"
Team:Paris/Transduction testing
From 2009.igem.org
iGEM > Paris > WetLab > Reception
Contents |
WetLab - Reception
In this section we have two kinds of experiments :
- Fusion : the construction required for the fusion of the vesicles that contain the message with the receiver.
- 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.
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: | digestion: | digestion: |
| verification digestion:
ok | verification digestion:
- | verification digestion:
- | verification digestion:
-
|
| ligation:
aidA CTerm (Kpn1/HindIII):: aidA NTerm (Kpn1/HindIII)
| ligation: | ligation: | ligation: |
| PCR colo :
ok | PCR colo :
- | PCR colo :
- | PCR colo :
- |
| miniprep:
done | miniprep:
- | miniprep:
- | miniprep:
- |
| sequencing :
unecessary | sequencing :
- | sequencing :
- | sequencing :
- |
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 :
- |
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).
| 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 :
- |
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 :
- |
