Here is a summary of what we did for our project, week by week.
You can go on our Human Practices labbook page to know more about how each event affected us to take a decision about the future of our project.
January
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February
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March
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April
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May
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June
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July
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August
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September
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October
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Week 1
Our team was being recruited. Nothing happened this week!
Week 2
Our team was being recruited. Nothing happened this week!
Week 3
Our team was being recruited. Nothing happened this week!
Week 4
01-25: Kick Off Meeting
Gathering the team members and distribute the tasks... The adventure begins!
Week 5
01-30: 2th brainstorming: on the track of crocodile antimicrobial peptides...
We’ve took charge of differents accounts of the association (facebook, twitter, email, bank account, google drive…), and made a listing of 45 potential subjects for the competition.
Week 6
02-06: 3th brainstorming meeting
We’ve selected 17 subjects that we considered the most originals, the most feasible, and the most interesting. We’ve imagined to make a dressing against nosocomial infections containing bacteria that produces antimicrobial peptides from crocodile. We’ve read that crocodile peptides are not toxic for humans at microbicide quantity.
Week 7
02-16: 4th brainstorming
Only 7 subjects left! To make a safe device, we’ve thought of switch on the dressing by using spores that can be activated with the temperature.
Week 8
02-23: 5th brainstorming
We made a list of our public engagement events. For our subject, we’ve focused our research on the originality, and we’ve noted that a similar dressing has already been made by an iGEM team before. So we imagined the use of anticoagulant molecules in the dressing. We’ve thought of a bandage for pets.
Week 9
03-02: 6th brainstorming
We’ve analysed the 7 subjects, and we’ve noted some technical issues. For the “crocodile peptides project”, we’ve raised several questions : what organisme should we use to produce antimicrobial peptides? We need to find an organism which is not susceptible to their antimicrobial activity. We need to use a special membrane to contain GMOs inside the dressing and not directly in contact to the skin.
Week 10
03-05: Team building at the restaurant
Our team met for the first time outside the INSA buildings and shared a delicious korean meal after brainstorming the whole afternoon!
03-09: 7th brainstorming meeting
Dropping of first device
Week 11
03-12: Brainstorming in a café
03-14: 8th brainstorming
Week 12
03-21: 9th brainstorming
Week 13
03-26: Brainstorming in a café
03-30: 10th brainstorming
Technical solutions
Week 14
04-06: 11th brainstorming
Second device proposition
04-07: choice of the subject
Week 15
04-13: 12th brainstorming
Week 16
04-19: Meeting with Marc Lemonnier, CEO of Antabio
Reception and storage of V. harveyi BB120 (WT) and V. harveyi JMH626 (ΔcqsA ΔluxQ ΔluxN) CmR at -80 °C.
Investigation of V. harveyi antibiotics resistances.
Week 24
06-14: Scope statement start
Third device proposition
06-15: 19th brainstorming
06-16: Skype with Greece iGEM team
Experiments
Amplifying pSB1C3
Reception of IDT gBlocks and storage
Preparative work with the cloning vectors : Amplification and stock of pSB1C3
Taking charge of the lab and of V. harveyi
Growth kinetic assay in order to determine the adequate NaCl concentration for V. harveyi growth.
Reception of IDT gBlocks and storage
Transformation of DH5α competent cells with pYFP, pDsRed, pPIZα, pBR322 and amplification of the plasmids. The plasmids are double digested with the appropriate restriction enzymes.
Week 25
06-19: Meeting with Doctors Without Borders, regional manager
We shared our survey about cholera on the social media.
06-22 Skype with NUS iGEM team
06-23: Meeting with all the team members and supervisors
Final choice of the global device purpose
06-23: First appearance of Sobki, our mascott!
Experiments
Plasmids preparation
Transformation of DH5α competent cells with pSB1C3 and amplification of the plasmids. The plasmids are double digested with the appropriate restriction enzymes.
Scale up of the preparative digestion, in order to have more DNA matrix.
Reception of the PCR primer.
Plasmids preparation
Transformation of DH5α competent cells with pGAP, pSB1C3 and amplification of the plasmids. The plasmids are double digested with the appropriate restriction enzymes.
Scale up of the preparative digestion, in order to have more DNA matrix.
Reception of the PCR primer.
Week 26
06-26: Skype with the iGEM team of Purdue
06-28: Meeting with the CEO of Sunwaterlife, building a collaboration
Production of C8-CAI-1 in minimal media, extration on LLE dichloromethane and analysis on NMR (fail)
Assembly of V. harveyi gene circuit : The first transformant
TA cloning (attempt #9): amplification of the IDT gblocks with KAPA2G polymerase , digestion of the PCR amplicons. Ligation with pGEM vector TA cloning vector.
Analysis of the transformants (attempt #8 and #9) by checking the restriction map.
The part 1 and 2 were successfully cloned in pBR322 and pGEM.
