Team:TUDelft/Conjugation Procedure

From 2009.igem.org

(Difference between revisions)
(Knockouts)
Line 1: Line 1:
{{Template:TUDelftiGEM2009_menu_M1_conj}}
{{Template:TUDelftiGEM2009_menu_M1_conj}}
-
=Experimental Procedures=
+
='''Experimental Procedures'''=
=='''Section 1: Helper Plasmid'''==
=='''Section 1: Helper Plasmid'''==
Line 80: Line 80:
'''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'''
-
 
+
On to the [[Team:TUDelft/Conjugation_Results | Experimental Results >>>]]
{{Template:TUDelftiGEM2009_end}}
{{Template:TUDelftiGEM2009_end}}

Revision as of 19:25, 19 October 2009

Experimental Procedures

Section 1: Helper Plasmid

Section 1: The Plan

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

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 (lambda red, NanoBio, knockout protocol used by Berkeley '06). Primers were designed following the standard 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
  • Verify that trbK expression blocks conjugation
  • Place trbK on standard backbone , sequence , and send to registry
  • Amplify and Transform BioBricks needed
  • 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 >>>