Team:Paris/Transduction testing fusion
From 2009.igem.org
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== Fusion == | == Fusion == | ||
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Fusion : the construction required for the fusion of the vesicles that contain the message. | Fusion : the construction required for the fusion of the vesicles that contain the message. | ||
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G3P , OmpAL , Jun, Fos , and AIDA protein. | G3P , OmpAL , Jun, Fos , and AIDA protein. | ||
- | + | [[Image:Paris_construc_7_aida.jpg |700px|center|plasmid = ]] | |
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+ | [[Image:Paris_construc_8_junfos.jpg |700px|center|plasmid = ]] | ||
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+ | Time required : A lOT !!!!! | ||
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+ | Experiments ran : | ||
+ | |||
+ | {| | ||
+ | |- style="background: #0a3585; text-align: center; color:white; font-weight:bold; " | ||
+ | |width= 300px| row 1 | ||
+ | |width=300px| row 2 | ||
+ | |width=300px| row 3 | ||
+ | |||
+ | |- style="background: #bebebe; text-align: center;" | ||
+ | |'''PCR :''' | ||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |'''PCR :''' | ||
+ | |'''PCR :''' | ||
+ | |||
+ | |||
+ | |- style="background: #d8d8d8; text-align: center;" | ||
+ | |'''Verification on gel :''' | ||
+ | |||
+ | ok | ||
+ | |||
+ | |'''Verification on gel :''' | ||
+ | |||
+ | ok | ||
+ | |||
+ | |'''Verification on gel :''' | ||
+ | |||
+ | ok | ||
+ | |||
+ | |- style="background: #bebebe; text-align: center;" | ||
+ | |'''Purification on gel :''' | ||
+ | |||
+ | ok | ||
+ | |||
+ | |'''Purification on gel :''' | ||
+ | |||
+ | ok | ||
+ | |||
+ | |'''Purification on gel :''' | ||
+ | |||
+ | ok | ||
+ | |||
+ | |||
+ | |- style="background: #d8d8d8; text-align: center;" | ||
+ | |'''digestion:''' | ||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |'''digestion:''' | ||
+ | |'''digestion:''' | ||
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+ | |- style="background: #bebebe; text-align: center;" | ||
+ | |'''verification digestion:''' | ||
+ | |||
+ | ok | ||
+ | |||
+ | |'''verification digestion:''' | ||
+ | |||
+ | ok | ||
+ | |||
+ | |'''verification digestion:''' | ||
+ | |||
+ | ok | ||
+ | |||
+ | |||
+ | |- style="background: #d8d8d8; text-align: center;" | ||
+ | |'''ligation:''' | ||
+ | |||
+ | |||
+ | |||
+ | |'''ligation:''' | ||
+ | |'''ligation:''' | ||
+ | |||
+ | |||
+ | |- style="background: #bebebe; text-align: center;" | ||
+ | |'''PCR colo :''' | ||
+ | |||
+ | ok | ||
+ | |||
+ | |'''PCR colo :''' | ||
+ | |||
+ | ok | ||
+ | |||
+ | |'''PCR colo :''' | ||
+ | |||
+ | ok | ||
+ | |||
+ | |||
+ | |- style="background: #d8d8d8; text-align: center;" | ||
+ | |'''miniprep:''' | ||
+ | |||
+ | clone : | ||
+ | |||
+ | |'''miniprep:''' | ||
+ | |||
+ | clone : | ||
+ | |||
+ | |'''miniprep:''' | ||
+ | |||
+ | clone : | ||
+ | |||
+ | |||
+ | |- style="background: #bebebe; text-align: center;" | ||
+ | |'''sequencing :''' | ||
+ | |||
+ | ok | ||
+ | |||
+ | |'''sequencing :''' | ||
+ | |||
+ | ok | ||
+ | |||
+ | |'''sequencing :''' | ||
+ | |||
+ | ok | ||
+ | |||
+ | |} | ||
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+ | |||
+ | |||
+ | |||
+ | ===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. | ||
+ | |||
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*<b>Surface expression of G3P</b> | *<b>Surface expression of G3P</b> | ||
Surface expression of G3P could be seen with antibodies.<br> | Surface expression of G3P could be seen with antibodies.<br> | ||
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. | 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. | ||
+ | |||
*<b>Floculation test</b> | *<b>Floculation test</b> |
Latest revision as of 15:44, 17 October 2009
iGEM > Paris > Reception > Testing > Fusion
Fusion
Fusion : the construction required for the fusion of the vesicles that contain the message.
G3P , OmpAL , Jun, Fos , and AIDA protein.
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
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).