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Team:Lyon-INSA-ENS/Realisation/Week12
From 2011.igem.org
Week 12
From Monday the 5th of September to Friday the 9th of September 2011
Monday
Transformations and controls for future tests
Miniprep of three individual clones of MC4100/piG6 and MC4100/piG2 isolated on 09/02 in order to chek the presence of the right plasmid.
Results of Flocculation Test and New Test
Crystal Violet coloration for the series of 09/02.
Start of a fourth 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, 25µM, 50µM, 100µM.
(three repetitions)
These liquid cultures are let for two days at 30°C and low stirring (75).
Adherence Test – pRcn-csgBAEFG Characterization
Start of three replica of 24 well plates in M63G medium + Amp with MC4100/piG6 without Co and with Co 10µM
(negative control) and MC4100/piG2 without Co and with Co 10µM, 25µM, 50µM, 100µM.
Incubation at 30°C for 48h.
Revealing of the well plates started on 09/02.
Measuring OD600 and Crystal Violet coloration.
Tuesday
Transformations and controls for future tests
TSS transformation of MC4100 with pIG27 (Cm) or pIG16 (Amp).
Digestion and electrophoresis of the previously extracted DNA of MC4100+pIG6 clones and MC4100+pIG2 clones. Comparison made with the parts put in the collection.--> Verification is OK.
Strain Collection
Storage of the strains :
-S25 = MC4100/piG6 (clone #3)
-S26 = MC4100/piG2 (clone #3)
Microscopy Test
Preparation
Start of 5mL liquid cultures of PHL1414/piG2 (AmpR), PHL1414/piG6 (AmpR) from solid cultures (09/02)
and S19/p127 (KanR/AmpR), S19/p116 (KanR/AmpR) from solid cultures (08/23).
Test
Preparation of plates for the following strains (two repetitions):
-S19/p127 KanR/AmpR
-S19/p116 KanR/AmpR = negative control
(18A-OmpR234 Characterization)
-PHL1414/piG6 AmpR = negative control without Co and with Co 10µM
-PHL1414/piG2 AmpR without Co and with Co 10µM, Co 25µM, Co 50µM, Co 100µM
(pRcn-csgBAEFG Characterization)
Incubation at 30°C overnight (for 15 hours).
CFA Test
Liquid culture of strains for the test.
Others
Culture of S22 in LB with Cm (NM522/pIG33) for conservation.
Wednesday
Transformations and controls for future tests
Results of tuesday transformations : no clone on the negative controls, several on the tests. Isolation of 4 clones per transformation in solid and liquid culture.
Results of Microscopy Test
Observation of the different slides with a fluorescent microscope.
There is a problem with S19/p127 and S19/p116 so we start again the same test for these two strains.
Results of Adherence Test - pRcn-csgBAEFG Characterization
Revealing of the well plates started on Monday.
Measuring OD600 and Crystal Violet coloration.
CFA Test
CFA test (4 plates) to check stickiness of strains with PHL1414, MC4100, NM522, S18, S20, S21, S23, S24, S25, S26 (10µL of overnight culture)
Others
Pouring of Amp plates.br/>
No growth of S22 (Too much antibiotic), new culture with the right dose of antibiotics.
Thursday
Transformations and controls for future tests
Extraction with Qiagen’s kit of:
- MC4100 pIG27 clones 1, 2, 3 and 4
- MC4100 pIG16 clones 1, 2, 3 and 4
- MC4100 pIG33 clones 1, 2 and 3
E+P digestion (Fermentas) and electrophoresis of all the isolated clones for the following transformations : MC4100+pIG27, MC4100 + pIG16, MC4100 + rcn-ompR234.
All clones are correct for pIG27 and pIG16. No clone is correct for rcn-ompR234 -> detection of an error in the ligation.
Plasmid Collection
Storage of clone 1 for pIG27 and pIG16.
Plating of those same clones on LB+antibiotic (Cm and Amp respectively ).
Results of Microscopy Test
Observation of the different slides with a fluorescent microscope.
Result of Flocculation Test
Test started on 09/05.
Crystal Violet coloration for the three series.
CFA Test
No significative results with this test
Others
No growth of S22 again. Culture of S22 with and without antibiotics.
Friday
Strain construction
Construction of the composite part rcn+ompR234 in the chassis MC4100. Because both part are in the plasmid pSB1C3, the ligation 3A is done with the plasmid Kanamycine :
pIG27 (ES) + pIG11 (XP) + pSB1K3 (EP)
An electrophoresis gel is done to verifie the digestion. Everything is ok.
A transformation following the TSS transformation protocol is done and the Petri dishes are incubated overnight.
Sequencing
Nanodrop quantification of the previous pIG27 miniprep : 48.0 ng/µL.
Preparation of tubes for sequencing (30µL at approximately 50-100 ng/µL).
The pIG27 miniprep is used as such.
pIG25 is diluted from 5µL of the collection (371.4 ng/µL) with 25µL water.
Others
No growth of S22 neither with antibiotic nor without antibiotic. We drop the conservation of the plasmid and restart the construction of pIG33 (fusion between promoter Rcn and ompR234).
Saturday
Strain construction
Results of the ligation from the 09/09/2011
- Petri Dish 800µl E.coli → 40 clones
- Petri dish 200µl E.coli → 12 clones
4 clones of each dish are purified.
Results of the transformation of pIG3 in PHL1414:
- Witness - (PHL1414 alone) → nothing
- PHL1414 + pIG3 → 1 clone
- PHL1414 +pIG16 → a lot of clones
Purification of the clone PHL1414+pIG3 and purification of 4 clones of PHL1414+pIG16
