From Monday the 5th of September to Friday the 9th of September 2011

Monday

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Transformations and controls for future tests

Miniprep of three individual clones of MC4100/piG6 and MC4100/piG2 isolated on 09/02 in order to chek the presence of the right plasmid.

Results of Flocculation Test and New Test

Crystal Violet coloration for the series of 09/02.

Start of a fourth test in M63G medium with :
-MC4100/piG6 (AmpR) without Co and with Co 10µM (negative controls)
-MC4100/piG2 (AmpR) without Co and with Co 10µM, 25µM, 50µM, 100µM.
(three repetitions)

These liquid cultures are let for two days at 30°C and low stirring (75).

Adherence Test – pRcn-csgBAEFG Characterization

Start of three replica of 24 well plates in M63G medium + Amp with MC4100/piG6 without Co and with Co 10µM
(negative control) and MC4100/piG2 without Co and with Co 10µM, 25µM, 50µM, 100µM.
Incubation at 30°C for 48h.

Revealing of the well plates started on 09/02.
Measuring OD600 and Crystal Violet coloration.

Tuesday

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Transformations and controls for future tests

TSS transformation of MC4100 with pIG27 (Cm) or pIG16 (Amp).

Digestion and electrophoresis of the previously extracted DNA of MC4100+pIG6 clones and MC4100+pIG2 clones. Comparison made with the parts put in the collection.--> Verification is OK.

Strain Collection

Storage of the strains :
-S25 = MC4100/piG6 (clone #3)
-S26 = MC4100/piG2 (clone #3)

Microscopy Test

Preparation
Start of 5mL liquid cultures of PHL1414/piG2 (AmpR), PHL1414/piG6 (AmpR) from solid cultures (09/02)
and S19/p127 (KanR/AmpR), S19/p116 (KanR/AmpR) from solid cultures (08/23).

Test
Preparation of plates for the following strains (two repetitions):
-S19/p127 KanR/AmpR
-S19/p116 KanR/AmpR = negative control
(18A-OmpR234 Characterization)
-PHL1414/piG6 AmpR = negative control without Co and with Co 10µM
-PHL1414/piG2 AmpR without Co and with Co 10µM, Co 25µM, Co 50µM, Co 100µM
(pRcn-csgBAEFG Characterization)
Incubation at 30°C overnight (for 15 hours).

CFA Test

Wednesday

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Transformations and controls for future tests

Results of tuesday transformations : no clone on the negative controls, several on the tests. Isolation of 4 clones per transformation in solid and liquid culture.

Results of Microscopy Test

Observation of the different slides with a fluorescent microscope.
There is a problem with S19/p127 and S19/p116 so we start again the same test for these two strains.

Results of Adherence Test - pRcn-csgBAEFG Characterization

Revealing of the well plates started on Monday.
Measuring OD600 and Crystal Violet coloration.

Others

Pouring of Amp plates.

Thursday

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Transformations and controls for future tests

E+P digestion and electrophoresis of all the isolated clones for the following transformations : MC4100+pIG27, MC4100 + pIG16, MC4100 + rcn-ompR234.
All clones are correct for pIG27 and pIG16. No clone is correct for rcn-ompR234 -> detection of an error in the ligation.

Plasmid Collection

Storage of clone 1 for pIG27 and pIG16.
Plating of those same clones on LB+antibiotic (Cm and Amp respectively ).

Results of Microscopy Test

Observation of the different slides with a fluorescent microscope.

Result of Flocculation Test

Test started on 09/05.
Crystal Violet coloration for the three series.

Friday

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Sequencing

Nanodrop quantification of the previous pIG27 miniprep : 48.0 ng/µL.
Preparation of tubes for sequencing (30µL at approximately 50-100 ng/µL).
The pIG27 miniprep is used as such.
pIG25 is diluted from 5µL of the collection (371.4 ng/µL) with 25µL water.