Team:TUDelft/Conjugation Procedure

From 2009.igem.org

(Difference between revisions)
(→Experimental Procedures)
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* Confirm wild R751 conjugation [https://2009.igem.org/Team:TUDelft/6_August_2009 <font color=limegreen>&#10004;</font>]
* Confirm wild R751 conjugation [https://2009.igem.org/Team:TUDelft/6_August_2009 <font color=limegreen>&#10004;</font>]
* Characterize conjugation efficiency [https://2009.igem.org/Team:TUDelft/6_August_2009 <font color=limegreen>&#10004;</font>]
* Characterize conjugation efficiency [https://2009.igem.org/Team:TUDelft/6_August_2009 <font color=limegreen>&#10004;</font>]
 +
Part 1B: oriTR knockout
Part 1B: oriTR knockout
* Design and order primers needed for &lambda;-red knockout [https://2009.igem.org/Team:TUDelft/7_August_2009#Calin <font color=limegreen>&#10004;</font>]
* Design and order primers needed for &lambda;-red knockout [https://2009.igem.org/Team:TUDelft/7_August_2009#Calin <font color=limegreen>&#10004;</font>]
* Acquire knockout plasmids [https://2009.igem.org/Team:TUDelft/10_August_2009#Calin <font color=limegreen>&#10004;</font>]
* Acquire knockout plasmids [https://2009.igem.org/Team:TUDelft/10_August_2009#Calin <font color=limegreen>&#10004;</font>]
-
* Knockout oriTR <font color=blue>**In Progress**</font>
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* Knockout oriTR <font color=blue><b>**In Progress**</b></font>
-
* Verify that conjugation stopped <font color=blue>**In Progress**</font>
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* Verify that conjugation stopped <font color=blue><b>**In Progress**</b></font>
* Characterize conjugation efficiency of Conjugation Testing Plasmid 1 with R751 &Delta;oriTR as helper
* Characterize conjugation efficiency of Conjugation Testing Plasmid 1 with R751 &Delta;oriTR as helper
* Send R751 &Delta;oriTR plasmid to registry
* Send R751 &Delta;oriTR plasmid to registry
 +
Part 1C: trbK knockout
Part 1C: trbK knockout
-
* Knockout trbK
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* Knockout trbK <font color=blue><b>**In Progress**</b></font>
-
* Electroporate into cells
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* Verify that conjugation takes place among R751 &Delta;trbK cells
-
* Verify that conjugation takes place among R+ cells using PlasmidG (see below)
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* Characterize conjugation efficiency
* Characterize conjugation efficiency
-
* If works send R751 oriT + trbK knockout plasmid to registry
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* Send R751 &Delta;oriT + &Delta;trbK plasmid to registry
 +
 
