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Team:TU-Delft/Notebook
From 2012.igem.org
Week 22
The lab work has finally started!
Week 23
On Tuesday we ordered the constructs that we designed to let them synthesize. An exciting moment! Last check: are all the illegal restriction sites removed, is the gene in frame, is it codon-optimized, do we have the prefix and the suffix... yes? GO!
Week 24
We transformed the competent cells with pUC19 cells. This led to an equivalent amount of colonies in the positive and negative plate. Competent cells were made again. The top-10 cells with plasmids were grown in liquid LB and with a midiprep the plasmids were purified. We checked the vectors with DpnI and prefix/suffix enzymes, this led to results as expected. This week we investigated the possibility of growing yeasts and bacteria in the same culture using bacteriostatins to regulate bacterial growth (which grows quicker). We inoculated bacteria and yeasts separately in liquid YPD with tetracyclin and observed the growth curve using OD600. This led to a nice correlation of concentration and growth speed for bacteria, where yeast cells weren’t affected.
Week 25
Synthesized banana receptors came in! Competent cells where checked and no growth was detected, we decided to transform cells anyway with the banana receptor vectors (IDT smartAMP backbone). This led to a positive outcome and we picked colonies and grew them and isolated the plasmids. Primers were designed to generate a knock-out cassette for the genes ATF1, FAR1 and GPA1 with the pUG73 and pUG72 vectors. Also primers were designed around the multiple cloning site of pRS to check inserts.
Week 26
Primers arrived. Restriction of the banana receptors was performed. Vector pRS305 and two banana receptors were cut with EcoRI and PstI. The product was put on gel and the gel band with the proper size was cut out and purified. Then a ligation step was performed and the ligation product was transformed in Top10 cells. This didn’t give colonies. We tried the ligation step again and the second time it did lead to colonies. A colony was picked, grown in selective liquid media and after a day the plasmid was purified. A PCR reaction using the primers which checks the insert was performed. This didn’t led to results. A PCR reaction using pFX polymerase to amplify the pUG73 cassette with knock-out flanks also didn’t lead to results after a few tries. New primers (shorter ones) were ordered and also the pRS305 primers were redesigned to anneal ~200 bp from the multiple cloning site for optimal sequencing. Preparation for transformation of yeast cells with the knock-out cassette was performed. Further the I7 and I7-olf10 genes where amplified with a PCR reaction which flanks the gene with a kozak sequence and BamHI and NdeI sites. This facilitates the implementation of the gene between the promoter and terminator. Also the synthesized banana receptor genes where cut, gel extracted and ligated into the backbone. This led to many false positives. We performed the ligation step again and this time we confirmed the insert through restriction.
Week 27
We found out that pRS305 isn’t usable in our system since the leucine gene is completely removed in our strain and there is no origin of amplification for yeast on our plasmid. Therefore we ordered pRS315 and we contacted Mark Chee to see whether he could send us the renewed pRSII415 vectors which are also Biobrick compatible. G alpha pieces A and B arrived (the company couldn’t make it as a whole, so it is chopped up in pieces A, B, C and D). They were transformed in Top10 cells, grown and purified. When performing a restriction check we came to the conclusion a mistake had been made. The second time (same story) the bands of the restriction check where fine. New primers arrived to make the knockout cassette. A PCR with pFX polymerase to make the knock-out cassette was performed again. This time Gpa1 was successfully amplified. Yeast cells were grown and transformed with help of Daniel Solis Escalante. The cells where transformed with the gel purified Gpa1 knock-out DNA. After 2 days 3 colonies where seen and the colonies where grown in deficient media.
Week 28
Mark was at a festival here, so it was a bit more quiet in the lab.
Week 29
We got microscopy training! Mark, Sietske, Lizah, Eftychia, Marcel, Yifan and Menno were there! Daniel Lam was kind to show us around in the microscopy section of the Nanotechnology building of the university. There we performed a shmoo test (adding alpha factor in a yeast environment), but due to long waiting times the yeast cells didn’t seem shmoo-ish. The I7-receptor was ligated into the IDT vector which had a constitutive promoter (GPD) and terminator. We performed a few rounds of PCR on the yeast knock-out colonies. To see whether our method of preparing was correct we checked also with EukIF and Euk563 primers. This led to a positive result. Varying annealing conditions didn’t lead to results.
Week 30
The colonies of the knock-out where checked again using PCR on the yeast colonies. After a few rounds bands where seen as expected. For this to occur we had to extract the genomic DNA first by beading. We stored the Mat a –gpa1 –fus1 strain. We further experimented with PCR condition to make an ATF1 and GPA1 knockout cassette (to complete the universal platform study). Also we cloned the banana receptor in the pRS415 vector, but no insert was seen. Because we concluded it is hard to distinguish between a single and double cut (very close together when put on a gel for extraction), we took one positive colony (with insert) and use that as template for further cloning.
Week 31
Mark was at Berlin and Warsaw here, so it was a bit more quiet in the lab.
