Lab book.
Prologue.
October 2012. Weronika came back from the iGEM 2012 Regional Jamborees. After a series of Poznan University of Technology Bioinformatics Students' Research Group meetings concerning synthetic biology Poznan-BioInf came into being.
Cold, cold November. We've participated in "Generation of the Future" programme organized by the Ministry of Science and Higher Education for students
needing financial support to take up a challenge in an international contest. And we're waiting...
...and waiting...
and waaaaaiting...
and at the end of April 2013 we've received decision that we are granted 200 000 zlotys.
May. we've officially signed the funding agreement and we're waiting for approval. Unfortunatelly two thirds of out team were working on end-of-term examination
and Bachelor's thesis so... we received BioBricks and we're waiting for decision that we can administer our funds.
That month We are looking for wet lab instructor and place, where we could spend summer. We are going to have meeting to one of our professor, who we are going
to ask about help. Finally we where send to Phd Przemyslaw Nuc, who we welcomed in our team.
And then came June. We are still waiting for agreement acceptation.
We've started to transform E. coli with biobirck form parts registry to test the efficiency of our strain and our protocols. Since now we are working on borrowed
laboratory equipments. During the tests we're looking for a suitable GFP. From now on we start to working with BBa_E0040, which exhibits bright green fluorescence.
July
1st week, 2nd week
We've started to trying to equip our lab. Because we are studying on two universities: Poznan University of Technology (where we officially belong)
and Adam Mickiewicz University, we had some problems with the administration. On Poznan University of Technology there is no wet laboratory specialized
at microbiological research. That's why we are force to work on Adam Mickiewicz University.
Problems have started to rise. Due to Polish law we can't make order as fast as we think and as we would like to.
Under all (every single one) of our orders we need four various signs. What is more, we are oblige to contact three various producers or
distributor to get valuation of their products. Unfortunately to us the holidays begins so we encounter obstructs with making an appointments.
3rd week
We shake off from the shock.
We are still trying to make an order at the biotechnological companies, so.. we are
waiting for the reply from distributors.
4th week
We are still trying to make an order at the biotechnological companies, so.. we are
gathering the signatures and forcing the offices of administration.
5th week
We are still waiting...
6th week
We're done with waiting.
Just kidding.
We feel more like administrative labours than scientists.. - not kidding.
We've receive the first part (one third) of our order of lab equipments.
We are seeing the light in the end of the tunnel, yey!
Now, we could calmly put everything into designing our oligos and synthetic genes.
We could start to planning the oligos not until we were certain, that we will get the second tranche of government fund.
7 week
This week we've isolated the chromosome E. coli strain DH5alpha. From bacterial chromosome we want to pull out:
1. arabinose promoter
2. rhamnose promoter
3. melibiose promoter
4. xylose promoter
Continously we are designing of our constructs. (redirection)
8 week
We received the first part of our oligos.
We were ably to make the PCR of our promoters, backbones with proper resistances.
Okey, a lot of PCR reaction with various primers. We wanted to be sure that reaction will go well, because
A lot of agarose gel, as well. And photos, a lot of agarose gel photos.
We are making the PCR form the AddGene plasmid, which we've ordered. On this plasmids we have:
1. phiC31 integrase
2. TP901 integrase
3. Bxb1 integrase
4. arabinose promoter
9 week
We've received the second part of oligos and synthetic genes.
We are able to make the PCR reaction of fluorescent proteins: BFP, GFP and mRFP of our design, optimize for E. coli bacterium with three
different tags, which should speed up the degradation of the proteins.
10 week
Let's start the Gibson assembly!
Protocol you can find here.
In the beginning we use the Gibson assembly kit and the Gibson assembly master mix. We've achieved nothing.
"
