Team:Paris Bettencourt/Notebook/Trojan Horse/Thursday 18th July.html

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Trojan Horse

18th July


Vincent and Aude

Glycerol stock of ELS-41 and ELS-13 and MGZ1 now renamed sT004, sT005, sT006 respectively:

sT004: RP437, F+ Litmus28i_J23115-B0032-GFP AmpR

sT005: XL1-Blue, M13K07 KanR

Making electrocompetent cells from strain sT004, sT005 and MGZ1 => Bad old water => could not obtain competent cells.

Conjugation :

sT004: RP437, F+ Litmus28i_J23115-B0032-GFP + conjugate with MGZ1.

F+ comes from XL1 Blue (Ortiz paper) => F+ is tetR

Protocol :

  1. From O/N cultures Dilute both strains 1/100 , in LB

  2. Wait for OD to reach O,2

  3. Prepare 4 tubes (in BD tubes) :

  • Tube 1 = 0,5mL LB with Strain 1 (Here MGZ1) + O,5mL LB = control

  • Tube 2 = 0,5mL LB with Strain 2 (here sT004) + 0,5mL LB = control

  • Tube 3 and 4= 0,5mL LB with Strain 1 (Here MGZ1) + 0,5mL LB with Strain 2 (here sT004

  1. Incubate 2 hours at 37°C

  2. Plate 100ul for controls, 100uL, 50ul, 20ul for mixed tubes on LB antiobiotics (here tet and spec)

  3. Incubate overnight at 37°C

Aude

Miniprep pT005 (Litmus28i_J23115-B0032-GFP) pT006 (M13K07) (Thermo Scientific Miniprep Kit)

pT005 : 280 ng /uL

pT006 : 61 ng/ul

Aude Vincent Sebastian and Yonatan

Work on the model

Define the players, the processes in place (more in Modeling file)

A description...

Anne and Clovis

Miniprep (sT002, sT003, NEB+pUC18) using the Thermo Scientific Miniprep Kit

1) Pellet 5ml of liquid culture (max speed, 1 min)

2) Discard supernatant

3) resuspend the cells in 250ul of resuspension olution

4) add 250ul of lysis solution, mix by inverting 4-6 times

5) add 350ul of neutralization solution

4) centrifuge for 5 min

5) transfer supernatant to spin column

6) centrifuge for 1 min

7) discard flow through

8) add 500 ul wash solution and centrifuge for 1 min , discard flow through(repeat this step)

9) centrifuge for 1 min to remove left over liquid

10) transfer the column on a 1.5ml tube

11) add 50ul of elution buffer and incubate for 2 min

12) centrifuge for 2 min

13) Nanodrop the concentration and freeze at -20°

pT.002: 53.9ng/ul -> redo with more liquid culture, it is only a low copy plasmid

pT.003: 53.8ng/ul -> redo with more liquid culture, it is only a low copy plasmid

pT.007: 214ng/ul

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