From 2011.igem.org

Week 13

From Monday the 12th of September to Sunday the 18th of September 2011

Monday

Strain construction

Extraction of PHL1414+pIG3, PHL1414+pIG16 (clone 1,2,3) , MC4100+piG34 200µl (clone 1,2), MC4100+pIG34 800µl (clone 1, 2).

Digestion by EcoRI and PstI.

Gel electrophoresis to verify the ligations. Inserts have the good length for pIG3, pIG16 so the transformation went well. Inserts of pIG34 200µl clone 1 and pIG34 800µl clone 1 are good so the ligation went well. We’re keeping in collection the pIG34 200µl clone 1 as pIG34 in the plasmids’ collection and MC400+pIG34 as S32 in the strains’ collection.

Cloning and transformation of rcn-ompR234 + pSB1C3 in MC4100.

Plasmid and strain Collection

Plating of S17 from the collection to extract a bigger quantity of pIG25 (pSB1C3 containing rcn-csgBAEFG).

Storage of the correct clone of:
PHL1414 + pIG3 (S30)
PHL1414+pIG16 (S31)
MC4100 + pIG34 (S32)

Storage of rcn-OmpR in psB1K3 (pIG34) from the previous miniprep.

Microscopy Test

Preparation
Sterilization of microscope cover slips in ethanol during a few minutes, then rinsed in sterilized water.

Test
Preparation of plates for the following strains (one repetition):
-PHL1414/piG3 AmpR = negative control without Co and with Co 25µM
-PHL1414/piG16 AmpR without Co, Co 10µM, Co 25µM, Co 50µM and Co 100µM

Incubation at 30°C overnight (during 16 hours).

Others

Pouring of 9 LB+Amp plates

Dilution of 5µL pIG25 (371.4 ng/µL) into 25µL water to obtain 3 additional tubes at concentration of about 50ng/µL for sequencing.
Sequencing (pIG25 and pIG27) sent.

Tuesday

Strain construction

Purification and culture of 2 clones from each Petri dish of the transformation of rcn-ompR234 + pSB1C3 in MC4100.
Extraction of piG33 800µl 1 and 2, and pIG33 200µl 1 and 2.
Digestion by EcoR1, Pst1 and gel electrophoresis for verification.

Plasmid and strain Collection

Start of a 5mL liquid culture of S17 in LB+Cm medium.
Minipreps of this culture to extract pIG25 in larger quantity. Digestion (E+P) and electrophoresis for control : the insert of pIG25 is not good so there was a mistake the first time it was controlled. More or less 500bp are missing. Back to the lab, let’s try to built it again to have it on time.
The inserts pIG33 800µl 1 and 200µl 2 are good. pIG33 800µl 1 becomes pIG33 and is put in the plasmids’ collection.
MC4100+pIG33 = S33 is stored.

Wednesday

Strain construction

The Ptrc strong promoter has been received from Genecust in pBluescriptIISK+: transformation in MC4100 and plating on LB+Amp medium.

Digestion by E+P of this plasmid and ligation into pSB1C3 overnight

Cloning of rcn-csgBAEFG in pSB1C3, transformation in pSB1C3 (pIG25)
Plating of PHL916 on LB ( sureE strain that should not recombine DNA ) in case the transformation of pIG25 into MC4100 fails.

Digestion of pIG20 with E and P.
Gel electrophoresis for control of the insert → good

Cloning of rcn-csgBAEFG in pSB1C3, transformation in pSB1C3.

Adherence Test - pRcn-OmpR234 Characterization

Start of a rcn-OmpR234 24 well plate with :
S27 ( rcn in pSB1C3 ) without cobalt and with cobalt (25µM).
S33 ( rcn-OmpR234 in pSB1C3 ) with increasing concentration of cobalt (0, 10, 25 and 100 µM).

Others

Plating of PHL644, PHL1256, PHL628 to be sent to other research teams, on request.

Thursday

Strain construction

None of the transformation of the day before has grown. Let’s try it again.

Fluorescence Test - rcn Characterization

Characterization of rcn-gfp with the kinetic studie.
3 people are repeating the same 4 erlen of culture with the M63G medium:
- NM522/p157 + spc + 25µM Co
- NM522/p157 + spc
- NM522/p115 + spc + 25µM Co
- NM522/p115 + spc
1 ml of spc (spectinomycine) is added
The culture is sed by 0,5mL of a overnight preculture.

Adherence Test - 18A-0mpR234 Characterization

Start a 24 well plates in M63G medium + Amp with PHL1414/piG3 (negative control) and PHL1414/piG16.
Incubation at 30°C for 48h.

Friday

Strain construction

The transformations from Wednesday have grown : selection of 5 clones for pIG36 and Ptrc +Cm, 10 for pIG25.

Miniprep (QIAGen), digestion (Fermentas E+P) and electrophoresis of all clones for pIG25, 4 for pIG36, 2 for Ptrc-Cm ( the other cultures had not grown ). One correct clone is found for pIG25 and pIG36, none is correct for Ptrc-Cm.

Adherence Test - pRcn-OmpR234 Characterization

Revealing of the plate : no effect from rcn-ompR234 is visible compared to the controls.

Others

Dissolution of 0.11g of Ampiciline sodium salt ( Sigma A0166-5G) into 10mL water and filtration to obtain a solution of Amp at 10mg/mL.

Saturday

Plasmid and strain Collection

Storage of the correct clones and extracted plasmids from the previous day : S34 (NM522 + pIG25), S35 (NM522+pIG36), pIG25, pIG37 (=pIG36 after transformation and extraction ).

Adherence Test - 18A-OmpR234 Characterization

Revealing of the plate : ompR234 doubles the adherence compared to negative controls.