Team:Paris/Production testing

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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

Digestion:
Verification digestion:

ok

Verification digestion:

ok

Verification digestion:

ok

-


Ligation:

pBAD-LacI

RBS-YFP

Ligation: Ligation: -
- Colony PCR :

ok

Colony PCR :

-

Colony PCR:

-

Miniprep:

ok

Miniprep:

ok

Miniprep:

ok

-
Sequencing :

-

Sequencing :

-

Sequencing :

-

Sequencing :

-

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


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 :

mRFP Cterm matrix : pSB1A3 Oligo :O59 and O60 TM :


mRFP Nterm plasmid : pSB1A3 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:

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

-

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.



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 :

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

-



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