Team:NAWI Graz/Notebook
Notebook
Week 0: The iGEM team TU Delft organized an european iGEM Meet-up, the team of Graz, of course, could not be missing. We sent a delegation to join the meet-up. Interesting presentations about synthetic biology and talks to other iGEM teams were a good start into a summer of lab work and opportunities for collaborations.
Week 1: 10. July - 16. July The real journey started in july, when we decided that team building would be a good opportunity to start motivated and dedicated into a summer of lab-work. That is why we made a trip to a so-called "Buschenschank", where you can enjoy very good self-made food and drinks. As the days went on, motivation increased and everyone felt they could meet the challenges ahead. During the first week, we took part in a safety training about good laboratory practice – correct waste disposal, clean and decent laboratory work. In addition to this lab safety training, we further had a safety seminar where all people who worked at the institute participated. There we got used to all institute related safety questions. Both safety seminars took place before officially starting with the lab work.
Week 2: 17. July - 23. July Due to our work in the lab, we had one person who was in charge for the safety precaution. Our “safety authorized person” organized a training for all team members including fire drill and evacuation, how to behave in case of an accident and how to render first aid. Slowly, everyone felt comfortable working in the lab. Since we were working in a large building, during the first weeks it was hard to find all the different rooms for the various devices. Besides this, we were also very happy to welcome our new lab buddy “thymio” - an open-source robot from EPFL University. At this point, we want to thank all our sponsors, who supported us with lab material and chemical substances. Last but not least, to celebrate the end of a busy semester, we organized a “Spritzerstandl” for all friends of iGEM. As a special surprise, we offered all students 3D-printed bacteriophages and microscopes made by a team colleague.
Week 3: 24. July - 30. July Finally, our primers and gBlocks from IDT arrived. We diluted a couple of primers, in fact many, many primers. After a while, all primers were ready for PCR---the first step of successful cloning. Also the biobricks we needed, provided by iGEM, were extracted from the plates and transformed into E. coli to duplicate them. Sounds easy, but E. coli is not always willing to take plasmid DNA. After some tries we finally convinced E. coli to duplicate our gBlocks and biobricks. The next and last step for this week was our Miniprep-season to get our biobricks and gBlocks in higher concentrations. Lab-work at the weekend can be exhausting, but the success of this hard work is even more rewarding.
Week 4: 31. July - 6. August This week was a very exciting one! Our robot was put into operation for the first time, a lot of work was done to install the electronics and to test the setup for the robot, the bioreactor and the arena. After finishing this, we tried to get the construction started. Shortly after we achieved a success - a milestone was reached! In week 4, we also started the interlab study and additionally, we got a new load of laboratory materials from our sponsors for further lab work - thanks again!
Week 5: 7. August - 13. August This week we performed an endless amount of PCRs, PCR-party :-), using Q5-polymerase with proof-reading activity from NEB (NewEnglandBiolabs) in order to prepare our DNA-fragments for cloning. We also had an extended 5-hour-meeting to discuss the progressing temperature-sensing-project. The fluorescence-measurement chamber could finally distinguish between active and inactive bacteria and we worked on comparing the data received from the reactor setup to measurements with a spectrophotometer. An important step for collaborations was the skype conference with the iGEM team of Chennai, India, where we talked about M9 media and its implications for bacteria cultivation when using the alkaline-induced alx-promoter. Moreover, we decided to establish wednesdays as weekly pizza-days and some of us enjoyed nature while hiking at Rettenbachklamm in Graz.
Week 6: 14. August - 20. August After many oePCRs to make Gibson-cloning more efficient, we unboxed our HiFi-Assembly Kit sponsored by NEB to perform said Gibson-cloning. Thanks to NEB for providing great chemically competent E.coli DH5α cells. Not having to make competent cells ourselves saved a lot of time. E.coli DH5α grew really well on our LB-agar plates. To make sure that only E.coli cells with our plasmid can survive on the agar-plates we added chloramphenicol. And actually we got many clones, which needed to be tested. This very “enjoyable” pipetting work was shifted to the next week. In the meantime, we were pushing for a first demonstrator of the robot in the arena using the temperature-sensitive bacteria we have received.
