Team:Paris/Addressing testing

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iGEM > Paris > WetLab > Addressing


WetLab - Addressing the message


global constructions :

The green color means the experiment was a success
plasmid = PSB3T5


Time required : A lot !!!!!



Experiments ran :

column 1 column 2 column 3 column 4
PCR :

pBAD: plate 2008


ClyA Cterm matrix : F4

Oligo :O10 and O31 TM :55°C


mRFP Nterm plasmid : pSB1A3

Oligo :O57 and O58 TM :


ClyA Nterm matrix : F4

Oligo : O32 and O9 TM :55°C


mRFP Cterm matrix : pSB1A3

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


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)


PBAD S/P ClyA Cter-RFP Nter PSB2K3 X/P (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 :

-

stock glycerol:

-

stock glycerol

-

stock glycerol

-

stock glycerol

-


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