Team:Paris/Transduction testing

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WetLab - Reception

In this section we have two kinds of experiments :


  1. Fusion : the construction required for the fusion of the vesicles that contain the message with the receiver.
  2. Transduction : the construction of the system that will be able de decypher the message.


The first part deals with G3P , OmpAL , Jun, Fos , and AIDA protein. the second with the transduction system of the fec operon (FecA, FecR, FecI).

Fusion

Fusion : the construction required for the fusion of the vesicles that contain the message.

G3P , OmpAL , Jun, Fos , and AIDA protein.



The green color means the experiment was a success


plasmid =


Time required : A lOT !!!!!


Experiments ran :


Column 1 Column 2 Column 3 Column 4


PCR :

AidA and Linker : Design, synthesize and order in two parts due to the seize.

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:

AidA CTerm Kpn1/HindIII

AidA NTerm Kpn1/HindIII

Digestion:

AidA complete E/P

PSB1A3 E/P

We did it to obtain a biobrick first

Digestion: Digestion:
Verification digestion:

ok

Verification digestion:

-

Verification digestion:

-

Verification digestion:

-


Ligation:

AidA CTerm (Kpn1/HindIII):: aidA NTerm (Kpn1/HindIII)


Ligation:

AidA complete E/P :: PSB1A3 E/P

Ligation: Ligation:
Colony pCR :

ok

Colony PCR:

ok

Colony PCR :

-

Colony PCR :

ok

Miniprep:

done

Miniprep:

done

Miniprep:

-

Miniprep:

done

Sequencing :

unecessary

Sequencing :

unecessary

Sequencing :

-

Sequencing :

unecessary

Stock glycerol:

-

Stock glycerol

S75

Stock glycerol

-

Stock glycerol

-




The green color means the experiment was a success


plasmid =


Time required : A lOT !!!!!


Experiments ran :


column 1 column 2 column 3 column 4


PCR :

Jun *: design and order to be a a mutant incapable to form homodimere.

pLac : previously done

SSOmpA : previously done

Fos : previously done

PCR : PCR : PCR :

Fos : design and order.

Verification on gel :

ok

Verification on gel :

-

Verification on gel :

-

Verification on gel :

ok

Purification on gel :

ok

Purification on gel :

-

Purification on gel :

-

Purification on gel :



Digestion:

pLac E/X

ssOmpA E/S

Jun E/X and E/P

PSB1A3 E/P

Digestion: Digestion: Digestion:

Fos E/X and E/P

PSB1A3 E/P

Verification digestion:

ok

Verification digestion:

-

Verification digestion:

-

Verification digestion:

ok


Ligation:

pLac on PSB2K3 E/X :: ssOmpA E/S

Jun E/P :: PSB1A3 E/P

Ligation: Ligation: Ligation:

Fos E/P :: PSB1A3 E/P

Colony PCR:

ok

Colony PCR :

-

Colony PCR :

-

Colony PCR :

-

Miniprep:

ok

Miniprep:

-

Miniprep:

-

Miniprep:

ok

Sequencing :

-

Sequencing :

-

Sequencing :

-

Sequencing :

-

Stock glycerol:

unecessary

Stock glycerol

-

Stock glycerol

-

Stock glycerol

-



G3P

Even though it has not been realized, we planned to do these experiments to verify that G3P is expressed into the bacterial surface and induces the membranal fusion.


  • Surface expression of G3P

Surface expression of G3P could be seen with antibodies.
Culture of bacteria expressing OmpA-linker-G3P construction on a membrane. Adding antibodies anti-G3P. Revelation should shows whether G3P is expressed at the surface of bacteria.


  • Floculation test

"Fusion" could be approached by a flocculation test.
Two separate bacterial population growth on media. A population (F-) expressing the OmpA-linker_G3P construction and a population (F +) which express sexual pilus (by expression of the episome F).
Then we growth bacteria without agitation and we look (compared to culture controls) the speed of sedimentation. Faster sedimentation is due to an interaction between bacterial populations showing an increase of contact time. At vesicle level, it induce membrane fusion.


Transduction

Transduction : the construction of the system that will be able de decypher the message.


the transduction system of the fec operon (FecA, FecR, FecI).



The green color means the experiment was a success


plasmid =


column 1 column 2 column 3 column 4


PCR :

pFec matrix: K12 DNA

Oligo: TM

FecI matrix: K12 DNA

Oligo: TM

FecR 1-85 matrix: K12 DNA

Oligo: TM

mRFP matrix: PSB1A3 (previous IGEM plate)

Oligo: 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:

Ter E/X

mRFP E/S

digestion: digestion: digestion:
verification digestion:

ok

verification digestion:

-

verification digestion:

-

verification digestion:

-


ligation:

mRFP E/S Ter E/X in Ter vector

ligation: ligation: ligation:
PCR colo :

ok

PCR colo :

-

PCR colo :

-

PCR colo :

-

miniprep:

ok

miniprep:

-

miniprep:

-

miniprep:

-

sequencing :

-

sequencing :

-

sequencing :

-

sequencing :

-




The green color means the experiment was a success


plasmid =





Time required : A lOT !!!!!



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 :

-


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