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Team:Paris/Addressing testing
From 2009.igem.org
iGEM > Paris > WetLab > Addressing
Adressing system
global constructions :
Time required : A lOT !!!!!
Experiments ran :
| Date | Authors | Article | ||
| [] | 1999 | Terry J. Beveridge | Structures of gram-negative cell walls and their derived membrane vesicles. | 10438737 |
| [] | 2003 | Miller SI & Guina T. | Bacterial vesicle formation as a mechanism of protein transfer to animals. | 14531993 |
| [] | 2005 | Kuehn MJ & Kesty NC. | Bacterial outer membrane vesicles and the host-pathogen interaction. | 16291643 |
| [] | 2006 | McBroom AJ & Kuehn MJ. | Outer membrane vesicle production by Escherichia coli is independent of membrane instability. | 16855227 |
| [] | 2007 | McBroom AJ & Kuehn MJ. | Release of outer membrane vesicles by Gram-negative bacteria is a novel envelope stress response. | 17163978 |
| [] | 2009 | Deatherage BL & Cookson BT. | Biogenesis of bacterial membrane vesicles | 19432795 |
| [] | 2009 | Purnick PE & Weiss R. | The second wave of synthetic biology: from modules to systems. | 19461664 |
From plate: mRFP
PCR :
ClyA Cterm plasmid : ? Oligo : TM :
ClyA Nterm plasmid : ? Oligo : TM :
mRFP Cterm plasmid : ? Oligo : TM :
mRFP Nterm plasmid : ? Oligo : TM :
Verification on gel
ok
Purification on gel
ok
digestion:
ClyA Cterm S/P RFP Nterm X/P PBAD E/S
ligation:
ClyA Cter-RFP Nter vector PSB1A3
PCR colo :
miniprep:
verifivation of the miniprep by digestion
sequencing [[ClyA]]
-ClyA Nterm= biobrick+sequencage+stock glycerol Done
-ClyA Cterm= biobrick+sequencage+stock glycerol Done
[[-RFP-]]
-RFP Nterm= biobrick+sequencage+stock glycerol Done
-RFP Cterm= biobrick+sequencage+stock glycerol Done
[[-ClyA Cter-RFP Nter-]]
-ClyA Cter-RFP Nter= biobrick+stock glycerol Done
[[-RFP Cter-ClyA Nter-]]
-miss ligation between 2 parts (j'ai fait plusieurs ligation le 7ou8/09, mais pas marché le 9/09 apres culture overnight, voir si les autres ont continué)
[[PBad-ClyA Cter-RFP Nter-]]
-failed many time with:
Pbad cut in S/P and ClyA Cter-RFP Nter in X/P
Pbad cut in E/S and ClyA Cter-RFP Nter in E/X
Export system
