Kick off event 19.06.13

Brainstorming 19.06.13 - 3.07.13

Coming up with different ideas for our iGEM project, considering the advantages and disadvantages and the final impact. In the end, we decided on something innovative and fun!

Start Work 4.07.13

Week 1:

PLANNING AND LAB WORK.
Planning of responsibilities and establishment of roles of all team members; starting the wiki; gathering more information on the project by doing research in literature; designing a logo for our product, coming up with a catchy slogan, and deciding on a name for the final project; designing initial experiments and learning many necessary lab techniques (some of the first lab tasks included making buffers, media, chemical competent cells, choosing biobricks for our cells, and doing transformations of the first chosen bricks.</p>

Week 2:

CONTINUATION OF LAB WORK AND BEGINNING OF MODELING.
Transforming all the biobricks and cloning to build our chosen pathways; after research in literature, deciding on 4 hydrolases : NagZ, PhoA, GusA and Aes as well as respective substrates to color them.
We encountered some difficulties during transformation of triple knockout cells with the hydrolases, so this step will have to be repeated in the following weeks with a different strain of bacteria.

Week 3-4 :

MORE LAB WORK AND MODELING.
Transforming and cloning; designing primers for the mutation of LuxR promoter to alter sensitvity; starting AHL diffusion experiments for characterization of the AHL diffusion.
The receiver cells react to AHL in liquid culture and on Agar plates (proof of principle for the receiver cells)

Week 5-6 :

LAB WORK, MODELING, UPDATING WIKI.
Working on AHL diffusion, particularly its efficacy; transforming a new strain of E.coli with the T7 polymerase to test the substrates; working on transforming the triple knockout cells; retransforming the LuxI (K805016) construct because the first one (C0061) din't have an RBS.

Week 7 Half-time:

LAB WORK, MANY UPDATES ON THE WIKI, MODELLING, WORKING ON HUMAN PRACTICES.
Great pictures for the AHL diffusion on single layer agar with GFP receiver cells; Pcons-LuxI (strong promoter) was transformed. The supernatant of the Pcons-LuxI overnight culture induces GFP in the receiver cell (proof of principle for AHL quorum sensing of sender and receiver cells);linear titration of the AHL concentration.PCR of the hydrolases for the biobricks.Do random mutagenisis of the Plux promoter for different affinities.The pLuxR mutation worked and we saw different fluorescent responses to an AHL concentration of [5uM].First Model created about the AHL diffusion on Agar.

Week 8:

LAB WORK , WIKI UPDATES, HUMAN PRACTICES, MODELING, SAFETY FORMS, ABSTRACT.
Mutagenized promoter selection by comparing the sensivities to the sensivity of the Wild-Type pLuxR promoter. Sender cell receiver cell experiment prooves that quorum sensing, as well as our sender and receiver cells work properly.We have the hydrolases as biobricks now. Clonning of the hydrolases in the receiver construct. Planning of final plasmid construct for the mine cells and receiver cells. Arrangement of hydrolases based on the results of the enzyme substrate tests. Flagging options is left by side for the moment.Abstract, safety forms and track selection are done for the deadline of 30th of August. First game grid create and analyzed. Booking of Lyon accomodation and train travel.

Week 9:

LAB WORK , WIKI UPDATES, HUMAN PRACTICES, MODELING.
Screening of mutated promoters to find one with higher or lower sensivity than the wild type, timelaps experiment of the game with the GFP construct. Searching for new NagZ substrate, cloning of final constructs, testing of the hydrolase set-up

Week 10:

LAB WORK, HUMAN PRACTISES,MODELING, WIKI UPDATES
We found some promoters with different sensivities and analyse them in the single cell analyser. Hugh sender and receiver cells experiment with 60 Agar plates to characterize the gameplay (tests with different concentrations and subtrate addition at different timepoints). Major WIKI layout updates and navigation changes. The randomized PCR mutagenisis did not work so well because we had a lot of wild type in our library. Fortunalety we find a promoter which is 10 000 times less sensitive. We will use this one and mutate it back to have less difference between both. The substrate tests had show some difficulties and the NagZ has to be recloned.

Week 11:

LAB WORK, MODELING, BIG WIKI UPDATES
We are really happy to announce that we obtain a sponsorship from Kontaktgruppe fuer Forschungsfragen We discover a leakiness in our construct and try to find the sourse to correct it. We created a "fake game" picture how the game would look like after playing if the leakiness problem wasn't there. Screening for new pLuxR mutants. Characterization experiments of the hydrolases and pLuxR mutant called G1. Materials page and avisors page on the Wiki.We send in the biobricks and ordered the T-Shirts. We finished our Video and start designing the Poster.

Week 12 : 13 Days left before the wiki freeze:

LAB WORK, MODELING, WIKI UPDATES
PhoA-His purification and SDS-PAGE. New cloning to diminuate the leakiness. Promoter screening of the new mutated promoters.Substrate tests. work on presentation , video and poster

Week 13 : 5 Days left before the wiki freeze:

LAB WORK, PRESENTATION, POSTER AND VIDEO, MODEL OPTIMIZATION
HIS tag blotting worked and we did the characterization for the gold medal, test new substrates, enzyme kinetics, work on presentation, train the talk, do a GFP - RFP game version

Week 15: We advance to the world championship

WIKI UPDATES, LAB WORK, POSTER FINETUNNING, ANALYTICAL SOLUTION FOR THE PLUXR
We tried diefferent attempts to shutdown the leakiness of the Plac promoter. Between glucose tests and negative feedback loop the loops seems to work well. This data we have to update everywhere. We also set-up a preliminary game if you do not find the appropriate PluxR promoter during the screening. Based on the feedback from regional jamboree we improve our project, wiki poster, model, human practice part and project attributions.

We thank our sponsors: