Team:Toulouse/Notebook/Calendar

- iGEM competition presentation and explanations
- Constitution of the team by interviewing different students from Université Paul Sabatier and INSA

The adventure begins for the Toulouse iGEM Team 2014!

- Thanks to weekly meetings, our team was able to discuss about new project ideas and publications.
- Many ideas were approached and debated regarding the feasibility thanks to the literature and supervisors including teachers and researchers
This is a list of the main projects:
- Let's save our trees with SubtiTree: the purpose is to use Bacillus subtilis as an optimized bacterium to detect, target the fungi and secrete fungicides to destroy the pathogen
- E. coli cancer: the concept was to create a bacterium able to colonize specifically the hypoxic regions of the tumor. The oxygen-sensitive bacterium would not be able to develop in the healthy zones
- Reduction of ruminants’methane production: the goal is to reduce the production of methane by ingesting a microorganism in the animal’s rumen. This bacterium would be capable of reducing the quantity of metabolic hydrogen and eliminating the methanogenic Archaes population

The discussion has been long to estimate the practicabilty of our final project. By the end of June, most of the ideas were eliminated but two of them remained: controlling the cancer or saving the trees. It became easily clear that SubtiTree was successfully completed whether regarding the scientific part or the ethical aspect. Indeed, our knowledge in medicine was not sufficient enough to evaluate the consequences of a bacterium injection in the human body for E. coli cancer. Therefore, the whole team agreed on focusing on a local issue as the preservation of the Canal du Midi plane trees. However, the final goal was based on the idea to allow our optimized bacteria to be efficient for all fungi infections in the environment. By this period, we evaluated our budget to approximately 40 k€ .

Preparation period for the laboratory work:

- Bibliography researches about our subject
- Cleaning the whole lab to allow good manipulations and avoid any contamination
- Inventory of necessary lab equipment and biobricks
- Summarize the iGEM protocols
- Prepare all growing media
- Preparing competent cells by optimizing protocols
- Transformation of RFP plasmid to practice

Communication and financial support:

- Increase in our communication strategy to find more sponsors specialized in environment and ecological matters
- Support of Adisseo, Toulouse White Biotechnology, LISBP, INSA Toulouse
- Organization of a weekly meeting each Friday with our supervisors to tackle any problem encountered

Weekly meeting with the instructors (06/20/2014)  Distribution of the non-scientific tasks regarding the wiki, communication, sponsorship, ethical problems, safety but also the lab work
 Discussion about the different construction parts of the project
 Need to check the absence of stop codon in fungicides sequences

Preparation period for the laboratory work:

- Ordering the list of necessary products for the laboratory and biobricks
- Checking and validation of the genes sequences
Communication and financial support
- BioSynSyS conference work: powerpoint, logo in order to present our SubtiTree project to a whole audience of scientific and researchers (http://biosynsys2014.sciencesconf.org/

Lab work:

- E. coli transformation with BBA_J004450 (pSB1C3)

Weekly meeting with the instructors (06/27/2014)  Concentration of antibiotics in media must be checked
 The transformation rate for pUC19 must be evaluated
 The transformation efficiency must be calculated regarding the quantity of DNA
 Possibility to contact the cities halls for the sponsorship
 Check the mechanism of action of each fungicide to complete the ethical part

Main activites
- Beginning of the laboratory work to create our optimized bacterium
- Research of sponsors and the communication thanks to the press

Communication and financial support:

- Participation at the BioSynSys conferences: presentation of SubtiTree
- We have put in place a newsletter system the first day of each month to keep all our sponsors and people who support us updated about the project, our financial state.

Lab work:

- Design of all the constructions needed with cloning and restriction enzymes to save some time and allow a better comprehension of our bacterium system Problem: A problem was reported in the use of the CYP. Indeed, the biobrick is not expressed properly in Bacillus subtilis strain. Therefore we had to look at the literature to find a new fungicide called GAFP-1.
- A bioreactor was conceived to reproduce the composition of the sap in the plane trees. The main goal was to identify the behavior of Bacillus strain in a medium composed of many different carbon sources.
- Competent cells and transformation practice using GFP and RFP.

