Team:TUDelft/Conjugation Procedure
From 2009.igem.org
Experimental Procedures
Section 1: Helper Plasmid
Part 1A:
Part 1B: oriTR knockout
- Design and order primers needed for λ-red knockout ✔
- Acquire knockout plasmids ✔
- Knockout oriTR **In Progress**
- Verify that conjugation stopped **In Progress**
- Characterize conjugation efficiency of Conjugation Testing Plasmid 1 with R751 ΔoriTR as helper
- Send R751 ΔoriTR plasmid to registry
Part 1C: trbK knockout
- Knockout 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 [http://partsregistry.org/wiki/index.php?title=Part:BBa_K175000 BBa_K175000] (trbC) and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K175001 BBa_K175001] (trbK)
- Amplify BioBricks needed [http://partsregistry.org/wiki/index.php?title=Part:BBa_E0840 BBa_E0840] (GFP generator), [http://partsregistry.org/wiki/index.php?title=Part:BBa_B0034 BBa_B0034] (strong rbs), [http://partsregistry.org/wiki/index.php?title=Part:BBa_J23100 BBa_J23100] (constitutive promoter), [http://partsregistry.org/wiki/index.php?title=Part:BBa_I714031 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