Team:INSA Toulouse/contenu/lab practice/notebook/calendar/carry




Carry Characterization

June 2013

  • Week 3 (24-30 June)
    Amplification of new biobricks for cloning:
    BBa_K081008: rbs Lux I,
    BBa_I0462: rbs Lux R term,
    BBa_C0061: cI,
    BBa_R0065: pLux
    BBa_K081014: rbs RFP term,

    We tested a new method for midipreps using kits: alkaline lysis with spin columns. Results were really better in terms of samples purity. Cloning can start!!!

July 2013

  • Week 4 (1-7 July)
    The first necessary construction for carry characterization is the following: Planning of cloning and study of the available methods for biobricks assembly.

    Assembly of first biobricks thanks to the iGEM 3A Assembly method:
    - strong prom/rbs + Lux I (698 bp) in PSB1K3
    - pLux + rbs RFP term (936 bp) in PSB1A2
    Restriction of parts, ligation and then transformation in DH5 α.

    2 mL liquid cultures of 4 clones for each cloning.
    Miniprep after 8 hours of incubation. The electrophoresis of the digested plasmids with EcoRI and PstI confirmed the presence of the wanted constructions for all clones (at 689 and 936 bp). A 1,5% gel was also run as reference to confirm the assembly. Small parts like promoters had been indeed added.

    Cloning for carry characterisation keeps going. New constructions have been considered useful to study AHL diffusion.

    New round of 3A cloning:
    - strong prom/rbs Lux I + rbs Lux R term (1634 bp) in PSB1C3
    - strong prom/rbs + rbs Lux R term (991 bp) in PSB1K3

  • Week 5 (8-14 July)
    2 mL liquid cultures of 6 clones for each cloning and then minipreps.
    0,8% gel was run to confirm cloning success.
    - correct insert for strong promoter strong rbs + Lux R terminator
    - cloning failed for strong prom/rbs Lux I + rbs Lux R term (1634 bp) in PSB1C3

    New try for cloning of 08/08.
    Two new assembly with 3A method:
    - strong promoter/rbs Lux R + pLux RFP in PSB1C3
    - rbs cI term + pLux RFP in PSB1K3

    2 mL liquid cultures of 6 clones for cI pLux RFP cloning and then minipreps.
    Bacterial carpets were obtained for the two other clonings: transformation with the remaining 5 µL ligation of 09/07.
    0,8% gel: Cloning of cI failed. It seemed to have a problem with the biobrick’s size. May be the biobrick from the plate is wrong.

    Stand by because of a lack of plasmids backbones...

  • Week 7 (22-28 July)
    Verification by restriction of constructions. There are many problems with LuxI, LuxR and strong promoter.

August 2013

  • Week 8 (29-4 August)
    We decide to use a new promoter (J23119) and also luxI gene (BBa-K081008) in order to concentrate our forces on the production and diffusion of AHL into gelose.

  • Week 9 (5-11 August)
    5/08: Transformation of J23119 and BBa-K081008 in DH5-1 cells.
    6/08: Minipreps and assembly of J23119+K081008 in pSB1C3.
    7/08: Minipreps and verification of constructions by restriction. Restrictions with AvrII (in order to cut into J23119 biobrick) and PstI. We have one clone with the good restriction profile !
    8/08: Overnight culture of Chromobacterium violaceum in 4 ml of LB with only 5 g/L of NaCl. Chromobacterium violaceum can detect AHL by producing violacein pigment.
    9/08: Test of AHL production by J23119+K081008.

  • Week 10 (12-18 August)
    Several experimentations to detect AHL production:
    - Incubation of Chromobacteirum + DH5-1 with J23119+BBA-K081008 on petri dishes during 24h at 35°C, 30°C and 25°C.

  • Week 11 (19-25 August)
    Test of diffusion of AHL in LB gelose with commercial product (3-oxohexanoyl-homoserine lactone, sigma K3007).
    Incubation 24 h at 25°C of Chromobacterium violaceum in presence of different amounts of AHL: 300 nmol, 30 nmol and 3 nmol.

  • Week 12 (26-31 August)
    Modelling of diffusion of AHL into LB solid medium with the results of Chromobacterium experiences.

September 2013

  • All month
    Modelling of diffusion of AHL into LB solid medium with the results of Chromobacterium experiences.

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