Team:Lyon-INSA-ENS/Realisation/Week8

From 2011.igem.org

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Characterization of OmpR234 with a <b>24 well plate</b> and a <b>flocculation test</b>.<br>
Characterization of OmpR234 with a <b>24 well plate</b> and a <b>flocculation test</b>.<br>
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<b>Test in CFA medium</b> to show the formation of biofilms by colouring them red using the following strains( 10µL each ) :<br>
<b>Test in CFA medium</b> to show the formation of biofilms by colouring them red using the following strains( 10µL each ) :<br>
PHL818 (positive control),NM522 (negative control), and test bacteria containing 18A promoter ( alone without the coding part ),our synthesized parts (Amp or Cm)<br>
PHL818 (positive control),NM522 (negative control), and test bacteria containing 18A promoter ( alone without the coding part ),our synthesized parts (Amp or Cm)<br>
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-> not very conclusive, a light circle of light around the bacteria.
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-> not very conclusive, a little circle of light around the bacteria.
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<b>Construction</b> of Prcn-GFP(LVA).<br>
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<b>Construction</b> of Prcn-GFP(LVA).<br/> <br/>
<b>Digestion</b> of the plasmid with Prcn (S+P) = linearization  -> error: the part goes out from the plasmid <br>
<b>Digestion</b> of the plasmid with Prcn (S+P) = linearization  -> error: the part goes out from the plasmid <br>
-> use of other enzyme tubes, with the same results, same with a simple digestion -> our enzyme tubes are contaminated<br>
-> use of other enzyme tubes, with the same results, same with a simple digestion -> our enzyme tubes are contaminated<br>
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  <h6 style="text-align :left"> Friday</h6><HR>
  <h6 style="text-align :left"> Friday</h6><HR>
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Repeat of the <b>flocculation test</b> for the characterization of OmpR234.<br>
Repeat of the <b>flocculation test</b> for the characterization of OmpR234.<br>

Revision as of 14:12, 13 September 2011







Week 8


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







Monday


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


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



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


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 little 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



Repeat of the flocculation test for the characterization of OmpR234.

Repeat of the CFA medium test.

Revealing of the four 24 well plates from thursday.








ENS assystem Biomérieux INSA INSA