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 **In Progress**
• Verify that conjugation takes place among R751 ΔtrbK cells
• Characterize conjugation efficiency
• Send R751 ΔoriT + ΔtrbK plasmid to registry

Part 1D: trbC knockout

• Knockout trbC
• Verify that no conjugation takes place in presence of Conjugation Testing Plasmid 1
• Send R751 ΔoriT + ΔtrbK + ΔtrbC plasmid to registry

Section 2: Message Plasmid

Part 2A: BioBrick Assembly

• Order DNA synthesis for
  • BBa_K175001 (trbK) ✔
  • BBa_K175000 (trbC)
• Verify that trbK expression blocks conjugation ✔
• Place trbK on standard backbone and send to registry ✔
• Amplify and Transform BioBricks needed
  • BBa_I13522 (pTet-RBS-GFP-term-term) ✔
  • BBa_I714031 (oriTR) ✔
  • BBa_J13002 (pTet-RBS) ✔
  • BBa_E0840 (RBS-GFP-term-term) ✔
• Assemble Conjugation Testing Plasmid 1: [promoter][GFP generator][oriT] ✔
• Verify Conjugation Testing Plasmid 1 works. ✔
• Sequence Conjugation Testing Plasmid 1. **In Progress**
• Assemble Conjugation Testing Plasmid 2 [promoter][rbs][trbK][rbs][GFP][oriT] ✔
• Verify Conjugation Testing Plasmid 2 works. **In Progress**
• Sequence Conjugation Testing Plasmid 2. **In Progress**

Part 2B: Full Communication testing

• Electroporate Conjugation Testing Plasmid 2 into some R751 ΔoriT cells creating InitiatorCells (select for presence of both message and helper plasmid)
• Add InitiatorCells to a culture of R751 ΔoriT + ΔtrbK and observe signal propagation, characterize rate of signal propagation. Look for lethal zygosis issues.
• 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