The first week in the wet lab started with the preparation of materials needed in future experiments. We made growth medium and agar plates containing antibiotics, which will be used to culture bacteria. Furthermore competent cells were prepared with the CaCl2 – method. We also resuspended the DNA of the biobricks necessary for our project as described in the iGEM guidelines. We managed to perform the first transformations with the competent cells and the resuspended DNA and thus concluded that the competent cells were functional.
This week we wanted to purify the plasmid DNA from the bacteria grown in the first week. To do this we inoculated growth medium with antibiotics and a single colony. These cultures were incubated overnight at 37°C followed by a minipreparation to purify the DNA from the bacteria. Due to the low DNA yield we decided to repeat the minipreparation. This repetition succeeded in producing higher DNA concentrations.
The goal this week was to bring different biobricks together in one plasmid (=ligation). We started with restriction of the biobricks that had been miniprepped the previous week. Some biobricks were successfully cut by the restriction enzymes whilst others weren’t. We performed gel purifications on the restricted fragments. However this didn’t result in high enough concentrations to start the ligation of biobricks.
This week we mainly focused on performing restriction of the biobricks (according to the standard assembly method this time) and gel purification of those restricted fragments. Most restrictions worked nicely but gel purification almost never resulted in measurable concentrations. We also transformed and miniprepped 2 new promoters we want to use. Furthermore, the primers we ordered from Eurogentec have arrived and we started with the PCR of INP, MelA, Ribolock and the 4 IGEM vectors.
This week we mainly tried to get enough DNA for performing restrictions. Since the miniprep kits usually give lower concentrations than the CTAB method, we decided to switch to the CTAB method. This method takes a little bit longer than minipreparations with a kit, but we expected to get higher amounts of DNA. However, this was not the case here. Since Thursday we also noticed the agar plates seemed to be contaminated and we decided to start all over again.
This week we started with minipreparations of the promoters and GFP that were transformed in TOP10 cells last week. This time, the CTAB method worked very well, resulting in high DNA concentrations. Due to this we were able to perform restrictions and ligations. Unfortunately we only found false positive colonies. Next week we hope to get beter results by switching back to the 3A assembly method.
We did multiple minipreparations and restrictions this week. The results of the restriction purification were bad. The high point of this week however was the ligation of two biobricks; the BAD promoter and GFP. Next week we will continue to ligate the other biobricks with the same protocol.
We concentrated on the basic ligations of 2 biobricks with the new technique that had been successful the week before. We also ligated all the promoters to GFP and transformed them into bacteria that are capable of taking in arabinose/lactose.
Week 9 and 10
The basic ligations were nearly finished so during the ninth week we started ligating 3 biobricks to eachother. We also measured the amount of fluorescence the promoters, ligated to GFP, produced after being induced.
This week we started ligating our biobricks into the chloramphenicol vector.Due to the successful ligation of HybB to GFP we were able to test the promoter strength.
The iGEM Wetlab team spent the final days of the competition trying to get the last biobricks ligated into the chloramphenicol vector.
The first week after the wiki freeze we tested multiple waters and concluded that deionised water showed the least fluctuations in its ice nucleation temperature. We also started a new set of experiments to further characterize INP and AFP.
We did more experiments to characterize INP and AFP. Qualitative and quantitative measurements were performed, the results of these experiments can be found on our results page. Finally, stocks of all different strains containing our biobricks were created.
We installed the Matlab software and Simbiology toolkit, we watched some tutorial ‘webinars’ from Mathworks. In a meeting with the advisors, we learned more about the purpose and basics of modeling in synthetic biology. After downloading the projects of previous year teams of the K.U.Leuven to understand the inner workings of their model.
We made our own model in Simbiology of the freeze-antifreeze system. We still have to implement the kinetic parameters of the whole system to predict the outcome of the wet lab experiments. We wait for the promoters linked to GFP, synthesized in the wet lab, to check the kinetics of the promoters.
Installation of maya, an amazing 3D-visualising program, provided by iGEM sponsor the mathworks. We checked the price and normally it costs $3,251! Time to watch tutorials... Friday-evening we had a funny evening dinner with our team
Place text of third week modelling category.
These week we focused on PowerPoint animations and project description. The PowerPoint animation should make the project overview more attractive. We’re wondering which software other teams are using for animations. 3D-animations are to difficult and time-consuming while PowerPoint animations are rather simple. We also worked on project description, describing all the biobricks we are going to use and also which one we can’t use anymore because they didn’t work in the lab.
Stimulation test on each part. Good results are obtained from Freeze and Celldeath part while more works are needed to optimize Antifreeze part.
We finish the description of biobricks, and tried sensitivity analysis. For lack of some data, e.g. CeaB, some assumptions have to be made to run the model.
