Revision as of 20:31, 19 October 2016

June

Organizational work. Fundraising - selling iGEM T-shirts to obtain finances for chemicals and kits.
Meetings with different biotech companies for sponsorships. Promoting the creation of the first Bulgarian iGEM team among different universities. Making list of the available equipment in different facilities that can be used for our future project.
Project proposals and discussions.
Voting and selection of the final topic. iGEM-BG has a project! ☺

Literature observation and discussions.
Collecting and cultivating different E.coli strains (DH5α, Top10, R. gammi), making glycerol stocks. Preparing antibiotic stocks and testing the optimal working concentrations.
Getting familiar with the lab equipment in our new lab (shakers, autoclaves, MiliQ system, laminar hoods, 3D-printer, PCR machines, gel imaging system). Familiarizing lab personal with the accompanying software products. Test of basic protocols (PCR, agarose gel, electroporation, making electrocompetent cells).
Demonstrational electroporation with several iGEM vectors (encoding for different chromoproteins) for general audience. Agarose gel electrophoresis and gel fragment purification for interested visitors.

Summer break + wiki research
Design and optimization of iGEM gBlocks for our proteins (SAHS2, MAHS, CASH and RvLEAM) + all primers for cloning and for colony PCR. In silicodesign of our constructs using SnapGene viewer and SerialCloner.
Waiting for IDT orders + preparation for The European researchers night

Test PCRs and cloning via CPEC (circular polymerase extension cloning) – no positive colonies were found. Further we try to clone our constructs via SLIC (sequence- and ligation-independent cloning) but again no positive colonies were found.
Literature research and selection of novel cloning strategy that is compatible with our cloning primers – selection of OEP (overlap extinction PCR).
PCR amplification of our CDSs and the LacI-tac control fragment. OEP, gel purification of the required bands, followed by electroporation. Positive clones have been identified via colony PCR.
Clone selections. Preparation of electrocompetent Rosetta gammi cells. Electroporation of the selected expression constructs in Rosetta gammi. Clone selections.

Initial expression tests – IPTG induction + PAGE (Polyacrylamide gel electrophoresis) in Rossetta gammi and Top10 and preparation of our poster
Freeze-thawing of cells followed by cell viability assays (Top10 and R. gami). These experiments are still in progress. iGEM presentation preparation. antibiotic stocks and testing the optimal working concentrations.

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