From Monday the 22th of August to Friday the 26th of August 2011

Monday

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Both NM522 and NM522/pIG24 grew on LB+Amp ( liquid and solid ) : antibiotic concentration in liquid was not sufficient, a more concentrated solution of Amp (10mg/mL which corresponds to 100X) is made and sterilized by filtration.
Creation of our own LB+Amp plates by plating 200µL of Amp on a LB plate.
Plating of several AmpS strains on the previous plates : PHL818, MC4100, NM522+pIG4, NM522 from the collection, NM522 from the previous LB+Amp plate, a new NM522 found in another lab.

Plating of 10µL of the new NM522 on LB

Dissolution of 0.08g of Spectinomycin ( C14H24N2O7 + 2 HCl + 5 H20 ) into 5mL water and filtration to obtain a solution at 10mg/mL.

QIAGen extraction of NM522 and NM522+pIG4 and electrophoresis : no plasmid is in the strains.

Transformation and plating of NM522 on LB with : pUC18 (=pIG6) (3 µL) + Amp, pIG25 (3 µL) + Cm
Transformation and plating of S19 on LB and the antibiotic Amp with : p157 (3 µL) + SpcR, p115 (3 µL) + SpcR, p127 (3 µL) + Kn, p116 (3 µL) + Kn

Revealing of 24 well plates and flocculation test from 08.19.11. The (2) has failed. The others plates are quite good.

Tuesday

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Transformations and controls for future tests

Start of 5mL liquid cultures of two clones for each plates obtained by the previous transformations, in order to check the presence of the right plasmids and start fluorescence tests.

Transformation and plating of NM522 on LB with : p157 (2 µL) + Spc, p115 (2 µL) + Spc, p56 (2 µL) + Amp, p10 (3 µL) + Amp, pIG30 (2 µL) + Cm, p127 (2 µL) + Kn, p116 (5 µL) + Kn

Strain Collection

Storage of S20.
Plating of S20 on home-made LB+Amp dishes and start of 5mL liquid cultures in LB+Amp.

Start of 50mL liquid culture of NM522/piG6 for a future extraction.
Storage of NM522/piG6 in the collection as S21.

Adherence Test Preparation

Start of 5mL cultures for 24 well plates in M63G medium, with 50µL antibiotic if appropriate, from 10µL of saturated cultures : PHL818, NM522, NM522+pUC18 (Amp), S18(Amp).

Others

Results of the LB+Amp tests from the previous day : MC4100, NM522+piG4, PHL818, the new NM522 don't grow.
NM522 and NM522 from the collection produce a few colonies -> the collection was contaminated by an AmpR strain.
NM522 in the collection (S11) is replaced by the new NM522.

Test of 2 other batches of LB+Amp dishes by plating the new NM522 on them.

Plating of NMYO on LB+Kan from an old solid culture.

Wednesday

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Transformations and controls for future tests

Start of 5mL liquid cultures of two clones for each plates obtained by the previous transformations, in order to check the presence of the right plasmids.

Plating of MC4100 on LB from an old solid culture (07/26) for future transformations.

Miniprep of :
- S19/p157
- S19/p115
- S19/p127
- S19/p116
Digestion by E and electrophoresis to control the presence of the two plasmids : the plasmid piG16 doesn’t seem to be in these strains!!

Plasmid Collection

Midiprep of pIG6 (=pUC18)
Digestion by E and electrophoresis to control the plasmid : one band at 2.5-3kb which is coherent.
Nanodrop quantification : c=83ng/µL, 260/280=2.02
Storage of 300µL of the plasmid at -20°C.

Start of 50mL cultures (500µL antibiotic if necessary and 100µL bacteria ) of NM522 + p10 (Amp),NM522 + p127 (Kan),NM522 + p56 (Amp),NM522 + p157 (Spc),NM522 + p115 (Spc)for future extractions.

Adherence Test - pRcn-OmpR234 Characterization

Start of 24 well plate adherence test in M63G medium with PHL818 (positive control), NM522 (negative control), S21+Amp without Co, S21+Amp+Co 0,1mM, S18+Amp without Co, S18+Amp+Co 0,1mM.

Fluorescence Tests

Measuring the fluorescence and the OD600 of the tubes from the transformation of tuesday.

Others

Result of the test of the 2 batches of LB+Amp : NM522 doesn't grow so they seem efficient.

Start of a 5mL preculture of NMYO from the previous dish

Thursday

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Transformations and controls for future tests

Start of 5mL liquid culture of MC4100 from solid culture (08/24).

Start of 5mL liquid culture for fluorescence of : S19/p127, S19/p116, NM522/p127, NM522/p116 in LB/2 and M63G : 30°C and slow agitation
Start of 5mL liquid culture for fluorescence of : S19/p115, S19/p157, NM522/p115, NM522/p157 in LB/2 and M63G : 37°C and fast agitation. For each strain, there are 4 tubes without Cobalt and with 3 concentrations of Cobalt

Transformations and plating of MC4100 on LB+Amp with : piG6 (3 µL), piG2 (3µL) and sterile water (3µL) for negative control.

Plasmid Collection

Midiprep of p56 and p157.
Digestion by E and electrophoresis to control the plasmid : the obtained bands are coherent for each plasmid.
Nanodrop quantification :
-p56: c = 15,1 ng/µL
-p157: c = 19,3 ng/µL
Storage of 300µL of the plasmid at -20°C.
p56 = piG31
p157 = piG32

Start of 50mL liquid cultures (500µL antibiotic if necessary and 100µL saturated cultures (08/24)) of NM522/piG30 (Cm), NM522/p127 (Kan), NM522/p116 (Kan) for future extractions.

Others

Pouring of Cm+Amp dishes

Preparation of new Spc at 10mg/mL from 0.15g ( C14H24N2O7 + 2 HCl + 5 H20 ) into 10mL water.

Friday

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Digestion of R1, R2, C1, N1, N2 minipreps by E and electrophoresis :

R2 : 2kb, not good
R1 : 2.8kb, validated.
N2 : 2kb, 5kb , not good
N1 : 8kb , not good
C1 : 3kb, 2.5kb , not good
Start of 2X5 mL cultures of NMYO ( 5mL LB, 50µL saturated NMYO, 50µL Kan)

Because no NiCoT plasmid was correct, the transformation was abandonned.