Week 7: 21. August - 27. August This week was extremely exciting, we examined our transformation-plates from Gibson-cloning for positive clones. For this purpose, a colonyPCR was carried out. This sounds like lots of pipetting work and it definitely is. Our main actor in this PCR-reaction is the DreamTaq-polymerase, but also many other ingredients need to be added for a successful PCR, like dNTPs, primers, the templates to test, and of course the polymerase-buffer to ensure that the polymerase feels comfortable during amplifying the DNA. And behold---we got positive clones in the form of bands in the right size on agarose gel! These positive clones were cultivated over night and the plasmid DNA was isolated by Miniprep. Finally, the DNA was ready to be sent for sequencing to “Microsynth”, which was so nice and offers us a lot of free sequencing labels. Slowly the pH-construct gets ready for expression! While the robot in the laboratory was already running fast (although controlled by the bacteria reacting to heat-shock fluorescence proteins that have been provided to us).
Week 8: 28. August - 3. September Week eight was a rather successful one. The pH-project finally showed first results - we more or less finished cloning our constructs - and we were able to start planning the experiments for the pH-dependent expression of fluorescence protein. Additionally, we pretty much finished our work on the temperature-inducible expression of Gfp with the heat shock promoter ibpa. On another front, a valuable team member left us for his semester abroad in South Korea. Starting this week, he will be performing mainly assignments that can be done over the internet. We’ll meet again at the Giant Jamboree in Boston.
Week 9: 4. September - 10. September Expression of the pH-construct posed some major challenges. First of all, buffering capacity of the M9 media made it difficult to change the pH-values in the bacterial cultures accordingly. Somehow a solution for our problems with pH-dependent expression must be found. For that reason we had a never ending meeting until late at night, which brought us to total desperation. To make lab-work more fun again we started drawing with E. coli on agar-plates harboring differently colored fluorescent proteins. Additionally, we filmed a video describing our project to be able to start a crowdfunding campaign.
Week 10: 11. September - 17. September
This week we had a great opportunity to present ourself and the iGEM project to new students at the Welcome Days organised of the TU Graz. This event is a three day long presentation of all general bac studies at the TU Graz together with representatives of all bigger student project groups. As a special surprise we tempted visitors with special petri shell pudding to our information booth. Really delicious! In addition we send our alx-promotor to our collaboration partners.
Week 11: 18. September - 24. September
After spending the summer holidays in the lab, many events and many hot pizza-days, the daily-university routines were almost back. Regular courses started again and exams had to be written.
Before studying peaks we organized an education event: We went visiting an Austrian High School to talk about iGEM in general, our project idea and synthetic biology. To make it even more understandable for the students how genetic engineering works, we performed some experiments with them like extracting DNA from strawberries.
Week 12: 25. September - 1. October
After some weeks of cloning, the results of the final cloning-work were outstanding - finally: Biobrick-Submission was the goal.
Attaching prefix and suffix to our biobricks and cloning them into our vector pSB1C3 sounds like simple restriction-cloning. In theory it is easy (especially due to Snapgene, a great, free programming tool, which helped us during the whole iGEM competition) a simulation of a whole restriction-cloning process is completed within minutes. Unfortunately the real practical part in the lab looks a little bit different - every step takes hours and many problems and pitfalls emerge. After many working steps finally a successful transformation turns up, gets cloned, after restriction-digest and ligation process but is still far from being finished. More often than after colonyPCR no clones with the biobrick in it were found. Instead only the vector without insert (biobrick) turned up. After a few night shifts in the lab we finally managed to get our biobricks, which were ready for submission. E.coli we defeat you, like we always did.
Week 13: 2. October - 8. October
A lot of thirsty fresh man had to be served at our last official Spritzerstandl, this time with “Sturm”, shortly fermented grape must, a local special in Austria. Nice and sweet tasting wine.
As chance would have it, these “Spritzerstandl” are the perfect method to introduce the iGEM competition to new students and other interested people. The first steps for an iGEM team NAWI_Graz 2018 were made this evening.
Week 14: 9. October - 15. October
The NerdNite took place! A little evening event for students and science interested people aiming on introducing kind of different topics in a humorous way while the audience kills it with a real late-bar feeling and drinking. And for the best of all we heard the best bee jokes ever this night! [please insert bee joke]
Week 15: 16. October - 22. October
We organised a real interesting event at our university. Together with doctors and postdocs of biochemistry, doctor students of medicine and a postdoc of biotechnology a representative of our team held a public discussion about the general awareness of genetic engineering and synthetic biology in general public. We had a quite nice talk about several themes like improving the general knowledge, explaining some legal developments in europe and future applications of genetic engineering. The publicity always had a chance to ask in between and got all their questions answered. In the end we served self-baked cakes and some drinks.
Week 16: 23. October - 29. October
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