Team:TUDelft/Conjugation Procedure

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Experimental Procedures

Section 1: Helper Plasmid
Part 1A:

  • Acquire R751 and/or RP4 plasmid
  • Electoporate plasmid into cells and confirm conjugation. See [http://openwetware.org/wiki/Conjugation conjugation protocol]
  • Characterize conjugation efficiency

Part 1B: oriT knockout

  • Knockout oriT using either [http://openwetware.org/wiki/Recombineering/Lambda_red-mediated_gene_replacement lambda red], [http://openwetware.org/wiki/IGEM:Peking/2007/Count:Knockout knockout protocol used by Peking '07] or [http://openwetware.org/wiki/Berk2006-ConjugationTeam 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 [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

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