Weekly meetings with the instructors (07/04/2014) and (07/11/2014)  iGEM efficiency kit does not work, we shall not use it anymore
 Organization of a timetable to check the stored and sterile equipment everyday
 Try a transformation in Bacillus subtilis

Communication and financial support:

- Our team was interviewed by many local newspapers such as 20minutes, Metronews, La Voix du Midi, La Dépêche but also by the local television channel France3 Midi-Pyrénées to present our draft to the community. Moreover, SubtiTree was approached in some radio programs like Virgin Radio Toulouse and France info.
- Support of ThermoFisher, New Englands BioLabs, Qiagen, Eurofins but also GenoToul, Labex Tulip, L’Université Paul Sabatier, CROUS.

Lab work:

- GFP and RFP digestion amplified by miniprep and gel electrophoresis
Problem: the digested GFP doesn’t appear on the gel contrary to RFP plasmid. We assumed a problem with the GFP miniprep. We tried the miniprep from the INSA Toulouse team from 2013 and the GFP plasmid was fully digested.
- Transformation and cryopreservation of biobricks BBa_K823002 (PlepA), BBa_K823003 (Pveg), BBa_K606061 (RBS SpoVG) and BBa_B0015 (double terminator), BBa_K823023 (pSBBS1C), BBa_K733013 (Pveg+ RBS) in E. coli
- Transformation of the Munich B. subtilis backbones
- Cloning of BBa_K606061 (RBS SpoVG) with BBa_K823003 (PvEG) and BBa_K823002 (PlepA)

Weekly meeting with the instructors (07/18/2014)  Transformation of the Eurofins genes
 Start assembling the biobricks for the fungicides
 Check all the cloning

Communication and financial support:

- Description of SubtiTree project and determination of the goodies for a crowdfunding campaign on Ulule website

Lab work:

- Transformation of BBA_1364002 (GAFP1) and BBA_1364003 (D4E1) fungicides genes
- Subculture of the clones for each gene
- PCR and migration on electrophoresis gel
- Transformation BBA_1364003 (D4E1) on pSB1C3 and BBA_1364002 (GAFP1) on pSB1C3
- Cloning BBA_1364002 (GAFP1) with BBa_B0015 (double terminator) and BBA_1364003 (D4E1) with BBa_K823003 (Pveg)

Others:

- Research for plane tickets and hotels in Boston for the Giant Jamboree
- New idea: analyze the plane tree sap to determine the composition

Main activites
- Finishing the cloning for the different parts of the bacterium
- Putting in place the fungicides, binding and chemotaxis tests

Communication and financial support:

- Financial supports of the Ministery (Ministère de l’Agriculture, de l’Agroalimentaire et de la Forêt) and Voies Navigables de France
- Interview with Johan Langot from Sciences Animation for a possible presentation at the Toulouse Festival of Science
- Launch of the crowdfunding campaign on Ulule

Lab work:

- Cloning BBa_K823003 (Pveg) + RFP and BBa_K823002 (PlepA) + RFP in pSBBs1C
- Test of every pSBBs vector : BBa_K823021 (pSBBS1C-lacZ), BBa_K823022 (pSBBS4S), BBa_K823023 (pSBBS1C), minipreps and cryopreservation of the best clones
- Checking of BBA_1364002 (GAFP1) + BBa_B0015 (double terminator) transformants (biobrick BBa_K1364007) and BBA_1364003 (D4E1) + BBa_K823003 (Pveg) biobrick BBa_K1364009
- Cloning of BBA_1364003 (D4E1) + pSBBS4S (BBa_K823022) and subculture of the colonies
- Cloning of BBa_K823003 (Pveg) + BBA_1364003 (D4E1) with BBa_B0015 (double terminator)
- Cloning of BBa_K823003 (Pveg) + RFP (BBa_K1364017) in pSBBS4S (BBa_K823022), subculture and minipreps
- Cloning of BBa_K823002 (PlepA) + RFP (BBa_K1364016) in pSBBs4S (BBa_K823022), subculture and minipreps
- Cloning of BBa_1364018 (Strong Promotor + RFP)
- Cloning of BBA_1364003 (D4E1) + BBA_1364002 (GAFP1) + Promotor BBa_K823003 (Pveg) biobirck BBa_K1364012 with gel extraction for BBA_1364003 (D4E1) + BBA_1364002 (GAFP1) and a PCR cleanup for BBa_K823003 (Pveg), subculture
- Cloning BBa_K823003 (Pveg) + BBA_1364002 (GAFP1) + BBa_B0015 (double terminator) on pSBBS4S in E. coli
- Transformation of binding gene with E. coli competent cells
- Transformation of BBa_K1364000, the chemotaxis gene in E. coli
Problem: the transformation worked but a cell layer appeared. A new subculture of the colonies was made as well as a new spreading on petri dishes.
- Spreading of BBa_K1162001 (EcAMP), miniprep and digestion
Problem: the digestion did not work  Issue with the quantity of DNA in the miniprep is assumed
- First experience of chemotaxis with a glucose chemoattractant

