Tuesday 30th
In the morning, we have checked the plates and obtained some pretty colonies!
We have inoculated 4 colonies per plate to be incubated overnight at 37C
In the afternoon, we planned the assembly of our BBa_J23100-BBa_B0034-TPH1-BBa_B0015. We have digested the gBlock1 with EcoRI and gBlock2 with PstI. Then we have performed the Gibson assembly of digested gBlock 1, gBlock 2, and linearized pSB1C plasmid, followed by the transformation.
Wednesday 1st
Bifidobacter
There was only one colony on our plates from the Gibson assembly! We suspect that this is because the overlap between the linearized pSB1C3 backbone and our synthetic constructs was not long enough. We plan to try the Gibson assembly of two gBlocks alone tomorrow, followed by ligation to the plasmid backbone. We will see if this works better!
We have also purified the DNA from the transformants of four promoters from Monday. Some parts from the kit were missing, so we spent 4 hours on making a miniprep! This is bad!
Interlab Study
In the afternoon, we have transformed the BBa_I13504 part from the 2014 distribution which we plan to use for our InterLab study.
Entrepreneurship
We are becoming entrepreneurs! Our vision is to commercialize our probiotics in an easy and accessible product to make sure anyone can benefit from our awesome engineered bacteria.
Our product of choice is chocolate bars! They will deliver the needed probiotics in your gut whilst enjoying a delicious and high quality product.
Thursday 2nd
Bifidobacter
This morning we have repeated the Gibson assembly of TPH1 gBlocks. This time we have decided to just assemble the two gBlocks using the assembly kit. Then we digested them with EcoR1 and Pst1, performed PCR clean-up using Wizard SV gel and PCR clean-up system, and ligated the assembled construct into the linearized pSB1C3. We finished the day by doing the transformation of DH5alpha cells.
Interlab Study
One colony was picked from the transformation of the 2014 BBa_I13504 part and we made an overnight culture.
Wiki Design
We had a first talk about the design of our wiki and came to the conclusion to play around and figure out the perfect design through trial, error and further discussions. But fortunately, the wiki is starting to take shape.
Entrepreneurship
We are finalizing the details of our product and business model, today we had a very intense brainstorming session, but we can proudly say that we have set the base for our new brand.
Friday 3rd
Bifidobacter
Bad news: Our colonies did not grow again
Good news: We finally run the diagnostic gel of our promoters and they all worked perfectly
Modelling
We made our first model for a pathway with COPASI
Lab
Entrepreneurship
Our business model is finished, now it’s time to design a pitch and start attracting investors.
Miniprep
Diagnostic digest
Week 4 (6th July – 12th July)
Monday 6th
Bifidobacter
As the Gibson Assemly refused to work for the second time, we had adapted a new strategy. We want to subclone our TPH1 biobrick that we already have in PSB1C3 into the BBa_K314103, an IPTG-inducible expression casette. Luckily, we already have a prep of that part from our last year's team!
Today, we tried to run the PSB1C3-TPH1 on a gel, extract it, and ligate it to the backbone containing the expression casette. Unfortunately, it turned out that the issues we had with the freezer last week killed our restriction enzymes and we did not obtain an expected band on the gel to extract!
Tuesday 7th
Bifidobacter
We are not giving up. Today we have executed the Monday's plan and transformed our cells with what we hope is a TPH1 ligated into IPTG-inducible cassete. Let's hope for some colonies tomorrow!
Wednesday 8th
Mind the Gut: Effectors
had a look at their TPH1 plates. Nothing has grown, again! We are deciding to change our strategy from gel extraction to the 2A assembly! We will subclone the TPH1 into the PSB1A3 backbone today and attempt transformation again. If we succeed with that, we will subclone it into the expression cassette on CAM-resistant backbone in the beginning of next week.
Improvement of Biobrick
After the RFP cultures did not grow on the Ampicilin medium we made made a new culture on chloramphenicol because we thought it might be a mistake in the registry. However, they did not grow on that plate either. A liquid control without antibiotics worked well, so at least we know that the cells (initially) are alive.
We think that the the transformation did not work. Maybe there is a problem with the DNA too.
We used the liquid cultures of the GFP we made on Tuesday to make minipreps and to use them with the promoters from the interlab-study.
High School Collaboration
The collaboration we have with UCL-academy is taking shape. We introduced them to the basics of wiki-design, modelling and let them help us with the ligation and subsequent transformation of the GFP constructs we made this morning.
Wiki
Little changes to the wiki. We managed to get a rough project description onto the wiki.
Thursday 9th
Bifidobacter
Some pretty colonies!!! Let's inoculate the overnights!
Friday 10th
Weekend 12th-13th
Week 5 (13th July – 19th July)
Monday 13th
We started the week with the meeting with Vitor Pinheiro, who gave us some great tips on our cloning schedule and strategies as well as on directed evolution systems that we could use. During the meeting we also came up with an excited idea of using synthetic amino acids as precursors for neurotransmitter synthesis!
