Team:Paris/Addressing testing

From 2009.igem.org

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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.
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PBAD ClyA RFP on PSB3T5 was transformed into Delta Tol bacteria , in this case we are supposed to see fluorescent vesicles when the medium contains 1 % arabinose , and to have no fluorescent on 1% glucose.
 +
 
 +
 
 +
To learn more, see the [[Team:Paris/Conclusion | Conclusion page]]

Latest revision as of 03:53, 22 October 2009

iGEM > Paris > WetLab > Addressing


WetLab - Addressing the message



Global constructions :


The green color means the experiment was a success


plasmid = PSB3T5




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 :


-

-

-

Verification on gel :

ok

-

-

-


Purification on gel :

ok

-


-

-


Digestion:

ClyA Cterm S/P


RFP Nterm X/P


pBAD vector : ? E/S


Digestion:

ClyA Cter-RFP Nter vector PSB1A3 E/X

-

-

Verification digestion:

ok

Verification digestion:

ok

-

-

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:

-


Colony PCR :

ok

Colony PCR :

ok

Colony PCR :

ok


Miniprep:

clone :

Miniprep:

clone :

Miniprep:

clone 3

clone 5


Sequencing :

ok

Sequencing :

ok

Sequencing :

ok

-


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

pBAD ClyA RFP : S89 (clone 3)

pBAD ClyA RFP: S90 (clone 5)



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 when the medium contains 1 % arabinose , and to have no fluorescent on 1% glucose.


To learn more, see the Conclusion page


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.



Experiments ran :


Column 1 Column 2 Column 3 Column 4
PCR :

TatABCE matrix :

- - -


Verification on gel :

ok

-

-

-

Purification on gel :

ok

-

-

-


Digestion:

none

Digestion:

none

Digestion:

none

Digestion:

none

Digestion checking:

none

Digestion checking:

none


Digestion checking:

none

Digestion checking:

none


Ligation:

none

Ligation:

none

Ligation:

none

Ligation:

none

Colony PCR :

none

Colony PCR :

none

Colony PCR :

none

Colony PCR :

none

Miniprep:

none

Miniprep:

none

Miniprep:

none

Miniprep:

none

Sequencing :

none

Sequencing :

none

Sequencing :

none

Sequencing :

none

Stock glycerol:

none

Stock glycerol

none

Stock glycerol

none

Stock glycerol

none


Retrieved from "http://2009.igem.org/Team:Paris/Addressing_testing"