(1)made assumptions for senstivity analysis of freeze part;exploit parameters for luxR,cI,luxI which we know; try the model in two stimulation time.
(2)choose different initial amount of gene level, 1.0,6.0,10.0;choose different stimulation time:for stimulation, time choice is conservative, 1.0,1000.0, 5000.0 while for sensitivity analysis, it covers wider range, 1.0,100.0,1000.0,10000000.0 (3)for series reaction, use rate-limiting reaction since no enough data.
Three models have been finished.
Full model is tested with different amounts of lactose and L-arabinose to check the overall working of the system.(1)evolution of three parts (2)the effect of lac and ara.
Wiki edit (1)text upload (2)correction on subsystem page.
The whole team gave the presentation in Amsterdam.
We used matlab code to run the model, and yet this work still needed to be optimized. we assumed that the formation of AHL complex and the transcription of INP gene happen in series. Compared to Simbiology, we divided simulation time into 10 intervals so that we can apply the changeable effect of AHL complex on the INP production. In the output, we can easily find the synthesis rate of INP is accelerated and reaches maximal value at around 5 hours.
The first day we divided the work. We decided to divide ourselves in smaller teams, one of these smaller teams started working on ethics, safety and education. We started with brainstorming; we were looking for an original idea for presenting the safety problems. After finding a good concept we read several papers. From Wednesday until Friday we worked really hard on writing a text about all the dangers our organism could entail. Tuesday, the 5th of July Eurogentec and Delphi Genetics gave us an interesting presentation about their companies and what they do. Afterwards we talked about our projects and they gave us interesting insides and offered to help us when needed. We are very thankful and I hope to visit their lab in the future. Eurogentec agreed to synthesize primers and the AFP gene for us, free of charge. Delphi Genetics also agreed to sponsor us, by giving us a 96 well-plate with DG1 competent cells. Thank you for that!
When we finished writing the final safety text, it was passed on to the design team to be put on the website. Afterwards, we started working on the ethics and education part. We wanted to create something outstanding and interactive which hadn’t been done before. That’s why we came up with the idea to make a small movie about Chris and Stefan who aren’t familiar with synthetic biology and GMO’s. We formed three different groups of people to work with: youngsters (app. twelve years old), student of several universities and seniors. Uitgeverij Cascade NV. (EOS, Scientific American, Psyche & Brein) agreed to sponsor us by giving us 200 magazines which we can use as promotional material!
This week, we’ve worked on our debate. The organization of this event is not going smoothly but we will not give up! Genzyme announced that they will be a Silver Sponsor of the K.U.Leuven iGEM team!
ULB agreed to collaborate with us!! The debate is starting to take shape! On Tuesday, Jeroen and Tom first went to Antwerp to collect the 200 magazines of 'Uitgeverij Cascade NV.' and afterwards they drove towards Gosselies, next to Charleroi. There, they received DG1 competent cells and promotional material from Philippe Gabant of Delphi Genetics. Thrombogenics announced that they will be a Jamboree Sponsor of the K.U.Leuven iGEM team! On Friday we had contact with the Edinburgh iGEM team and hopefully we will send them the INP-gene in the beginning of next week! After the weekly team meeting, Tom went to Genzyme in Geel, to collect 100 bags and 100 gimmicks, Thanks Genzyme =]
On Tuesday, Raf Roelands provided us with tons of gimmicks from Eurogentec: 100 pens and lots of mints. Our goodiebags are finally starting to take shape. We also applied to be featured in the BBC documentary about synthetic biology, which would be an honour if we would be the chosen one! Our wetlab team faced major problems with the INP gene, but we will soon receive a plasmid containing the INP from Professor Steven E. Lindlow from the University of California, Berkeley and from Professor Gregory Gloor from the University of Western Ontario, for which we are very grateful. We will also send this plasmid to the Edinburgh team (or make sure that the labs also send it to them) because they also have problems with the INP gene. On Wednesday, most of us were lined up for a short introduction movie for our 'BBC film' application. The project with the children is approaching! Only two more weeks and we will make them our little scientists, let their imagination run wild! Now we're working hard on the organisation of the debate. ULB has found two professors who want to come as speakers to our debate.
We are still working on the debate. We have four speakers: Prof. Bruno André, Prof. Jacques van Helden, Prof. Filip Rolland and Prof. Johan de Tavernier. Though it is a little tricky to get a moderator. Sir Rob Heirbaut wasn' able to come on the first of September, hopefully sir Jeroen Tuytten will give us a positive response! We made a detailed program for our workshop with kids. The workshop will be next week! Also a booklet about our project has been made in English and Dutch, for the boardmeeting of the K.U.Leuven. We received lots of gimmicks from imec (see our facebook). IBM announced to be a platinumsponsor, which deserves a BIG thank you!