Week 31
08-01: 25th brainstorming
Experiments
Expressing VhCqsA (aborted) + Cloning of VcCqsA
Production of C8-CAI-1 in minimal media, extration on LLE dichloromethane and analysis on NMR aborted : no growth of bacteria
Step 2 : reconstruction of CqsS Rc by ligating Vh2 to Vh1 in pBR322.
Storage of the transformants with Vh1 and Vh2 in glycerol at -80°C.
Amplification, digestion of pBR322-Vh1, pGEM-Vh2 and ligation in E. coli TOP10 competent cells (attempt A)
Transformation of Vh3 (attempt #10) in E. coli DH5α competent cells
Analysis of the transformants (attempt #A and #10) by checking the restriction map.
Week 32
08-07: Skype with the iGEM team of Boston
Experiments
Expressing VhCqsA (4th time) + VcCqsA cloning
Production of C8-CAI-1 in minimal media, extration on LLE dichloromethane and analysis on NMR (fail)
PCR on VcCqsA
Step 2 CqsS Rc assembly in progress…still no success for Vh3
Amplification, digestion of pBR322-Vh1 pGEM-Vh2 and ligation in E. coli TOP10 competent cells (attempt B and C)
Transformation of Vh3 (attempt #11 to #13) in E. coli TOP10 competent cells using an alternative restriction enzyme : PstI-HF restriction enzyme is remplaced by SpeI.
Week 33
08-16: Skype with the iGEM team of Groningen
08-17: “iGEM on ice”
Experiments
Cloning of VcCqsA
From ligation to transformation : transformants grew.
Step 2 CqsS Rc assembly in progress, preparation of the conjugation
Analysis of the transformants (attempt #13) by checking the restriction map : the part 3 is successfully cloned in pSB1C3. The transformant is stored in glycerol at -80°C.
Amplification of pBBR1MCS-4 and pBBR1MCS-5 for cloning of Vh3 and for a conjugation assay.
Ligation of Vh3 in pBBR1MCS-4 and transformation in E. coli TOP10 (attempt α).
Amplification, digestion of pBR322-Vh1 pGEM-Vh2 and ligation in E. coli TOP10 competent cells (attempt D to F)
Production of C8-CAI-1 in minimal media, extration on LLE dichloromethane and analysis on NMR (fail)
Analysis of the transformants (attempt #F) by checking the restriction map : the part Vh1+Vh2 is successfully cloned in pBR322. The transformant is stored in glycerol at -80°C.
Amplification, digestion of pBR322-Vh1+Vh2, pSB1C3-Vh3. Ligation and transformation (attempt #1 and #2) in E. coli TOP10 competent cells.
Ligation of Vh3 in pBBR1MCS-4 and transformation in E. coli TOP10 (attempt β and γ)
Vh3 part validation : diacetyl production assay by NMR detection with E. coli BL21.
Week 35
08-31: 27th brainstorming
Experiments
Expressing VhCqsA (6th time) + Bioluminescence
Production of C8-CAI-1 in minimal media, extration on LLE dichloromethane and analysis on NMR (fail)
Optimisation of Vibrio harveyi JMH626 bioluminescent assay
2nd diacetyl production assay by NMR detection with E. coli BL21.
Success of the conjugation assay of pBBR1MCS-4.
Week 36
09-04: Start of our Ulule campaign
09-07: 28th brainstorming
09-08: Farewell party
09-09: Meeting with Marie-Pierre Escudié
Experiments
Expressing VhCqsA (7th time)
Production of C8-CAI-1 in minimal media, and analysis on MS (fail) extration on LLE dichloromethane and analysis on NMR (fail)
3rd Diacetyl production assay by NMR detection with E. coli MG1655.
Amplification of pBBR1MCS-4 and pBBR1MCS-5.
Week 37
09-14: 29th brainstorming
Experiments
Bioluminescence assay
Trial on the expression of C8-CAI-1 with induction on bioluminescence on liquid media : fail
4th diacetyl production assay by NMR detection with E. coli MG1655 in M9 media supplemented with xylose and pyruvate.
Transformation of E. coli TOP10 competent cells with GFP from iGEM kit
Ligation and transformation of pBBR1MCS-4 to RFP from iGEM kit in E. coli TOP10 competent cells for conjugation to V. harveyi.
Trial on the expression of C8-CAI-1 with induction on bioluminescence on plate : success
Results of the 4th Diacetyl production assay by NMR detection: a small peak of diacetyl is visible.
5th diacetyl production assay by NMR detection with E. coli MG1655 in M9 media supplemented with xylose and pyruvate.
Results of ligation of RFP in conjugative plasmid: all transformants are red. Two transformants are stored at -80°C in order to be used for conjugation assay.
Preparative work for the ligation of Vh3 in pBBR1MCS-4 with fresh material
Week 39
09-27: 31th brainstorming
09-29: European Researcher's Night
Experiments
Observation by microscopy of V. harveyi (rfp) transformant
Ligation of pBBR1MCS-4 and Vh3 at 16°C overnight, and transformation in competent E. coli (Stellars)