Week 32
A banana receptor and HKUST I7-olf10 transformed yeast cells were checked on presence of plasmid by PCR on the yeast colonies. This led to positive results. To continue we performed a transformation with the niacin receptor, another banana receptor in –far1 knockout yeast and a wildtype strain. To get a nice representation and conclusion of our knockout strain –far1, -gpa1 we’ve performed a PCR using genomic DNA of the yeast. Further we have begun cloning yeast Biobrick genes mOrange and Venus YFP to get multiple color outputs. First a PCR reaction with pFX polymerase and the biobrick is performed to add res. Enz. Sites to the gene.
Week 33
We used a technique called TOPO cloning to attach the PCR product (elongated color gene) into a backbone for easier cutting. After ligation these products are transformed. This resulted in colonies for mOrange, but not for YFP. The EGFP-TOPO is cut but very vague bands could be seen when put on gel. Therefore EGFP is amplified again in Top10 cells and isolated.
Week 34
EGFP in Topo is cut with NdeI and BamHI and ligated into two vectors, pRS415II between promoter GPD and terminator CYC1 and in IDT-smartAmp between promoter FUS1 and terminator ADH1. Transformation of the ligation product led to colonies which were checked on inserts using EGFP primers. Further, yeast cells containing I7-olf10 receptor and one of the banana receptor plasmid where grown and plasmids were isolated (using zymolase) and used for PCR reaction to see whether the receptor gene was still intact. This wasn’t the case, the insert has shortened from 1500 to ~500 nt. With the banana receptors 3 bands could be distinguished (original, somewhat shortened and the ~500 nt band). We have decided to immediately after transformation inoculate a grown colony of yeast and freeze it after a day. From there we only have to use that strain.
Week 42
Succeding better localization in the membrane!
brainstorming session
Week 23The diffusion model is complete and will be adapted according to the experiment design. The biochemical model structure has been designed, the modellers will be busy trying to fit the model to data this week and replicate the pheromone response The Bioinformatics model for checking the ligand binding is in it's design phase, we are hoping to get it ready in three weeks time
Week 31
The reduced model of the pathway is complete and the responses are similar to those obtained from the paper. The next week will be spent doing some analysis of the model and then look to incorporate stochasticity into the model
Week 32
Last week the structural modeling got a nice boost! A member of the SARA institute helped us out in setting up Virtual Machines to execute our Molecular Dynamics Simulations. With the results of these simulations, certain point-mutations in the DNA sequence of the receptor and specific chemicals, we will be able to determine how the ligand will be docked in the niacin receptor. From there, we might be able to engineer a receptor with higher affinity for our ligand.
Week 37
Modelling wiki pages under construction!!
Week 41
Getting a Gold Medal from the European Jamboree in Amsterdam we are back to work! Our Snifferometer starts getting form!
Week 43
Before the 2nd wiki freeze:
Snifferomyces working: check
Snifferomyter working: check
Research around ethics done: check
wonderful!
Week 22
2-6-2012 Eftychia, Yifan and Mark representing our group partipate at De Nacht van Kunst & Wetenschap!!! It was great experience. We had 3 games: "smelling game" where people had to guess each odor and write down their predictions, the "lego receptor" showing how the receptor can match only with a specific molecule and one more game showing the fluorescence produced when a compound comes in contact with yeast. It was so exciting that more and more people were willing to know more about our project!
Week 23
This Monday Esengul, Marcel and Sietske traveled to the south of the country to go to the opening of the Biobased Kidshouse at the Floriade. We did a workshop with the children where they could make a necklace with their own DNA in it. It was a very, very rainy day but the faces of the children when they saw their own DNA precipitate, was all worth it!
Week 28
Our first real week! We are working on our Llowlab plan, which is a part of Lowlands where the theme is 'FASTEN YOUR FUTURE!' it is a floating laboratory where Lowlands provides a stage for sustainable initiatives, new techniques and vigorous discussions. So we are going to show them Synthetic Biology! And with a estimated amount of visitors at 45.000 for Lowlands, it must be a big success! For more info on Llowlab you can go to: http://www.llowlab.nl/
Week 29
Our second summer holiday week of iGEM! We are trying really hard to get enough funding and having a lot of video-moments with Frank Theys.
Week 30
Week 31
Check out the logo that Lizah prepared this week for our team that will participate in LlowLab!!
Week 32
We are really working on our Llowlab project now! It has to be finished since we are leaving on thursday next week. It's a lot of sawing, sanding, painting! On friday we had a nice dutch friday-afternoon-drink, we played darts, had a drink and really enjoyed ourselves! You might have seen some nice pictures on facebook, like the dart the Isabelle threw in the bulls eye, unfortunately her next to throws ended up next to the dartboard..
Week 33
There is a little bit of stress now ending the Llowlab game! Some mistakes have been made with painting and the spray paint stopped while working on the last few blocks! Wednesday Menno and Mark were working on the game until half past 8! But it all turned out well and thursday at noon we'll leave at noon to Biddinghuizen, were Llowlab will take place.
Week 34
The fun is over, well, Lowlands is over. We think we made a good impression at Llowlab. We introduced many people to synthetic biology by offering a crash course supported by the game we designed and the T-Shirt. Now back to the lab to get evidence of the nice story we told.
Week 43
Inteeviewing Doctors about our TB diagnostic tool.
