Team:TUDelft/Conjugation Procedure

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==Experimental Procedures==
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=Experimental Procedures=
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'''Section 1: Helper Plasmid'''<br>
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=='''Section 1: Helper Plasmid'''==
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Part 1A:
Part 1A:
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*Acquire R751 and/or RP4 plasmid
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* Acquire R751 plasmid [https://2009.igem.org/Team:TUDelft/31_July_2009#Calin <font color=limegreen>&#10004;</font>]
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*Electoporate plasmid into cells and confirm conjugation. See [http://openwetware.org/wiki/Conjugation conjugation protocol]  
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* Confirm wild R751 conjugation [https://2009.igem.org/Team:TUDelft/6_August_2009 <font color=limegreen>&#10004;</font>]
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*Characterize conjugation efficiency
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* Characterize conjugation efficiency [https://2009.igem.org/Team:TUDelft/6_August_2009 <font color=limegreen>&#10004;</font>]
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Part 1B: oriT knockout
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*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]
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Part 1B: oriTR knockout
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*Electroporate into cells
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* Design and order primers needed for &lambda;-red knockout [https://2009.igem.org/Team:TUDelft/7_August_2009#Calin <font color=limegreen>&#10004;</font>]
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*Verify that conjugation stopped
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* Acquire knockout plasmids [https://2009.igem.org/Team:TUDelft/10_August_2009#Calin <font color=limegreen>&#10004;</font>]
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*Electroporate PlasmidG as well and characterize conjugation efficiency
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* Knockout oriTR <font color=blue>**In Progress**</font>
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*If works send R751 oriT knockout plasmid to registry
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* Verify that conjugation stopped <font color=blue>**In Progress**</font>
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* Characterize conjugation efficiency of Conjugation Testing Plasmid 1 with R751 &Delta;oriTR as helper
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* Send R751 &Delta;oriTR plasmid to registry
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Part 1C: trbK knockout
Part 1C: trbK knockout
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*Knockout oriT + trbK
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* Knockout trbK
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*Electroporate into cells
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* Electroporate into cells
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*Verify that conjugation takes place among R+ cells using PlasmidG (see below)
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* Verify that conjugation takes place among R+ cells using PlasmidG (see below)
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*Characterize conjugation efficiency
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* Characterize conjugation efficiency
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*If works send R751 oriT + trbK knockout plasmid to registry
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* If works send R751 oriT + trbK knockout plasmid to registry
Part 1D: trbC knockout
Part 1D: trbC knockout
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*Knockout oriT + trbK + trbC
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* Knockout oriT + trbK + trbC
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*Electroporate into cells and create culture for communication (cultureCom)  
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* Electroporate into cells and create culture for communication (cultureCom)  
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*Verify that no conjugation takes place in presence of PlasmidG
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* Verify that no conjugation takes place in presence of PlasmidG
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*If works send R751 oriT + trbK + trbC knockout plasmid to registry
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* If works send R751 oriT + trbK + trbC knockout plasmid to registry
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'''Section 2: Message Plasmid'''<br>
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=='''Section 2: Message Plasmid'''==
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* Synthesize trbK gene [https://2009.igem.org/Team:TUDelft/7_August_2009#Calin <font color=limegreen>&#10004;</font>]
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* Verify that trbK expression blocks conjugation [https://2009.igem.org/Team:TUDelft/19_August_2009#Calin <font color=limegreen>&#10004;</font>]
Part 2A: BioBrick Assembly
Part 2A: BioBrick Assembly

Revision as of 17:44, 19 October 2009

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

Retrieved from "http://2009.igem.org/Team:TUDelft/Conjugation_Procedure"