Team:TUDelft/Conjugation Procedure
From 2009.igem.org
(→Experimental Procedures) |
(→Experimental Procedures) |
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Part 1A: | Part 1A: | ||
* Acquire R751 plasmid [https://2009.igem.org/Team:TUDelft/31_July_2009#Calin <font color=limegreen>✔</font>] | * Acquire R751 plasmid [https://2009.igem.org/Team:TUDelft/31_July_2009#Calin <font color=limegreen>✔</font>] | ||
- | * Confirm wild R751 conjugation [ | + | * Confirm wild R751 conjugation [[Team:TUDelft/Conjugation_Results | <font color=limegreen>✔</font>]] |
- | * Characterize conjugation efficiency [ | + | * Characterize conjugation efficiency [[Team:TUDelft/Conjugation_Results | <font color=limegreen>✔</font>]] |
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** [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>✔</font>] | ** [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>✔</font>] | ||
** [http://partsregistry.org/wiki/index.php?title=Part:BBa_K175000 BBa_K175000] (trbC) | ** [http://partsregistry.org/wiki/index.php?title=Part:BBa_K175000 BBa_K175000] (trbC) | ||
- | * Verify that trbK expression blocks conjugation [ | + | * Verify that trbK expression blocks conjugation [[Team:TUDelft/Conjugation_Results | <font color=limegreen>✔</font>]] |
* Place trbK on standard backbone [https://2009.igem.org/Team:TUDelft/11_August_2009#Calin <font color=limegreen>✔</font>], sequence [https://static.igem.org/mediawiki/2009/9/9e/V92371145_E_VF_QS.txt <font color=limegreen>✔</font>], and send to registry <font color=limegreen>✔</font> | * Place trbK on standard backbone [https://2009.igem.org/Team:TUDelft/11_August_2009#Calin <font color=limegreen>✔</font>], sequence [https://static.igem.org/mediawiki/2009/9/9e/V92371145_E_VF_QS.txt <font color=limegreen>✔</font>], and send to registry <font color=limegreen>✔</font> | ||
* Amplify and Transform BioBricks needed | * Amplify and Transform BioBricks needed | ||
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** [http://partsregistry.org/wiki/index.php?title=Part:BBa_E0840 BBa_E0840] (RBS-GFP-term-term) <font color=limegreen>✔</font> | ** [http://partsregistry.org/wiki/index.php?title=Part:BBa_E0840 BBa_E0840] (RBS-GFP-term-term) <font color=limegreen>✔</font> | ||
* Assemble Conjugation Testing Plasmid 1: [promoter][GFP generator][oriT] <font color=limegreen>✔</font> | * Assemble Conjugation Testing Plasmid 1: [promoter][GFP generator][oriT] <font color=limegreen>✔</font> | ||
- | * Verify Conjugation Testing Plasmid 1 works. [ | + | * Verify Conjugation Testing Plasmid 1 works. [[Team:TUDelft/Conjugation_Results | <font color=limegreen>✔</font>]] |
* Sequence Conjugation Testing Plasmid 1. <font color=blue><b>**In Progress**</b></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>✔</font> | * Assemble Conjugation Testing Plasmid 2 [promoter][rbs][trbK][rbs][GFP][oriT] <font color=limegreen>✔</font> |
Revision as of 20:58, 19 October 2009
Experimental Procedures
This page contains the step-by-step plan followed by the conjugation team and each steps current status.
Section 1: Helper Plasmid
Section 1: The Plan
Part 1A:
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
Knockouts
The λ-red knockout system was selected as the procedure for doing the knockouts. It had previously been used by several people and demonstrated to work ([http://openwetware.org/wiki/Recombineering/Lambda_red-mediated_gene_replacement lambda red], [http://openwetware.org/wiki/NanoBio:_Protocol_for_gene_knockout NanoBio], [http://openwetware.org/wiki/Berk2006-ConjugationTeam knockout protocol used by Berkeley '06]). Primers were designed following the standard [http://openwetware.org/wiki/NanoBio:_Primer_Design procedure]. The following primers were used:
trbK_KO_FWD
trbK_KO_REV
oriTR_KO_FWD
oriTR_KO_REV
Linear fragments were created using pKD4 (KAN) as a template using Platinum® Pfx DNA Polymerase. See Results page for more info.
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 ✔
- Place trbK on standard backbone ✔, sequence ✔, and send to registry ✔
- Amplify and Transform BioBricks needed
- [http://partsregistry.org/Part:BBa_I13522 BBa_I13522] (pTet-RBS-GFP-term-term) ✔
- [http://partsregistry.org/wiki/index.php?title=Part:BBa_I714031 BBa_I714031] (oriTR) ✔
- [http://partsregistry.org/wiki/index.php?title=Part:BBa_J13002 BBa_J13002] (pTet-RBS) ✔
- [http://partsregistry.org/wiki/index.php?title=Part:BBa_E0840 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
On to the Experimental Results >>>