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Team:Valencia Biocampus/Notebook
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== '''Old Notebook''' == | == '''Old Notebook''' == | ||
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'''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. | '''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. | ||
Revision as of 07:28, 25 July 2012
Calendar
Old Notebook
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.
The [modelling sub-team] has properly arranged the components of the circuit. It has been checked again and it has been proved that the circuit works correctly.
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.
The [modelling sub-team] already had all the necessary components but they have encountered that, contrary to what was thought, the TL-084 amplifier was a 14-pin amplifier (not 8) which integrated itself the four operational assembled in the previous session. Therefore, it has been necessary to modify the circuit in order to get a correct assembly. Capacitors have been also replaced because the values chosen in the previous session were not correct. Once these problems have been solved, they have proceeded to an experimental testing. The experimental verification have not work due to various failures in the circuit assembly.
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).
The [modelling sub-team] has assembled the circuit. The required amplifier is a TL-084 but this component hasn´t been available, so that operational amplifiers 741 have been used. Due to time constraints, they haven´t performed any experimental test, so the circuit has been left ready for the next session.
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.
The [modelling sub-team] has assembled and tested the circuit. Once assembled, it has been also made a circuit consisting of one LED that mimicked the behavior of GFP protein, to excite the photodiode and see its response. Using the LED as light source, the circuit worked properly. However, the intensity of the light emitted by the GFP protein is significantly lower than that gave by the LED. Therefore, they have changed the intensity of the light emitted and have found that the fluorimeter amplified very little light. To solve this problem, it has been decided to use a second stage, incorporating a new operational amplifier. Here they have encountered another problem. The value of the output noise was higher than the output signal itself. For this reason it was decided to discard this circuit and return to the initial transimpedance amplifier.
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).
The [modelling sub-team] has made a meeting to establish the steps required to construct the fluorimeter, its configuration and its components. First, it has been thought to use a transimpedance amplifier, as its principal advantage is the low noise output. Despite being the best circuit, they have tried to simplify using only one stage with an operational amplifier. Thus, in the end it was agreed to make a first circuit formed by an AO-741 in non-inverting configuration.
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. They also have chosen the components required to assemble the fluorimeter. They will need leds with an excitation wavelength and an emission wavelength similar to that of proteins (AsRed2, AmCYan1, ZsGreen1, ZsYellow1), optic filters and a photodiode.
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.
