While we were working on our project, we had connected with several other iGEM teams. We worked really well with a student team from Aachen, Germany. They had a similar topic as us, so collaboration was easier due to common ground. Firstly, we had a few conference calls with them and after exchanging our experience with already conducted research and preparations for iGEM, we worked on possible ways to solve the problems we all pointed out. Furthermore we discussed other ways of collaboration. We helped them with their Human Practises by filling out their questionnaire on lab organisation and also conducted several tests for them.
They were investigating the degree of GMO pollution in the laboratories we worked in, which is indeed a valuable problem to explore. When dealing with something with such power to transform the world as synthetic biology has, safety is very important, both in the outside world when we try to apply our ideas to everyday lives and in the lab as well. GMO pollution in the lab is an important question because it concerns our safety and the safety of other people we interact with, so we were glad to be able to participate in their study.
We conducted several swab tests on different surfaces with agar chips by placing 3 on each selected surface for 5 seconds and then placed them in petri dishes. They were incubated at 37°C and after 24 hours we received the following results:
The agar chips were maid according to the protocol they have sent us:
•prepare 250 ml of LB medium with 2% Agar (2.5 g NaCl, 2.5 g Peptone/Tryptone, 1.25 g Yeast extract, 5 g Agar),
•sterilize the black chip-mould by wiping it with 70 % Ethanol,
•fill the mould with hot LB and let it cool down for 1-10 minutes,
•use a sterile (wiped with Ethanol) scalpel to cut along the markings,
•move agar chips into sterile petri dishes with a spatula,
•store at 4 °C.
We collaborated with Aalto_helsinki team as well. We contacted them because they were working on a similar project we were working on: producing boifuel with the tools of synthetic biology. While they focused on producing propanol, we focused on producing butanol.
We kept in touch regularly trought e-mail, but also held a skype conference, where we talked about our projects and the ways we could help each other. We both ordered gene fragments from IDT and talked about that expreience and we gave each other tips on how to best accomplish other tasks of iGEM. At the time, Slovenia HS has already done some work on our Human Practices, such as presentations for students and appearences in media, while Aalto-Helsinki was just starting to prepare their Human Practices, so we talked and echanged information and ideas about that. On teh other hand, aalto-Helsinki already had experience creating wiki pages (their team won the award for best wiki in 2014), while Slovenia HS was beginning to learn, so we were glad to also be able to ask some questions about creating wiki.
We did some work for them in the laboratory as well. Aalto-Helsinki sent us one of their BioBricks (BBa_K1655001) for validation and to be able to compare their results with ours. This BioBrick is a GFP that can be fused into any protein's aminoterminal. We first attempted to validate this brick by fusing it with one of our genes (CtfB) that was already tested and proved to be functional and active.
Restriction of plasmids with CtfB and double terminator as back inserts and plamids with GFP as back vectors. The inserts and vectors were isolated from the gel and ligated.
Colony PCR after ligation of CtfB and double terminator after the GFP. Longer products are succesful.The fused construct was then ligated as a back insert into a plasmid with a strong promoter and a strong RBS (BBa_K608002).
Restriction of plasmids with GFP, CtfB and double terminator as back inserts and plasmid with promoter and RBS as back vector. After they have separated better the insert and vector were isolated from the gel and ligated.
Colony PCR after ligation into a plasmid with promoter and RBS. Longer products are succesful.
We received positive results with colony PCR and anlytical restriction, but no flurescence was detected under UV light and we were unable to determine the functionality of the fused protein with SDS-PAGE and Western blot.
SDS-PAGE of the construct containing GFP and CtfB. Gene activity was not confirmed.
Therefore, another experiment was completed. The BioBrick with GFP was ligated as a back insert into the same vector with strong promoter and a strong RBS as before.
Restriction of plasmids with GFP as back inserts and plamids with promoter and RBS as back vectors. The inserts and vectors were isolated from the gel and ligated.
Colony PCR after ligation of GFP into the plasmid containing promoter and RBS. Succesful bands are circuled.
An SDS-PAGE was again completed, but no flurescence was observed under UV light.
SDS-PAGE of construct containing GFP. Gene is active as evidend by the bands around 25 kDa.