Calendar

MG1655 del(EnvZ) Strain

August 2013

Week 9 (5-11 August)
In order to use this strain for light sensors characterization, we need to delete the envZ gene. For this we did a transduction of the strain with phages previously mixed with the del(EnvZ) strain from the Keio collection.
The strains from this collection have individual kan cassette replacements of non-essential genes flanked by FRT sites allowing excision using the FLP recombinase.
6/09
overnight culture of the Keio Del(EnvZ) Strain in LB and CaCl2

7/09
dilution of the overnight culture of del(envZ) strain until DO=1
a mix of bacterias with P1 phages is spread on soft LB agar for the night at 30°
overnight culture of the MG1655 strain

8/09
pick up of phage with bacterias
dilution of the overnight culture of MG1655 in LB and CaCl2 until DO=1
mix of bacterias (MG1655) with phages
incubation a night at 37°c

9/09
verification of lysis plaque
Week 10 (12-18 August)
Then we needed to remove the Kn cassette with the FLIP recombinase, in order to integrate with a selection factor Kn.

12/09
Transformation with the pFLP plasmid.
One night on LB agar plate at 30°C

13/09
Pick 4 clones and cultivate in liquid 2hours at 30°C then 3 hours at 42°C.
Dilution and spread on LB Agar.

14/09
Pick isolated clones on LB Agar, LB Cm, LB Ap and LB Kn.
Let one night at 42°C.
Right clones are Cm S, Ap S and Kn S.

15/08
Clones are ok.
Waiting for the primers to verify with PCR.
Week 11 (19-25 August)
19/08
PCR verification: ok ! The del(envZ) deFRT Kn is constructed!! Thanks to the wonderful help of Caroline Schiavon

20/08
Then in order to integrate modules in the genome with integration plasmids, the strain needs to be Streptomycine resistant for selection.
The strain has the Streptomycine resistance gene but with a mutation. With several spread on LB agar Strp, the strain can recover the resistance.
Spread on LB Str 50ng during a night at 37°c.

21/08
Several clones have grown but the right Strepto concentration was 200ng/µL.
Spread on LB Str 200ng during a night at 37°C

22/08
One clone on the plate
Subcloned the clone on spread on LB Str 200ng over the night at 37°C

23/08
Few other clones have grown
Subcloned of these clones and spread on LB Str 200ng at 37°C

25/08
Competent cells of the new strain: MG1655 del(envZ) deFRT Kn StrpR.
Transformation with the plasmid pMS58
Spread on LB Cm over the night at 30°C
Week 12 (26-1 September)
26/08
streak 4 clones of the previous transformation on LB Cm at 42°C one night

27/08
Streak 4 clones of the first integration on LB Cm at 42°C one night

28/08
Streak 4 clones of the second integration on LB Strp 42°C one night

29/08
Sub-cloned few clones on LB Cm, LB Str and LB Kn at 42°C one night
Right clone are StR CmS et KnR

30/08
Our clones were StrR, but unfortunately they were CmR and KnS…
Restart of the integration from the beginning:
Making competent cells
In parallele, spread of the MG1655 del(envZ) deFRT Kn StrpR strain on LB Cm, LB Str and LB Kn at 42°C