Week 11

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

Monday

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 LB/2 medium.
In the M63G medium, 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 in M63G medium of PHL818, MC4100, MC4100/piG2 (AmpR)
and MC4100/piG6 (AmpR).

Results of Adherence Test

Revealing of the 24 well plates started on Friday.
Measuring OD600 (only for the M63 well plate because the results of the other plate weren't exploitable) and Crystal Violet coloration.

Microscopy Test Preparation

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

Tuesday

Transformations and controls for future tests

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.

Flocculation Test

Preparation:
Start of 5mL liquid cultures in M63G medium + Amp of S18 and S21 from solids cultures (08/29).

Test:
Start of a second test in M63G medium with:
-MC4100/piG6 (AmpR) = negative control without Co and with Co 10µM.
-MC4100/piG2 (AmpR) without Co, with Co 10µM and with Co 100µM.
-S21 (AmpR) = negative control without Co and with Co 10µM.
-S18 (AmpR) without Co, with Co 10µM and with Co 100µM.
These liquid cultures are let over the weekend at 30°C and low stirring (85).

Adherence Test

Start a repetition of two 24 well plates in M63G medium with PHL818 (positive control),
MC4100 (negative control), MC4100/piG6 (pUC18) without Co and with Co 10µM,
MC4100/piG2 (rcn - csgBAEFG in pUC18) without Co and with Co 10µM.
Incubation at 30°C for 48h.

Fluorescence Test

Measuring the spectro fluorescence and the OD600 for the tubes from Thursday 08.25.

Wednesday

Transformations and controls for future tests

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

Transformations and controls for future tests

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 selections.

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.

Because of this problem, new transformation and plating of a new strain MC4100 with piG6 (Amp)
and piG2 (Amp). Negative control : MC4100 on Amp.

Start of 2.5mL cultures for extraction and checking of the individual clones of PHL1414+piG6 and PHL1414+piG2.

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

Collaboration

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

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+pIG3 (Amp), NM522+pIG16(Amp).

Results of Adherence Test – pRcn-csgBAEFG Characterization

Revealing of the 24 well plates started on Tuesday.
Measuring OD600 and Crystal Violet coloration.

Microscopy Test

Preparation of plates for the following strains (two repetitions):
-S19p127 KanR/AmpR
-S19p116 KanR/AmpR = negative control
(18A-OmpR234 Characterization)
-MC4100/piG2/p150 KanR/AmpR
-MC4100/piG6/p150 KanR/AmpR = negative control
-PHL1414/piG2 AmpR
-PHL1414/piG6 AmpR = negative control
(pRcn-csgBAEFG Characterization)

Incubation at 30°C overnight (for 23 hours).

Friday

Transformations and controls for future tests

Digestion of the previous minipreps by E and electrophoresis -> all clones are correct

Isolation of three individual clones from the transformations of MC4100/piG6 and MC4100/piG2 (09/01) for a future Adherence Test.

Plasmid Collection

Storage of clone 3 from PHL1414+pIG2 (S23) and clone 2 from PHL1414+pIG6(S24)
Plating of those same clones on LB+Amp medium.

Flocculation Test

Start of a third 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, 50µM, 100µM.
(two repetitions)
-S21 (AmpR) without Co and with Co 10µM (negative controls)
-S18 (AmpR) without Co and with Co 10µM, 50µM, 100µM.

These liquid cultures are let over the weekend at 30°C and low stirring (75).

Adherence Test

Start of 24 well plates in M63G : PHL818 (positive control), NM522 (negative control), NM522+piG3(Amp), NM522+piG16(Amp). Two same plates are done by two different operators.

Results of Microscopy Test