Team:Paris/Production testing

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iGEM > Paris > WetLab > Production


Contents

WetLab - Production

  1. The delay
    1. Part I : Testing delay phase I
    2. Part I : Functional testing of the part I
    3. Part II : Testing delay phase II
    4. Part II : Functional testing of the part II
  2. The destabilization of the membrane

The delay

We separate the construction on two plasmids : Part I and Part II.

Part I : Testing delay phase I

The green color means the experiment was a success


plasmid = PSB3T5



Experiments ran :


Column 1 Column 2 Column 3 Column 4
PCR :

pBAD: plate igem


LacI: plate igem


Terminator: plate igem


RBS: plate igem


YFP: plate igem

- - -
Verification on gel :

ok

- - -
Purification on gel :

ok

- - -



Digestion:

pBAD S/P


lacI E/S


terminator E/X


RBS S/P


YFP X/P

Digestion:

pBAD-LacI E/S


RBS-YFP E/S

Digestion:

RBS-YFP-ter E/X

-
Verification digestion:

ok

Verification digestion:

ok

Verification digestion:

ok

-


Ligation:

pBAD-LacI

RBS-YFP

Ligation: Ligation: Ligation:
Colony PCR :

ok

Colony PCR :

ok

Colony PCR :

ok

Colony PCR:

ok

Miniprep:

clone

Miniprep:

clone

Miniprep:

clone

Miniprep:

clone

Sequencing :

ok

Sequencing :

ok

Sequencing :

ok

Sequencing :

ok

Stock glycerol:


Stock glycerol

Cly A(Cter)-(Nter)RFP: S72(clone 8)

Stock glycerol

-

Stock glycerol

-

Part I : Functional testing of the part I

PBAD LacI LVA- YFP was transforme into Top ten Bacteria and grew on medium :


1% arabinose

1% glucose

LB pure


To learn more, see the Conclusion page



Part II : Testing delay phase II

The green color means the experiment was a success


plasmid =



Experiments ran :


Column 1 Column 2 Column 3 Column 4
PCR :

terminator: igem plate

pLac: igem plate


TetR


pTet:


mRFP


- - -
Verification on gel :

ok

-

-

-

-

-

-

Purification on gel :

ok

-

-

-


Digestion:

pLac S/P


TetR X/P


terminator E/X


pTet S/P


mRFP X/P


pSB3T5 E/P

Digestion:

plac-TetR E/S

pTet-mRFP X/P

Digestion:

pLac-tetR-ter S/P


Digestion:

pLac-tetR-ter-pTet-mRFP E/P


Digestion checking:

ok

Digestion checking:

ok

Digestion checking:

ok

Digestion checking:

ok


Ligation:

pLac-tetR


pTet-mRFP


Ligation:

pLac-TetR-ter

Ligation:

pLac-ter-pTet-mRFP

Ligation:

pLac-ter-pTet-mRFP-pSB3T5


Colony PCR:

ok

Colony PCR :

ok

Colony PCR:

ok

Colony PCR :

ok

Miniprep:

ok

Miniprep:

ok

Miniprep:

ok

Miniprep:

ok

Sequencing :

ok

Sequencing :

ok

Sequencing :

ok

Sequencing :

ok

Stock glycerol:

-

Stock glycerol

-

Stock glycerol

-

Stock glycerol

-

Part II : Functional testing of the part II

Ptet RFP Plac TetR was tranforme into Top10 bacteria and grew on media :


0,1% IPTG .

1% glucose.

LB pure.


To learn more, see the Conclusion page


The destabilization of the membrane

The green color means the experiment was a success


plasmid = PSB3T5



Experiments ran :


Column 1 Column 2 Column 3 Column 4
PCR :

pTet: igem plate

OmpAs: ToRII: pINK2 g3p: pINTg3p


'
Verification on gel :

ok

- - -


Purification on gel :

ok

- - -


Digestion:

pTet S/P

OmpAs X/P


TolRII X/P


g3p X/P


Digestion:

pTet-OmpAs S/P

Digestion:

none

-


Digestion checking:

ok

Digestion checking:

ok

Digestion checking:

none

-


Ligation:

pTet-OmpAs

Ligation:

none


Ligation:

none

-
Colony PCR :

ok

Colony PCR:

none

Colony PCR:

none

-
Miniprep:

ok

Miniprep:

none

Miniprep:

none

-
Sequencing :

ok

Sequencing :

none

Sequencing :

none

-


Stock glycerol:

-

Stock glycerol

none

Stock glycerol

none

-



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