Phase 2: Plasmid Construction and Bacterial Transformations

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'''2.  Ligated ComQX/P/A and Tsr fragments from Phase 1 into pSB1A3 plasmids'''
'''2.  Ligated ComQX/P/A and Tsr fragments from Phase 1 into pSB1A3 plasmids'''
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::*Used SpeI and XbaI restriction sites
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::*Cut at EcoRI, XbaI, and PstI restriction sites (cutting at XbaI site reduces background)
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::*pSB1A3 contains amp resistance
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::*Concentration of digested vector and inserts determined.  Ligations were set up for between 40:150 to 40ng:300ng ratio vector:insert
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'''3.  Bacterial transformation'''
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::*Used ''E.coli'' strain RP8611 (null for all MCP receptors, provided by John S. Parkinson, University of Utah)
::*Verified transformation for all fragments and sequenced genes (results positive in all cases).
::*Verified transformation for all fragments and sequenced genes (results positive in all cases).
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'''3.  Significance:
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'''4.  Significance:
::*We now have all the fundamental components for the project ahead
::*We now have all the fundamental components for the project ahead
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::*Can proceed in constructing the chimera using ComP and Tsr gene fragments
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::*Can proceed in constructing the hybrid quorumtaxis receptor using ComP and Tsr genes
::*Will need to perform quick-change mutagenesis for ComA to remove internal SpeI site
::*Will need to perform quick-change mutagenesis for ComA to remove internal SpeI site
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[[Rice University 2006|Back to Rice iGEM]]

Latest revision as of 06:25, 2 November 2006

1. Isolated ptetRBS and pSB1A3 plasmids from BioBricks

  • Attached tet promoter with Ribosome Binding Site to GFP gene, transformed XL-1 E. coli and observed positive expression



2. Ligated ComQX/P/A and Tsr fragments from Phase 1 into pSB1A3 plasmids

  • Cut at EcoRI, XbaI, and PstI restriction sites (cutting at XbaI site reduces background)
  • pSB1A3 contains amp resistance
  • Concentration of digested vector and inserts determined. Ligations were set up for between 40:150 to 40ng:300ng ratio vector:insert



3. Bacterial transformation

  • Used E.coli strain RP8611 (null for all MCP receptors, provided by John S. Parkinson, University of Utah)
  • Verified transformation for all fragments and sequenced genes (results positive in all cases).



4. Significance:

  • We now have all the fundamental components for the project ahead
  • Can proceed in constructing the hybrid quorumtaxis receptor using ComP and Tsr genes
  • Will need to perform quick-change mutagenesis for ComA to remove internal SpeI site



Back to Rice iGEM

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