Team:NAWI Graz/Notebook

Notebook

Week 1: 3rd of July - 9th of July

The iGEM team TUDelft 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 with other iGEM teams, were a good start into a summer of work and offered us the opportunity to form collaborations.

Week 2: 10th of July - 16th of July
The real journey started in july, when we decided that team building would be a good opportunity to start motivated 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 3: 17th of July - 23th of 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 3D-printed bacteriophages and microscopes made by a team colleague.

Week 4: 24th of July - 30th of 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 absorb 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-session, to get our biobricks and gBlocks in higher concentrations. Lab-work during the weekend can be exhausting, but the results of this hard work were worth it.

Week 5: 31st of July - 6th of 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 afterwards 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 6: 7th of August - 13rd of 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 7: 14th of August - 20th of August
After many oePCRs to make Gibson-cloning more efficient, we unboxed our HiFi-Assembly Kit sponsored by NEB to perform said Gibson-cloning. Special 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 following week. In the meantime, we tried our first demonstration of the robot in the arena using the temperature-sensitive bacteria we had received.

Week 8: 21st of August - 27th of August
This week was extremely intriguing, 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 to offer us a lot of free sequencing labels. Slowly the pH-construct got ready for expression! Meanwhile, 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).