On Tuesday we organized a workshop about synthetic biology and DNA for 36 children (9-11 years old). After discussing with them what scientists do and what DNA and synthetic biology is, we extracted DNA from tomatoes together with them, which they found very amusing! Then we asked them to draw whatever they wanted to create with synthetic biology. Afterwards they received their own Eppendorf tube filled with the extracted DNA. Rob TV was present at the workshop and filmed the whole workshop. A preview can be seen on [http://robtv.be/nieuws/oud-heverlee/tienjarigen-ontdekken-dna] but we will upload it ourselves with English subtitles. Pictures, movies and the drawings will be uploaded soon. Furthermore Beneo-Remy and reMYND agreed to become a Jamboree sponsor. VWR agreed to be our Platinum sponsor (we even receive new sets of microliter pipettes) ! The first persons registered for our debate on the 1st of September! We also received pop-ups from the K.U.Leuven to properly dress our debate room. The K.U.Leuven furthermore donates 100 additional bags and 200 beach balls, to expand our goodie bags! We received a plasmid with the inaZ gene (form of the INP-gene) from Prof. Gregory Gloor and we will send it to the Edinburgh team on Monday!
This week we worked on the last details for the debate. We made a scheme with all the different tasks and divided them between the K.U.Leuven and ULB team members. For next week we have to print the info kits and the questionnaires. We also need to search for presents for the panel members and moderator. As soon as we know how many people will come, we can make the goodie bags. Alice made a presentation which she will give on the debate. UCB announced that they will be an Affiliated sponsor of our team ! Tom also designed the sweaters that we will wear on the debate and in Amsterdam. We’re still working on a text for the human practices (the goal of our bacteria).
This week we were very busy with the debate: Next to updating the info-kit and creating a flyer to thank our sponsors (and printing them), we assembled 150 goodie bags to give away after the debate. We also received 8 large boxes filled with free lab consumables from VWR international! Most of our time and energy, we spent on optimizing everything for the debate. The debate went well: it was very interactive, interesting and gave us lots of new idea's for our project! Especially Prof. Vanhelden had some remarks about the our safety documents, so we hope that he will send us these asap, so that we can still make some changes or at least update the text with his remarks/questions. Otherwise we'll probably implement his thoughts on our debate page.
This week we worked on the ethics and education part. We wrote a small text for the children’s workshop to put on our wiki team page. Tom, Alice and Katrien gave their opinion about how they experienced working with children, explaining what DNA and synthetic biology is. Furthermore, we made graphs from the questionnaires distributed at our debate on September the first. These results will be the starting point for our text on the wiki, pictures and movies will be added. Tom put an extended project description online, with links to the BioBricks that we used. You can also find links to additional information about AFP and INP and CeaB in nature (really interesting)! Tom also created preliminary figures for the extended project, which probably will be 'pimped' by Chris and Stefan.We started with a global outline for our presentation and poster at the Jamboree in Amsterdam. Tom also had a meeting with Hilde Devoghel, and we are invited to develop a small stand for the Children's University the 15th of October and give a workshop on the week of science and day of sciende next year. We'll for sure look into the feasibility of it.
The end is getting closer and we still have quite some work to do. Beginning this week Tom found another sponsor: Technopolis. It is an edutaining institution, we are going to give two more workshops after Amsterdam. Besides re-reading everything on our wiki, we are also started with preparing the presentation for Amsterdam and the poster. As you can see, there is still a lot of work to do!
Last week of iGEM... We continued to work on the presentation for Amsterdam. The whole team is working really hard to get everything ready before the wiki freeze.
After European Jamboree
Now the wiki's unfrozen we continued our project. We wrote a text on possible applications for E.D.Frosti and wrote a reflection on the debate organized by the Rathenau Institue during the European Jamboree in Amsterdam. Due to all the media attention, we also drew the attention of the general public: Two highschools wanted to work together with our team: One scientific class wanted to thoroughly discuss our E.D. Frosti project during their science courses and they wanted to put in in their scienceblog, which they created to explain cool scientific facts to the general public. An economy class is creating a fictional marketing model in which a company produces E.D. Frosti. The 18th of October, we went to the European Commission to interview Dr. Lino Paula about law and patents on synthetic biology. We extended our safety text: we thought about how to prevent horizontal gene transfer and additional safety mechanisms. On Friday the 28th of October, we have an appointment with the Johan Bollue who is the sports league director of the Royal Belgian Ice Hockey Federation and LTP instructor of the International Ice Hockey Federation. In this meeting we discuss the potential applications of E.D. Frosti and how it could save energy in cooling systems in ice rinks.