Bifidobacter
There wasn't much time for lab work left but we managed to do some preps and set the overnight digestions
Interlab Study
We inoculated cultures with the GFP gene in liquid cultures for the Interlab Study. The cultures were left to grow over night. We also prepared plates with kanamycin which we are going to use for 3A assembly of our promoters and effectors. We made a miniprep of another GFP culture from last week
Tuesday 14th
Interlab Study
We used the cultures we inoculated yesterday for making a miniprep. Just to be safe we had made two cultures and after figuring out the quirks of the nanodrop it turned out that one had a DNA concentration more than twice that of the other. We digested the minipreps and the promoters from the Interlab Study with Xba1, Spe1 and Pst1 to ligate them together in the next step. We checked if the digestion of GFP (Interlab Study) worked but we could not see any bands so we'll try again tomorrow with an Ethidium Bromide gel and use more DNA. We started the ligation of promoters to GFP anyway, transformed and let these cultures grow overnight.
Bifidobacter
We have checked the concentrations of preps from yesterday and they were really low! The diagnostic digests confirmed that there was no DNA in our preps. In the mean time we decided to do the gel extraction od Ptac and GFP we got from our other team members. We digested the Ptac with S and P and GFP with X and P and run it on the gel. The Ptac was there, but GFP wasn't. 
Wednesday 15th
Interlab Study
We ran the same digestion as yesterday but this time with lots of DNA and a ton of Ethidium Bromide and it worked!!!
We could see the bands we expected in one of our samples (second well from the ladder). The other one seemed not to be digested. Now we are going to stick only to the good one. We transformed cells with the ligation from yesterday and let them grow overnight.
Mind the Gut: Promoters
We also started working on the promoters of our actual project Gad-A+RBS, Gad-A and PyeAr digested them and ligated them into a backbone with Cam resistance.
Mind the Gut: Effectors
Today we have decide to clone our TPH1 biobrick into the expression cassette using three different approaches: 3A assembly, gel extraction, and amplified insert assembly. We were not able to get hold of the kanamycin backbone we needed for subcloning the expression cassette, but we succeeded with two other approaches and managed to get to the stage of PCR/gel extraction clean ups.
Thursday 16th
Public Engagement
We have contacted many people involved in research, mental health charities and artists who could be interested in our project. Today we got a lot of feedback. We want to use their input to start off our public engagement part of the project. It is starting to take shape...
Interlab Study
The cultures for the interlab study grew (yeah!) and we inoculated them to liquid cultures and let them grow over night.
Mind the Gut: Promoters
We transformed cells with our promoters (Gad-A+RBS, Gad-A and PyeAr) in chloramphenicol resistance.
Mind the Gut: Effectors
We have done ligation and transformation of both gel extracted- and PCR amplified- TPH1 with PSB1C3 carrying expression cassette. In order to maximize the chances of success, we have carried each step according to long protocols, including dephosphorylation of backbone and heat inactivation of both restriction enzymes and T4 ligase. Because of that, we have only managed to finish with the transformation by the end of the day!
Friday 17th
Mind the Gut: Promoters
The cultures we transformed with our own promoters grew and we now need to check whether some have actually taken up the promoters or whether they just self-ligated. So we miniprepped them and wished them a nice weekend!
Mind the Gut: Effectors
We have repeated the mini preps and diagnostic digestions of TPH1 in PSB1A3, but no success again! :( What happened to our inserts?
Entrepreneurship
We had an end of week talk and shared some ideas about the entrepreneurship part of our project finding out more about regulations to consider in the retail of probiotics some of which seem a bit strange to us, so far...
Weekend 19th-20th
Week 5 (20rd July – 24th July)
Monday 20rd
Mind the Gut: Effectors
We have picked colonies of our TPH1 in the expression casette and transformed some new parts: BBa_J04450 on PSB1K3 from the distribution that we will need for amplification of kanamycin-resistant backbone, and our antiTNA silencing RNAs that arrived from IDT!
Human practices
This afternoon we went to the Dragon cafe, a creative space hosted by the Mental fight club, and met with some amazing people to discuss how we could present our project to the public, particularly to those who have experienced psychological problems or are interested about them.
Tuesday 21st
Mind the Gut: Effectors
We got some pretty red colonies of the BBa_J04450, but no colonies for antiTNA :( We will retry with overnight ligation at 16C to increase ligation efficiency. We have also overrun the gel confirming out TPH1exp biobrick...what an unlucky day
Mind the Gut: Effectors
Wednesday 22nd
Finally!!!! We have our second biobrick ❤ ❤ ❤ : the composite part consisting of BBa_K413103, BBa_B0034, TPH1 and BBa_B0015:
We have also got a confirmation for successful transformation of BBa_J04450 from the registry:
Thursday 23rd
Friday 24th
Weekend 27th-31th
