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Team:Valencia Biocampus/Notebook
From 2012.igem.org
Notebook
23th July The [yeast sub-team] has obtained the E.Coli with the ligation YAP1-YEplac181.
20th July: The [yeast sub-team] has made a maxi-prep of one of the vectors because it was something unclear. They have also made the tranformation of E. coli with the result of the ligation reaction. Thebacteria sub-team has repeated the measurement of bac5 E.coli fluorescence after some irradiation tests and caring about the viability of the cells after the radiation, and it seems they need to be harder with their cultures.
19th July: The [yeast sub-team] has already done the ligation reaction with the first construcction (YAP1) and the final vector for yeast. They will start working with yeast next week!! The bacteria sub-team has designed an instrument to irradiate UV light to bac5 transformed E.coliin controlled time pulses and distances and has been testing it. Also, they have been measuring the fluorescence of aerobiosi/anaerobiosi grown bac3 transformed E.coli.
18th July: Today, the [yeast sub-team] has made a big digestion of the plasmid for yeast and the first construction. They have left everything ready for the ligation reaction. And,great news for the [bacteria sub-team!!!]; after glycerining bac1 E.coli transformed cells they have started to measure the fluorescence of the proteins codified by the different inserts constructions in the defined media. They already have achieved good results in 3 out of 5 constructions tests!
17th July: In this sleepy day, the [bacteria sub-team] has picked a colony from a bac1 strain triple groove culture in order to grow it in LB+Amp media. Moreover, they have set up cultures with the other constructions to a selective media to get it grown in exponencial phase.
The [yeast sub-team] has done the minipreps of the second construction. They have run an electrophoresis gel with the maxipreps which were done the previous day, previously digested, and the minipreps of the second construccion, previously digested as well. Maxipreps were great but minipreps were not, so that they will sequence the second construction in order to confirm the integrity of it.
16th July: All the students have had a "wiki meeting" to plan how we want to design our wiki. The [bacteria sub-team] has set up a triple groove of bac1 vector transformed cells.
The [yeast sub-team]has carried on the maxipreps of the expression vectors for yeast. They will be checked tomorrow!
15th July: The [yeast sub-team] transformed E. coli with the second construction. The cell cultures carrying the yeast expresion vector have been incubated in 15 mL of LBA.
13th July: Today, the [bacteria sub-team] members carried on two mini-preps to get Bac1 construction purified. The plasmid with the interest construction was digested using the restriction enzymes EcoRI and PstI and the cell colonies were plated by triple groove. Furthermore, glycerined cultures have been made to keep them in stock.
The [yeast sub-team] did several minipreps to check the integrity of both vectors and the second construction. Unluckily, the last one was damaged, so next week they will start from the beggining with this construction.
12th July: Today, the bacterial cultures made the previous day grew succesfully, the [bacteria sub-team] members stringed several colonies and grew them in LB+Amp liquid media. Last construction (Bac1) arrived so E.coli has been trasnformed with it using the [Transformation Protocol Using Heat Shock]and has been plated in LB+Amp plates.
The [yeast sub-team] checked the 'maxis' done the day before by running an electrophoresis after digestion with EcoRI and PstI. Also, we have grown the strains with the Saccharomyces expression vectors and the yeast second construction for the following day.
11th July: The [bacteria sub-team]has set up some bacterial cultures, they have plated using triple groove in order to get isolated colonies which would be picked and grown in a selective media (37ºC, 200 rpm in a shaking stove) the following day. Moreover, they have made new media: LB and LB-agar.
The [yeast sub-team] has performed the maxipreps of their two constructions and they were stored in the fridge.
10th July: In this sunny day, the [bacteria sub-team] and the [yeast sub-team] have made an electrophoresis using the DNA which has been digested with EcoRV over-night. The results obtained were unuseful because the enzyme EcoRV was used to introduce the insert into the vector pUC57 so the cleavage site was not present anymore (the enzyme EcoRV cuts resulting in blunt ends).
9th July: Today, the [bacteria sub-team] and the [yeast sub-team] have made several mini-preps in order to purify the DNA constructions. The next step has been to test if the mini-preps have been successful. To do it, the sub-teams have digested these constructions (using restriction enzymes: EcoRI, PstI) and they have made an agarose gel in order to do an electrophoresis with the digested DNA. After that, they have revealed the gel and they have found out that the results were unexpected, so they have decided to make another digestion, in this case over-night and using only EcoRI, and carry out the subsequent electrophoresis tomorrow.
Moreover, the [poster sub-team] have made a brain-storming to start designing the logo for the team and the have drawn the first sketches.
8th July: Today, the [bacteria sub-team] and the [yeast sub-team] have transfered our transformed E.coli colonies to liquid media to make them grow over-night to manipulate them tomorrow.
6th July: Today, the [bacteria sub-team] and the [yeast sub-team] have transformed E.coli with the different constructions using the [Transformation Protocol Using Heat Shock]and have plated them in LB+Amp plates.
5th July: Today, the [bacteria sub-team] has finished all the culture media (for each construction). The bacteria sub-team has had lunch with the yeast sub-team and we have discussed some aspects about the project. Also, [yeast sub-team] has made the experimental protocol for next week.
4th July: Today, the [bacteria sub-team] has started to make up their bacteria media (L.B. and defined media).
3th July: Today, the [modelling sub-team] has made a meeting in order to share the deduced equations and decide the definitive ones. Later, it has been decided to search information which could be useful to characterize the behavior of E. coli systems and S. cerevisiae system
21th June: Today, the [modelling sub-team] has made a meeting where the advisors have explained the bases of modelling for synthetic biology. After that, the students have been responsible of deducing the equations that characterize our project.
