"
Team:TUDelft/Conjugation Procedure
From 2009.igem.org
(Difference between revisions)
| Line 1: | Line 1: | ||
| - | + | {{Template:TUDelftiGEM2009}} | |
==Experimental Procedures== | ==Experimental Procedures== | ||
'''Section 1: Helper Plasmid'''<br> | '''Section 1: Helper Plasmid'''<br> | ||
| Line 43: | Line 43: | ||
'''For more information on the cloning strategy of constructing the conjugation plasmids and knockouts check the [[Team:TUDelft/Conjugation_Cloning|cloning strategy]] page''' | '''For more information on the cloning strategy of constructing the conjugation plasmids and knockouts check the [[Team:TUDelft/Conjugation_Cloning|cloning strategy]] page''' | ||
| + | |||
| + | {{Template:TUDelftiGEM2009_end}} | ||
Revision as of 10:20, 7 October 2009
Experimental Procedures
Section 1: Helper Plasmid
Part 1A:
- Acquire R751 and/or RP4 plasmid
- Electoporate plasmid into cells and confirm conjugation. See conjugation protocol
- Characterize conjugation efficiency
Part 1B: oriT knockout
- Knockout oriT using either lambda red, knockout protocol used by Peking '07 or knockout protocol used by Berkeley '06
- Electroporate into cells
- Verify that conjugation stopped
- Electroporate PlasmidG as well and characterize conjugation efficiency
- If works send R751 oriT knockout plasmid to registry
Part 1C: trbK knockout
- Knockout oriT + trbK
- Electroporate into cells
- Verify that conjugation takes place among R+ cells using PlasmidG (see below)
- Characterize conjugation efficiency
- If works send R751 oriT + trbK knockout plasmid to registry
Part 1D: trbC knockout
- Knockout oriT + trbK + trbC
- Electroporate into cells and create culture for communication (cultureCom)
- Verify that no conjugation takes place in presence of PlasmidG
- If works send R751 oriT + trbK + trbC knockout plasmid to registry
Section 2: Message Plasmid
Part 2A: BioBrick Assembly
- Order DNA synthesis for BBa_K175000 (trbC) and BBa_K175001 (trbK)
- Amplify BioBricks needed BBa_E0840 (GFP generator), BBa_B0034 (strong rbs), BBa_J23100 (constitutive promoter), BBa_I714031 (oriT-R).
- Assemble: [oriT][promoter] and keep this for later
- Assemble PlasmidG: [oriT][promoter][GFP generator] + plasmid backbone with different resistance than helper plasmid and low copy number.
- Assemble [oriT][promoter][rbs][trbC][rbs][trbK] and keep this intermediate assembly product for possible use in integration stage
- Assemble [oriT][promoter][rbs][trbC][rbs][trbK][GFP generator] and keep this intermediate assembly product for possible use in integration stage
- Assemble PlasmidCKG: [oriT][promoter][rbs][trbC][rbs][trbK][GFP generator] + plasmid backbone with different resistance than helper plasmid and low copy number.
Part 2B: Full Communication testing
- Electroporate PlasmidCKG into some cells from cultureCom creating initiatorCells
- Select for presence of both message and helper plasmid
- Mix initiatorCells with cells not containing any plasmids and characterize conjugation efficiency
- Add initiatorCells to cultureCom and observe signal propagation, characterize rate of signal propagation.
- If signal propagation observed, do victory dance.
For more information on the cloning strategy of constructing the conjugation plasmids and knockouts check the cloning strategy page
