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. Unfortunately, two thirds of our team are working on end-of-term exams
and Bachelor's theses 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 a meeting with one of our professor, who we are going
to ask about help. Finally, we've been sent to PhD Przemyslaw Nuc, who has agreed to join our team.
And then came June. We are still waiting for the agreement's acceptation.
We've started to transform E. coli with Biobricks 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.
June.
1st-4th week
We've started to trying to equip our lab. Because we study 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 forced to work on Adam Mickiewicz University.
Problems have started to rise. Due to Polish law we can't make any order as fast as we think and as we would like to.
Every single one of our orders needs to be signed by four various officials. What is more, we are obliged to contact three various manufacturers or
distributors of every product/service to get pricings of their products. Unfortunately to us, the holidays begin so we're going to encounter some serious trouble with making appointments.
July.
5th-6th week
We're shaking 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.
7th 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.
8th week
We are still waiting...
August.
9th-10th week
We're done with waiting.
Just kidding.
We feel more like office workers and lawyers than scientists.. - not kidding.
11th-12th week
We've received the first part (one third) of our order of lab equipments.
We are seeing the light in the end of the tunnel, yay!
Now, we could calmly put everything into designing our oligos and synthetic genes.
We could not start to planning the oligos until we'd been certain we would get the second tranche of the government funds.
Semptember.
11 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
We are not expecting to end the project in time, so we're redesigning our constructs.
12 week
We've received the first part of our oligos.
We are able to make the PCR of our promoters & backbones with proper resistances.
Okay, a lot of PCRs with various primers. We want to be sure that reactions go well.
A lot of agarose gel. And photos, a lot of agarose gel photos.
We are making the PCR form the AddGene's plasmids, we've prevoiusly ordered. On this plasmids we have:
1. phiC31 integrase
2. TP901 integrase
3. Bxb1 integrase
4. arabinose promoter
13 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.
14 week
Let's start the Gibson assembly! We suggest reading the RFC 57 protocol.
We're going to use the NEB's Gibson Assembly Kit and the Gibson Assembly Master Mix.
Epilogue.
It did not work. The positive control we've been supplied with by NEB did not work despite very good quality of our electrocompetent strains. We're able to transform them with any operational construct in our possesion, including the test plasmid pUC19, but the assembly of NEB's control succeeds neither with their kit, nor with our own mixture based on RFC 57 and original Gibson's papers.
We're desperate.
As shown in the gel photo, the NEB positive control incubated overnight (1) does not differ from the sambple incubated for 60' (2) - and there is no closed plasmid in them. Samples no. 3, 4 show a comparison of 60'-incubated Gibson assembly on our own mix and fragments used respectively ("pSB1A3-ara-sfGFP-tagX"). Samples no. 5, 6 show a similar comparison of a construct "pSB1A3-ara-sfGFP-R5" on NEB's original mix. Samples no. 7, 8 are ladders - 1kb and 100bp.
Last day of the lab work. We've tried to assemble the constructs with the CPEC method. It did not work either. Brace yourselves... WikiFreeze is coming.
"
