From Monday the 29th of August to Friday the 2nd of September 2011

Monday

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Transformations and controls for future tests

Nanodrop quantification of several midipreps :
p116 : 726.3 ng/µL
piG30 : 251.6 ng/µL
p115: 488.4 ng/µL
p10 : 276.2 ng/µL
p127 : 673.5 ng/µL

Digestion and electrophoresis of p115 (from S19 and NM522), p116(from S19 and NM522), p127(from S19 and NM522), p10(from NM522), p157 (from S19 and NM522), 2 clones each.

Start of solid cultures of S18 and S21 from the collection

Plating of PHL1414 from the collection

Results of Flocculation Test

Test started on 08/26.
No result in the medium LB/2.
In the medium M63G, a biofilm is obtained for MC4100/piG2 and there is no biofilm for the negative control. For this series, Crystal Violet coloration.

Adherence Test Preparation

Start of 5mL liquid cultures of PHL818, MC4100, MC4100/piG2 (AmpR) and MC4100/piG6 (AmpR).

Microscopy Test Preparation

Sterilization of glass slides at 180°C for 4 hours.

Tuesday

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Transformations and controls for future tests

Plasmid Collection

Results of Flocculation Test

Others

Start a repetition of two 24 well plates : PHL818 (positive control), MC4100 (negative control), MC4100/piG6 (pUC18) without Co and with Co, MC4100/piG2 (rcn - csgBAEFG in pUC18) without Co and with Co.
Start of 5mL liquid cultures for transformations : PHL1414, MC4100, MC4100+pIG2
TSS Transformation and plating of PHL1414 with pIG2, MC4100 with R1 and R1+p150, MC4100+pIG2 with p150.
Measuring the spectro fluorescence and the OD600 for the tubes from Thuesday 08.25.2011

Wednesday

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Transformations and controls for future tests

Plasmid Collection

Results of Flocculation Test

Others

Obtention of a few clones on all previous transformations except on MC4100+R1 ( too many clones, suspicious )

TSS Transformation and plating of PHL1414 with pIG6 (Amp), MC4100 with pIG6+p150(Amp+Kan),R1(Cm), pIG26(Cm) and pIG26+p150(Cm+Kan). Negative controls : PHL1414 and MC4100 on every type of antibiotic previously used.

Thursday

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Transformations and controls for future tests

Plasmid Collection

Results of Flocculation Test

Others

Revealing of the 24 well plates from Tuesday 08.30.11.

The negative control for the transformations of MC4100 on Cm grew : start of several tests ( plating on solid medium with Cm, liquid cultures in LB+Cm) to identify the origin of the failure of the negative control.
Isolation of 4 individual clones on other antibiotic selctions.

Plating of PHL1414+pIG6 and PHL1414+pIG2 on LB+Amp for TUdelft collaboration

Start of 2.5mL cultures for extraction and checking of the individual clones

The tests for the failure of the negative transformation control showed that our starting MC4100 strain was resistant to Cm -> all transformations using this strain are eliminated.

Miniprep of PHL1414 + pIG2 and PHL1414+pIG6 ( 4 clones each )

Friday

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Transformations and controls for future tests

Plasmid Collection

Results of Flocculation Test

Others

Digestion of the previous minipreps by E and electrophoresis -> all clones are correct
Storage of clone 3 from PHL1414+pIG2 (S23) and clone 2 from PHL1414+pIG6(S24)
Plating of those same clones on LB+Amp medium.

Start of flocculation tests :
-MC4100+piG6 ( with and without cobalt ), MC4100+piG2 ( with and without cobalt ),in M63G+Amp at 30°C, 75rpm.