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Revision as of 10:17, 7 October 2017

Croc'n Cholera
iGEM UPS-INSA Toulouse 2017

Notebook

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

1 2 3 4 5 6 7
8 9 10 11 12 13 14
15 16 17 18 19 20 21
22 23 24 25 26 27 28
29 30 31 1 2 3 4

February

29 30 31 1 2 3 4
5 6 7 8 9 10 11
12 13 14 15 16 17 18
19 20 21 22 23 24 25
26 27 28 1 2 3 4

March

26 27 28 1 2 3 4
5 6 7 8 9 10 11
12 13 14 15 16 17 18
19 20 21 22 23 24 25
26 27 28 29 30 31 1

April

26 27 28 29 30 31 1
2 3 4 5 6 7 8
9 10 11 12 13 14 15
16 17 18 19 20 21 22
23 24 25 26 27 28 29
30 1 2 3 4 5 6

May

30 1 2 3 4 5 6
7 8 9 10 11 12 13
14 15 16 17 18 19 20
21 22 23 24 25 26 27
28 29 30 31 1 2 3

June

28 29 30 31 1 2 3
4 5 6 7 8 9 10
11 12 13 14 15 16 17
18 19 20 21 22 23 24
25 26 27 28 29 30 1

July

25 26 27 28 29 30 1
2 3 4 5 6 7 8
9 10 11 12 13 14 15
16 17 18 19 20 21 22
23 24 25 26 27 28 29
30 31 1 2 3 4 5

August

30 31 1 2 3 4 5
6 7 8 9 10 11 12
13 14 15 16 17 18 19
20 21 22 23 24 25 26
27 28 29 30 31 1 2

September

27 28 29 30 31 1 2
3 4 5 6 7 8 9
10 11 12 13 14 15 16
17 18 19 20 21 22 23
24 25 26 27 28 29 30

October

1 2 3 4 5 6 7
8 9 10 11 12 13 14
15 16 17 18 19 20 21
22 23 24 25 26 27 28
29 30 31 1 2 3 4

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

You can read the result of his testimony here.

04-20: 13th brainstorming

Week 17

04-23: First drafts of Sobki

04-26: 14th brainstorming

Week 18

05-02: 15th brainstorming

Week 19

05-11: 16th brainstorming

05-13: Paul & Margaux officialy joined our team!

Week 20

05-17: 17th brainstorming

05-18: iGEM distribution kit received

Week 21

05-25: Parts design day at Brice's

Week 22

05-28: Official announcement of our subject

We officialy the subject of our project on social media!

05-31: Exposciences

We took part to the Exposciences event.

06-01: Interventions in schools

Experiments

Design and orders

  • Design of the gBlocks and launching of the synthesis order for IDT
  • Design of the PCR oligos

Design and orders

  • Design of the gBlocks and launching of the synthesis order for IDT
  • Design of the PCR oligos

Week 23

06-06: Meeting with the CEO of Sunwaterlife

You can read the result of his testimony here.

06-08: 18th brainstorming

06-09: Intervention in school

Experiments

Taking care of V. harveyi

  • 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

You can read the result of her testimony here.

06-20: Cholera survey

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

You can read the result of his testimony here.

06-30: First steps of our ethical matrix

06-29: 21th brainstorming

06-30: Tropicool!

Experiments

Cloning VhCqsA

  • Amplification of the IDT gblocks and optimisation of the PCR cycle condition for VhCqsA part
  • Digestion of the PCR amplicons and ligation with the appropriate cloning vector.

Assembly of V. harveyi gene circuit : starting the sub-cloning work

  • Amplification of the IDT gblocks and optimisation of the PCR cycle condition for VhCqsA part
  • Digestion of the PCR amplicons and ligation with the appropriate cloning vector.
  • Cloning attempt #1 and #2 to sub-clone  Vh1, Vh2 and  Vh3 in their respective vector.  

Week 27

07-06: 22th brainstorming

07-07: Delft European iGEM Meetup

Experiments

Cloning VhCqsA

  • Optimization of PCR cycle
  • Cloning of VhCqsA into pSB1C3 : positives clones grew 

Assembly of V. harveyi gene circuit : continuing the sub-cloning work

  • Assembly of V.harveyi gene circuit: Analysis of the transformants of V.harveyi module (attempt #1 and #2) by checking of the restriction map.
  • New attempt (attempt #3) to sub-clone Vh1, Vh2 and  Vh3 in their respective vector.  

Week 28

07-13: 23th brainstorming

07-14: Meeting with Doctors Without Borders, cholera specialist

Experiments

Assembly of V. harveyi gene circuit : continuing the sub-cloning work

  • Assembly of V.harveyi gene circuit: Analysis of the transformants of V.harveyi module (attempt #1 and #2) by checking of the restriction map.
  • New attempt (attempt #3) to sub-clone Vh1, Vh2 and  Vh3 in their respective vector.  

Week 29

07-20: 24th brainstorming

07-21: Parisian meetup

Experiments

Expressing VhCqsA (2nd)

  • Production of C8-CAI-1 in minimal media, extration on LLE dichloromethane and analysis on NMR (fail)
  • SDS page fo the protein overexpression of our clones (fail)

Assembly of V. harveyi gene circuit : A new strategy

  • Amplification of the IDT gblocks, digestion of the PCR amplicons.
  • Amplification and digestion of the sub-cloning vectors.
  • Ligation and transformation in E. coli STELLAR competent cells (attempt #8).

Week 30

07-23: Skype with the iGEM team of Greece

07-27: Meeting at TWB

You can read the result of their testimony here.

07-29: Team building in Pyrenees

Experiments

Expressing VhCqsA (3rd)

  • 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)

Week 34

08-24: 26th brainstorming

Experiments

Confirming VcCqsa clone + Expressing VhCqsA (5th time)

  • Transformant were good
  • 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.
  • Preparation of the clones for sequencing.

Week 38

09-21: 30th brainstorming

09-22: Meeting with UNICEF

You can read the result of Alama Keita's testimony here.

Experiments

Bioluminescence assay

  • 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)

Week 40

09-05: 32th brainstorming

09-04: High school practical work

Week 41

Week 42

Week 43

Week 44