From Monday the 12th of September to Friday the 16th of September 2011

Monday

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Strain construction

Extraction of PHL1414+pIG3, PHL1414+pIG16 (clone 1,2,3) , MC4100+piG34 200µl (clone 1,2), MC4100+pIG34 800µl (clone 1, 2).

Digestion by EcoRI and PstI.

Gel electrophoresis to verify the ligations. Inserts have the good length for pIG3, pIG16 so the transformation went well. Inserts of pIG34 200µl clone 1 and pIG34 800µl clone 1 are good so the ligation went well. We’re keeping in collection the pIG34 200µl clone 1 as pIG34 in the plasmids’ collection and MC400+pIG34 as S32 in the strains’ collection.

Cloning and transformation of rcn-ompR234 + pSB1C3 in MC4100

Plasmid and strain Collection

Plating of S17 from the collection to extract a bigger quantity of pIG25 ( pSB1C3 containing rcn-csgBAEFG )

Storage of the correct clone of:
PHL1414 + pIG3 (S30)
PHL1414+pIG16 (S31)
MC4100 + pIG34 (S32)

Storage of rcn-OmpR in psB1K3 (pIG34) from the previous miniprep.

Adherence Test Preparation

Others

Pouring of 9 LB+Amp plates

Dilution of 5µL pIG25 (371.4 ng/µL) into 25µL water to obtain 3 additional tubes at concentration of about 50ng/µL for sequencing.
Sequencing (pIG25 and pIG27) sent.

Microscopy Test

Preparation
Sterilization of microscope cover slips in ethanol during a few minutes, then rinsed in sterilized water.

Test

Preparation of plates for the following strains (one repetition):
-PHL1414/piG3 AmpR = negative control without Co and with Co 25µM
-PHL1414/piG16 AmpR without Co, Co 10µM, Co 25µM, Co 50µM and Co 100µM

Incubation at 30°C overnight (during 16 hours).

Tuesday

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Strain construction

Purification and culture of 2 clones from each Petri dish of the transformation of rcn-ompR234 + pSB1C3 in MC4100.
Extraction of piG33 800µl 1 and 2, and pIG33 200µl 1 and 2.
Digestion by EcoR1, Pst1 and gel electrophoresis for verification.

Plasmid and strain Collection

Start of a 5mL liquid culture of S17 in LB+Cm medium
Minipreps of this culture to extract pIG25 in larger quantity. Digestion (E+P) and electrophoresis for control : the insert of pIG25 is not good so there was a mistake the first time it was controlled. More or less 500bp are missing. Back to the lab, let’s try to built it again to have it on time.
The inserts pIG33 800µl 1 and 200µl 2 are good. pIG33 800µl 1 becomes pIG33 and is put in the plasmids’ collection.
MC4100+pIG33 = S33 is stored.

Adherence Test Preparation

Others

Wednesday

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Strain construction

The Ptrc strong promoter has been received from Genecust in pBluescriptIISK+: transformation in MC4100 and plating on LB+Amp medium.

Digestion by E+P of this plasmid and ligation into pSB1C3 overnight

Cloning of rcn-csgBAEFG in pSB1C3, transformation in pSB1C3 (pIG25)
Plating of PHL916 on LB ( sureE strain that should not recombine DNA ) in case the transformation of pIG25 into MC4100 fails.

Digestion of pIG20 with E and P
Gel electrophoresis for control of the insert → good

Cloning of rcn-csgBAEFG in pSB1C3, transformation in pSB1C3

Plasmid and strain Collection

rcn-OmpR234 adherence test

Start of a rcn-OmpR234 24 well plate with :
S27 ( rcn in pSB1C3 ) without cobalt and with cobalt (25µM)
S33 ( rcn-OmpR234 in pSB1C3 ) with increasing concentration of cobalt (0, 10, 25 and 100 µM)

Others

Plating of PHL644, PHL1256, PHL628 to be sent to other research teams, on request.

Thursday

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Strain construction

None of the transformation of the day before has grown. Let’s try it again.

Adherence Test Preparation

Caracterisation of rcn-gfp with the kinetic studie.
3 people are repeating the same 4 erlen of culture with the M63G medium:
- NM522/p157 + spc + 25µM Co
- NM522/p157
- NM522/p115 + spc + 25µM Co
- NM522/p115 + spc
1 ml of spc (spectinomycine) is added
The culture is sed by 0,5mL of a overnight preculture.

Friday

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