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/piG6 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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Transformations and controls for future tests

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 is too small, not correct.

Adherence Test Preparation

Others

Wednesday

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Transformations and controls

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

Plating of PHL916 on LB ( sureE strain that should not recombine DNA ) in case the transformation of pIG25 into MC4100 fails.

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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Friday

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