Experimental Procedures

Section 1: Helper Plasmid

Part 1A:

• Acquire R751 plasmid ✔
• Confirm wild R751 conjugation ✔
• Characterize conjugation efficiency ✔

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

• Synthesize trbK gene ✔
• Verify that trbK expression blocks conjugation ✔

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