Team:Paris/Transduction testing fusion

From 2009.igem.org

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[[Image:Paris_construc_8_junfos.jpg  |700px|thumb|left|plasmid = ]]
[[Image:Paris_construc_8_junfos.jpg  |700px|thumb|left|plasmid = ]]
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Time required : A lOT !!!!!
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Experiments ran :
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{|
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|- style="background: #0a3585; text-align: center; color:white; font-weight:bold; "
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|width= 300px| row 1
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|width=300px| row 2
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|width=300px| row 3
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|- style="background: #bebebe; text-align: center;"
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|'''PCR :'''
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|'''PCR :'''
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|'''PCR :'''
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|- style="background: #d8d8d8; text-align: center;"
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|'''Verification on gel :'''
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ok
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|'''Verification on gel :'''
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ok
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|'''Verification on gel :'''
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ok
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|- style="background: #bebebe; text-align: center;" 
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|'''Purification on gel :'''
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ok
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|'''Purification on gel :'''
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ok
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|'''Purification on gel :'''
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ok
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|- style="background: #d8d8d8; text-align: center;"
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|'''digestion:'''
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|'''digestion:'''
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|'''digestion:'''
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|- style="background: #bebebe; text-align: center;" 
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|'''verification digestion:'''
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ok
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|'''verification digestion:'''
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ok
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|'''verification digestion:'''
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ok
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|- style="background: #d8d8d8; text-align: center;"
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|'''ligation:'''
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|'''ligation:'''
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|'''ligation:'''
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|- style="background: #bebebe; text-align: center;" 
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|'''PCR colo :'''
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ok
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|'''PCR colo :'''
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ok
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|'''PCR colo :'''
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ok
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|- style="background: #d8d8d8; text-align: center;"
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|'''miniprep:'''
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clone :
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|'''miniprep:'''
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clone :
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|'''miniprep:'''
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clone :
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|- style="background: #bebebe; text-align: center;" 
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|'''sequencing :'''
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ok
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|'''sequencing :'''
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ok
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|'''sequencing :'''
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ok
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|}
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Revision as of 11:46, 17 October 2009

iGEM > Paris > Reception > Testing > Fusion

WetLab

Main - Addressing - Production - reception -
Reception


Fusion

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

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

plasmid =


plasmid =



Time required : A lOT !!!!!



Experiments ran :

row 1 row 2 row 3
PCR :



PCR : PCR :


Verification on gel :

ok

Verification on gel :

ok

Verification on gel :

ok

Purification on gel :

ok

Purification on gel :

ok

Purification on gel :

ok


digestion:



digestion: digestion:
verification digestion:

ok

verification digestion:

ok

verification digestion:

ok


ligation:


ligation: ligation:


PCR colo :

ok

PCR colo :

ok

PCR colo :

ok


miniprep:

clone :

miniprep:

clone :

miniprep:

clone :


sequencing :

ok

sequencing :

ok

sequencing :

ok



G3P

Bien que non réalisé, nous avions prévus de faire ses manipulations afin de vérifier que la G3P ets bien exprimer à la surface bactériène et que cela induisse la fusion memebranaire.

  • 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.

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