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Team:Lyon-INSA-ENS/Realisation/Week13
From 2011.igem.org
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<p style=" line-height : 1.5em"> | <p style=" line-height : 1.5em"> | ||
| - | <FONT COLOR="red"> <b> | + | <FONT COLOR="red"> <b>TStrain construction</b> </FONT> <br/><br/> |
| + | Purification and culture of 2 clones from each Petri dish of the transformation of rcn-ompR234 + pSB1C3 in MC4100.<br/> | ||
| + | Extraction of piG33 800µl 1 and 2, and pIG33 200µl 1 and 2.<br/> | ||
| + | Digestion by EcoR1, Pst1 and gel electrophoresis for verification.<br/> | ||
</p> | </p> | ||
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<FONT COLOR="blue"><b>Plasmid and strain Collection</b></FONT> <br/><br/> | <FONT COLOR="blue"><b>Plasmid and strain Collection</b></FONT> <br/><br/> | ||
Start of a 5mL liquid culture of S17 in LB+Cm medium<br/> | Start of a 5mL liquid culture of S17 in LB+Cm medium<br/> | ||
| - | Minipreps of this culture to extract pIG25 in larger quantity. Digestion (E+P) and electrophoresis for control : the insert is | + | 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.<br/> |
| + | The inserts pIG33 800µl 1 and 200µl 2 are good. pIG33 800µl 1 becomes pIG33 and is put in the plasmids’ collection.<br/> | ||
| + | MC4100+pIG33 = S33 is stored.<br/><br/> | ||
</p> | </p> | ||
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<p style=" line-height : 1.5em"> | <p style=" line-height : 1.5em"> | ||
| - | <FONT COLOR="red"> <b> | + | <FONT COLOR="red"> <b>Strain construction </b> </FONT> <br/><br/> |
The Ptrc strong promoter has been received from Genecust in pBluescriptIISK+: transformation in MC4100 and plating on LB+Amp medium.<br/><br/> | The Ptrc strong promoter has been received from Genecust in pBluescriptIISK+: transformation in MC4100 and plating on LB+Amp medium.<br/><br/> | ||
Digestion by E+P of this plasmid and ligation into pSB1C3 overnight<br/><br/> | Digestion by E+P of this plasmid and ligation into pSB1C3 overnight<br/><br/> | ||
| + | Cloning of rcn-csgBAEFG in pSB1C3, transformation in pSB1C3 (pIG25) <br/> | ||
Plating of PHL916 on LB ( sureE strain that should not recombine DNA ) in case the transformation of pIG25 into MC4100 fails. <br/><br/> | Plating of PHL916 on LB ( sureE strain that should not recombine DNA ) in case the transformation of pIG25 into MC4100 fails. <br/><br/> | ||
| + | Digestion of pIG20 with E and P<br/> | ||
| + | Gel electrophoresis for control of the insert → good<br/><br/> | ||
| + | Cloning of rcn-csgBAEFG in pSB1C3, transformation in pSB1C3 | ||
</p> | </p> | ||
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<p style=" line-height : 1.5em"> | <p style=" line-height : 1.5em"> | ||
| + | |||
| + | <FONT COLOR="red"> <b>Strain construction </b> </FONT> <br/><br/> | ||
| + | None of the transformation of the day before has grown. Let’s try it again.<br/><br/> | ||
| + | |||
| + | </p> | ||
| + | |||
| + | <p style=" line-height : 1.5em"> | ||
| + | <FONT COLOR="purple"><b> Adherence Test Preparation </b></FONT> <br/><br/> | ||
| + | Caracterisation of rcn-gfp with the kinetic studie.<br/> | ||
| + | 3 people are repeating the same 4 erlen of culture with the M63G medium:<br/> | ||
| + | - NM522/p157 + spc + 25µM Co<br/> | ||
| + | - NM522/p157<br/> | ||
| + | - NM522/p115 + spc + 25µM Co<br/> | ||
| + | - NM522/p115 + spc<br/> | ||
| + | |||
| + | 1 ml of spc (spectinomycine) is added<br/> | ||
| + | The culture is sed by 0,5mL of a overnight preculture.<br/><br/> | ||
| + | |||
</p> | </p> | ||
Revision as of 19:25, 15 September 2011
Week 13
From Monday the 12th of September to Friday the 16th 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.
Adherence Test Preparation
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.
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/piG6 AmpR without Co, Co 10µM, Co 25µM, Co 50µM and Co 100µM
Incubation at 30°C overnight (during 16 hours).
Tuesday
TStrain 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.
Adherence Test Preparation
Others
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
Plasmid and strain Collection
rcn-OmpR234 adherence test
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.
Adherence Test Preparation
Caracterisation 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
- 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.
Friday
