Team:Paris/Addressing testing
From 2009.igem.org
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'''global constructions :''' | '''global constructions :''' | ||
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<center><font style="color:#00BF00;font-weight:bold; font-size:13px">The green color means the experiment was a success</font></center> | <center><font style="color:#00BF00;font-weight:bold; font-size:13px">The green color means the experiment was a success</font></center> | ||
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[[Image:Paris_construc_1_PCR.jpg |800px|center|plasmid = PSB3T5]] | [[Image:Paris_construc_1_PCR.jpg |800px|center|plasmid = PSB3T5]] |
Revision as of 22:18, 21 October 2009
iGEM > Paris > WetLab > Addressing
WetLab - Addressing the message
global constructions :
Time required : A lot !!!!!
Experiments ran :
Column 1 | Column 2 | Column 3 | Column 4 |
PCR :
pBAD: plate 2008
Oligo :O57 and O58 TM :
Oligo :O59 and O60 TM :
| PCR :
- | PCR :
- | PCR :
- |
Verification on gel :
ok | Verification on gel :
- | Verification on gel :
- | Verification on gel :
- |
Purification on gel :
ok | Purification on gel :
- | Purification on gel :
- | Purification on gel :
- |
Digestion:
ClyA Cterm S/P
| Digestion:
ClyA Cter-RFP Nter vector PSB1A3 E/X | Digestion:
- | Digestion:
- |
Verification digestion:
ok | Verification digestion:
ok | Verification digestion:
- | Verification digestion:
-
|
Ligation:
ClyA Cter-RFP Nter vector PSB1A3 | Ligation:
PBAD E/S ClyA Cter-RFP Nter PSB1A3 E/X (x2)
| Ligation:
- | Ligation:
- |
PCR colony :
ok | Colony PCR :
ok | Colony PCR :
- | Colony PCR :
-
|
Miniprep:
clone : | Miniprep:
clone : | Miniprep:
- | Miniprep:
-
|
Sequencing :
ok | Sequencing :
ok | Sequencing :
- | Sequencing :
- |
Stock glycerol:
ClyA CTer: S47(clone 3) and S48(clone 7) ClyA Nter: S72 RFP Cter: S55 (Clone 3) RFP Nter: S56 (Clone 3)
| Stock glycerol
Cly A(Cter)-(Nter)RFP: S72(clone 8) | Stock glycerol
- | Stock glycerol
-
|
Functional Testing:
PBAD ClyA RFP was transformed into Top10 bacteria in order to localize the fluorescence, we are supposed to have a superior fluorescence in the membrane.
PBAD ClyA RFP on PSB3T5 was transformed into Delta Tol bacteria , in this case we are supposed to see fluorescent vesicles went the medium contains 1 % arabinose , and to have no fluorescent on 1% glucose.
Export system
We finally thought that it won't be neccesary to overexpress the Tat system, nevertheless we have run a few experiments before starting to focus on others parts of the project.
Time required : A week.
Experiments ran :
Column 1 | Column 2 | Column 3 | Column 4
|
PCR :
TatABCE matrix : | PCR : | PCR : | PCR : |
Verification on gel :
ok | Verification on gel :
- | Verification on gel :
- | Verification on gel :
- |
Purification on gel :
ok | Purification on gel :
- | Purification on gel :
- | Purification on gel :
-
|
Digestion:
TatABCE | Digestion: | Digestion: | Digestion: |
Verification digestion:
ok | Verification digestion:
- | Verification digestion:
- | Verification digestion:
-
|
Ligation:
TatABCE | Ligation: | Ligation: | Ligation: |
Colony PCR :
ok | Colony PCR :
- | Colony PCR :
- | Colony PCR :
- |
Miniprep:
STOPPED | Miniprep:
- | Miniprep:
- | Miniprep:
- |
Sequencing :
- | Sequencing :
- | Sequencing :
- | Sequencing :
- |
Stock glycerol:
- | Stock glycerol
- | Stock glycerol
- | Stock glycerol
- |