Weekly meeting with the instructors(08/01/2014)  Possible problem with the restriction enzymes regarding the digestion: new recommendations
 Discussion about EcAMP cloning which presents some issues
 Discussion about the transfer of pSB1C3 from E. coli to Bacillus strain

Communication and financial support:

- Participation at the “Passion Jeune” competition organized by a French bank foundation named Fondation Crédit Agricole Toulouse

Lab work:

- Check the cloning of BBa_K823003 (Pveg)+BBA_1364002 (GAFP1) + BBa_B0015 (double terminator) on pSBBs4S and cryopreservation
- Transformation of BBa_K823003 (Pveg) +BBA_1364002 (GAFP1) + BBa_B0015 (double terminator) (biobrick BBa_ K1364008) in BBa_K823022 (pSBBS4S)
- Cloning of BBa_K1364001, the binding gene + BBa_K823003 (Pveg) on pSB1C3 and pSBBS 4S
Problem: the band is at 1500 bp instead of 1300 bp on the gel
- Cloning of BBa_K1162001 (EcAMP) + BBa_K823003 (Pveg) + BBa_K606061 (RBS SpoVG), miniprep, PCR and digestion
Problem: the fragment of DNA is too small (149 bp) to be seen on the gel and was probably lost during the PCR cleanup. Instead of that, we used the heat enzymes inactivation at 95°C for 15 minutes.
- Cloning of Promotor + BBA_1364003 (D4E1) + BBA_1364002 (GAFP1) in pSB1C3 and pSBBS4S and cryopreservation
- Elaboration of an efficient fungicide test protocol
- Test of the fungus growth on PDA medium + test of growth for Bacillus subtilis liquid culture on PDA

Weekly meeting with the instructors(08/08/2014)  Ask iGEM headquarters if all the biobricks must be on pSB1C3 plasmid
 Use another strain of E. coli (DH5-1 instead of DH5 alpha) to have a faster growth
 Check which quantity of Bacillus subtilis is necessary to naturally destroy the fungus

Communication and financial support:

- Publication of more articles about our project in Labiotech, Développement durable (Networkvisio), Midi Libre, l’Indépendant, DigiSchool, La voix du Midi Lauragais, LURIO Addl.

Lab work:

- Evaluation of the bacterial decrease rate from 10°C to 4°C with a sporuling strain of Bacillus subtilis.
- Chemotaxis test with different concentration of glucose on a 0.3% agar.
- Miniprep of pSBbs4S with binding gene and transformation in Bacillus subtilis.
Problem: no digestion was visible on the gel  the cloning failed
- Transformation of BBa_K1374010 (Pveg + RBS SpoVG + EcAMP) + BBa_K1364012 (RBS + Gafp1 + D4E1 + double terminator), subculture
- Subculture of Bacillus subtilis clones transformed by BBa_K823003 (Pveg) + BBA_1364002 (GAFP1) + BBa_B0015 (double terminator) in pSBBs4S, cryopreservation
 Fungicide test

Communication and financial support:

- Ethics: Interview with a specialist in ethical sciences, Vincent Grégoire Delory to complete our reflexion about the ethical issues in SubtiTree project.
- Wiki: hiring a web designer, Adrien Nicod, to improve the aesthetic effect and the logo.

Lab work:

- Subculture and transformation in Bacillus subtilis of BBa_K1364011 in pSBBS4S (BBa_K823022) and cloning of BBa_K1364011 on pSBBS1C (BBa_K823023).
Problem: no clone had the insert on pSBBS1C  the cloning had to be done again - Miniprep of BBa_K1364011 in BBa_K823023 (pSBBS1C) + Transformation in B. subtilis.
- Cloning of BBA_1364003 (D4E1) , BBA_1364002 (GAFP1) , BBA_1364003 (D4E1) + BBA_1364002 (GAFP1) on BBa_K823023 (pSBBS1C)
- Transformation BBa_1364004 (in pSBBS4S in E.coli
- Cloning of BBa_K1364014 (Pveg + RBS SpoVG + EcAMP + GAFP1 +D4E1 + double terminator) in K823022 (pSBBS4S) and in BBa_K823023 (pSBBS1C), miniprep and digestion by restriction enzymes to see the size of the insert on a 1% gel
Problem: no clone had the insert on pSBBS1C  the cloning had to be done again

