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Team:Paris/Addressing testing
From 2009.igem.org
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 :
ClyA Cterm matrix : F4 Oligo :O10 and O31 TM :55°C ClyA Nterm matrix : F4 Oligo : O32 and O9 TM :55°C mRFP Cterm matrix : ? Oligo :O59 and O60 TM : mRFP Nterm plasmid : ? Oligo :O57 and O58 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 RFP Nterm X/P pBAD vector : ? E/S
| 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 colo :
ok | PCR colo :
ok | PCR colo :
- | PCR colo :
-
|
| 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: |
| PCR colo :
ok | PCR colo :
- | PCR colo :
- | PCR colo :
- |
| miniprep:
STOPPED | miniprep:
- | miniprep:
- | miniprep:
- |
| sequencing :
- | sequencing :
- | sequencing :
- | sequencing :
- |
