From Monday the 1st of August to Friday the 5th of August 2011

Monday

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24 well plate with LB/2 or M63 medium to test bacterial adherence in response to cobalt :
positive control with an adherent strain PHL818 ( MG1655 malT::Tn10 adr101=OmpR234 ), negative control with a non adherent strain NM522, and our synthesized part Rcn-CsgBAEFG (Amp) with increasing concentrations of cobalt ( in CoCl2 form).

Flocculation test for the same strains with LB/2 or M63G medium.

Miniprep of the transformed colonies with the following plasmids : Prcn / Pcurli-GFP / Pcurli - Operon curli.
-> none of the isolated clones had the Prcn or Pcurli-GFP plasmid, start of liquid culture of new clones.
-> two clones had the Pcurli-curli operon part : storage in the collection

Tuesday

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In LB/2, the flocculation tests show flocculation for the adherent strain (PHL818), none for the negative control. Our strain shows a small flocculation that increases with the concentration in cobalt.
No flocculation in M63 medium for our strain : LB/2 will be used only for the next tests.

New flocculation tests with a wider range of cobalt concentration in LB or LB/2 and using the Rcn-CsgBAEFG (Cm) synthesized part

Revealing of the 24 well plate from the previous day, using 2 different methods : methyl violet (to visualise the formation of the biofilm at the surface of the well ) or direct OD600 measure.

Miniprep of the clones started the previous day
-> two clones have the Prcn part and one Pcurli-GFP.

Storage of PHL818, PHL1273, Rcn-CsgBAEFG (Cm) = S3 = S17, Rcn-CsgBAEFG (Amp) = S4 = S18

Wednesday

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Measurement of the OD600 of the LB/2 plate, which gives an adherence percentage for each strain.

Revealing of the M63 medium 24 well plate using the methyl violet ( 1 well) or OD600 method ( 3 wells ).

The flocculation tests from the previous day give better results in LB/2 compared to LB , but they're not significative. We'll use LB/2 for future tests.

Nanodrop quantification of some plasmids in the collection.

Thursday

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Start of new 24 well plates, to study:

• the effect of cobalt (in increasing concentration),
• the difference between Amp or Cm strains, -> it works better in Amp
• the effect of the presence of antibiotic, -> it works better with Antibiotic
• the effect of prior treatment of the plate with EDTA, -> it is better when plate is rinse
• the effect of EDTA (in increasing concentration) on the strain. -> no useful

Characterization of OmpR234 with a 24 well plate and a flocculation test.

Test in CFA medium to show the formation of biofilms by colouring them red using the following strains( 10µL each ) :
PHL818 (positive control),NM522 (negative control), and test bacteria containing 18A promoter ( alone without the coding part ),our synthesized parts (Amp or Cm)
-> not very conclusive, a light circle of light around the bacteria.
Construction of Prcn-GFP(LVA).
Digestion of the plasmid with Prcn (S+P) = linearization -> error: the part goes out from the plasmid
-> use of other enzyme tubes, with the same results, same with a simple digestion -> our enzyme tubes are contaminated
digestion of the plasmid with strong RRB-GFP (X+P) -> OK

Storage of MC4100 (Str) = S7, NM522 = S11, NMYO = S13 and NM522/NicoT (Amp) = S14

Friday

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Repeat of the flocculation test for the characterization of OmpR234.

Repeat of the CFA medium test.