 +
 
Part 1D: trbC knockout
Part 1D: trbC knockout
-
* Knockout oriT + trbK + trbC
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* Knockout trbC
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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 Conjugation Testing Plasmid 1
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* Verify that no conjugation takes place in presence of PlasmidG
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* Send R751 &Delta;oriT + &Delta;trbK + &Delta;trbC plasmid to registry
-
* If works send R751 oriT + trbK + trbC knockout plasmid to registry
+
=='''Section 2: Message Plasmid'''==
=='''Section 2: Message Plasmid'''==
-
* Synthesize trbK gene [https://2009.igem.org/Team:TUDelft/7_August_2009#Calin <font color=limegreen>&#10004;</font>]
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Part 2A: BioBrick Assembly
 +
* Order DNA synthesis for
 +
** [http://partsregistry.org/wiki/index.php?title=Part:BBa_K175001 BBa_K175001] (trbK) [https://2009.igem.org/Team:TUDelft/7_August_2009#Calin <font color=limegreen>&#10004;</font>]
 +
** [http://partsregistry.org/wiki/index.php?title=Part:BBa_K175000 BBa_K175000] (trbC)
* Verify that trbK expression blocks conjugation [https://2009.igem.org/Team:TUDelft/19_August_2009#Calin <font color=limegreen>&#10004;</font>]
* Verify that trbK expression blocks conjugation [https://2009.igem.org/Team:TUDelft/19_August_2009#Calin <font color=limegreen>&#10004;</font>]
 +
* Amplify and Transform BioBricks needed
 +
** [http://partsregistry.org/wiki/index.php?title=Part:BBa_E0840 BBa_E0840] (GFP generator) <font color=limegreen>&#10004;</font>
 +
** [http://partsregistry.org/wiki/index.php?title=Part:BBa_B0034 BBa_B0034] (strong rbs) <font color=limegreen>&#10004;</font>
 +
** [http://partsregistry.org/wiki/index.php?title=Part:BBa_J23100 BBa_J23100] (constitutive promoter) <font color=limegreen>&#10004;</font>
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** [http://partsregistry.org/wiki/index.php?title=Part:BBa_I714031 BBa_I714031] (oriT-R) <font color=limegreen>&#10004;</font>
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* Assemble Conjugation Testing Plasmid 1: [promoter][GFP generator][oriT] <font color=limegreen>&#10004;</font>
 +
* Verify Conjugation Testing Plasmid 1 works. <font color=limegreen>&#10004;</font>
 +
* Sequence Conjugation Testing Plasmid 1. <font color=blue><b>**In Progress**</b></font>
 +
* Assemble Conjugation Testing Plasmid 2 [promoter][rbs][trbK][rbs][GFP][oriT] <font color=limegreen>&#10004;</font>
 +
* Verify Conjugation Testing Plasmid 2 works. <font color=blue><b>**In Progress**</b></font>
 +
* Sequence Conjugation Testing Plasmid 2. <font color=blue><b>**In Progress**</b></font>
-
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).
 
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*Assemble: [oriT][promoter] and keep this for later
 
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*Assemble PlasmidG: [oriT][promoter][GFP generator] + plasmid backbone with different resistance than helper plasmid and low copy number.
 
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*Assemble [oriT][promoter][rbs][trbC][rbs][trbK] and keep this intermediate assembly product for possible use in integration stage
 
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*Assemble [oriT][promoter][rbs][trbC][rbs][trbK][GFP generator] and keep this intermediate assembly product for possible use in integration stage
 
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*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
Part 2B: Full Communication testing
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*Electroporate PlasmidCKG into some cells from cultureCom creating initiatorCells
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* Electroporate Conjugation Testing Plasmid 2 into some R751 &Delta;oriT cells creating InitiatorCells (select for presence of both message and helper plasmid)
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*Select for presence of both message and helper plasmid
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* Add InitiatorCells to a culture of R751 &Delta;oriT + &Delta;trbK and observe signal propagation, characterize rate of signal propagation. Look for lethal zygosis issues.
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*Mix initiatorCells with cells not containing any plasmids and characterize conjugation efficiency
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* If signal propagation observed, do victory dance.
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*Add initiatorCells to cultureCom and observe signal propagation, characterize rate of signal propagation.
+
 
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*If signal propagation observed, do victory dance.
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'''For more information on the cloning strategy of constructing the conjugation plasmids and knockouts check the [[Team:TUDelft/Conjugation_Cloning|cloning strategy]] page'''
'''For more information on the cloning strategy of constructing the conjugation plasmids and knockouts check the [[Team:TUDelft/Conjugation_Cloning|cloning strategy]] page'''
 +
 +
{{Template:TUDelftiGEM2009_end}}
{{Template:TUDelftiGEM2009_end}}

Revision as of 18:05, 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 **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
    • [http://partsregistry.org/wiki/index.php?title=Part:BBa_K175001 BBa_K175001] (trbK) ✔
    • [http://partsregistry.org/wiki/index.php?title=Part:BBa_K175000 BBa_K175000] (trbC)
  • Verify that trbK expression blocks conjugation ✔
  • Amplify and Transform 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 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


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