Communication and financial support:

- Beginning of the creation and redaction of the different parts of the wiki by the Toulouse iGEM Toulouse

Lab work:

- Cloning of BBa_K1364014 in BBa_K823022 and in BBa_K823023
- Cloning GAFP1 + BBa_K823023 (pSBBS1C) in Bacillus subtilis with a change in the protocol: pre-induction of the culture with 0.125 µg/ml of chloramphenicol one hour after the addition of DNA, and let grow for another hour.
Problem: no colony
- Liquid culture of Bacillus + BBA_1364002 (GAFP1) ; Bacillus + BBA_1364003 (D4E1) + BBA_1364002 (GAFP1) ; Bacillus + BBA_1364003 (D4E1) for future fungicide tests
- Miniprep of BBa_ K1364011 in BBa_ K823023 (pSBBs1C) and transformation in Bacillus subtilis strain.
- Fungicide test of BBa_K1364011 in pSBBs4S, Promotor + BBA_1364002 (GAFP1) + Terminator (biobrick BBa_K1364008) in pSBBS4S, Promotor + BBA_1364003 (D4E1) + Terminator (biobrick BBa_K1364009) in pSBBS4S, Promotor + BBA_1364002 (GAFP1) + BA_1364003 (D4E1) + Terminator ( biobrick BBa_K1364013) in pSBBS4S
- Transformation of a new vector PKL 190 (threonine integrative) in Bacillus strain

Weekly meeting with the instructors(08/29/2014)  Boston accommodation must be booked
 Discussion about the contamination seen on the fungicide tests : the issue seems to come from the sterile water which contained contaminants
 Objectives : chemotaxis, binding and fungicides tests have to be performed again

Main activites
- Finish the tests of the different modules
- Establish the final editorial parts for the wiki
- Design of the wiki
- Sequencing the different assembled parts of our bacterium

Communication and financial support:

- Different parts of the wiki by the Toulouse iGEM Toulouse
- Reservation of Boston accommodation

Lab work:

- Chemotaxis test and modelling
- Fungicides tests on EcAMP-A, EcAMP-B, EcAMP-C, EcAMP-D, MaxiOpA, MaxiOpB, MaxiOpC, MaxiOpD from 2ml of overnight cultures. Only with 25000 conidia with both fungi.
- PCR on pSBBS4S + BBa_K1162001 (EcAMP)
Problem: failed on both diluted plasmid and colony
- Spreading of BBa_K1364014 + BBa_ K823022 in threonine medium and in medium without threonine

Weekly meeting with the instructors(09/05/2014)

 The first sequencing was not successful  new sequencing with one primer/tube
 Chemotaxis test: increase the concentration in bacterium, try a new protocol by capillarity
 End of the binding test: IT WORKS PERFECTLY! SubtiTree has the good phenotype and presents a better binding property than the wild type Bacillus subtilis strain.
 Fungicides test: contaminants with the fungus. Need to ask for another culture of our fungi.

Communication and financial support:

- Telephone interview for an article in a French national review named Fluvial which will be published at the end of September.

Lab work:

- PCR on D4E1 colony with different primers
- Culture and miniprep of EcAMP to get more DNA + analytic digestion
- Transformation of K1364014+K1364006 in E. coli
- Transformation of GAFP1 + D4E1, GAFP1 and EcAMP+GAFP1+D4E1 on Bacillus subtilis + colony PCR + threonine test
NB : good results for the maxi operon and GAFP1 but not for GAFP1 + D4E1 CA
- Cloning of K606013+pEX_K4, 823022+PlepA_RFP, 823022+ Pveg_RFP
- Chemotaxis tests: different times of LB + agar addition in the LB medium (after 3 minutes, 5 minutes, 8 minutes, 10 minutes…) are performed  No difference of the results after spreading on the plates.
- New test of chemotaxis: capillary between two Eppendorf tubes
- Spreading of the fungi on a medium which mimics the sap

Weekly meeting with the instructors(09/11/2014)

 Try to use the GFP or RFP to follow the chemotaxis test.
 Make a new glass process for the chemotaxis capillary test.
 Possibility of contacting an INRA laboratory to get pictures of the binding test with confocal microscope.
 Sequencing the binding construction.
 Try different temperatures for the fungicide tests: 20°C, 25°C, and 37°C to reduce the fungus growth.