Construction and Testing

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Revision as of 08:57, 26 October 2007

Contents

Construction

Construction Chart


Testing/Results

Testing Constructs

Making use of flurorescent proteins(GFP and RFP) as reporter genes - use of indicuble promoters to...

Comparing basal and induced expression levels for the quorum sensing promoters

CONSTRUCT AIMS
UW testing constructs1AB.png The quorum-sensing promoters: How active is 'unactivated' Plas and Plux?
  • preliminary test for the basal levels of activity of the positively-controlled quorum sensing promoters
UW testing constructs1CD.png The quorum-sensing constructs: How off is 'off' and how on is 'on'?</br>
  • test for levels of expression in the unenhanced versus enhanced state
  • xR must be expressed in same strain as Px; xI can be expressed in a different strain since its AHL is diffusable
  • both strains grown in a single culture tube


Testing functionality of the various promoters

CONSTRUCT AIMS
UW testing constructs2A1A2.png The Omp promoter
  • postively-controlled promoter activated by endogenous OmpR protein (sensitive to salt concentrations via EnvZ protein)
  • test for activity using high salt levels in EnvZ+ strain
  • test for activity using the photo-active red system in EnvZ- strain
UW testing constructs2B.png The Tet promoter and operator
  • negatively-controlled promoter, repressed by TetR and derepressed by the inducer tetracycline
  • test for levels of expression during repressed versus derepressed state
UW testing constructs2CD.png The quorum-sensing promoters: Plas and Plux
  • as above
UW testing constructs2E.png The PlasOcI promoter-operator fusion
  • negatively- and positively-controlled promoter in the final half adder design, repressed by cI and enhanced by LasR
  • testfor activity in the repressed versus enhanced state
  • test in addition to above construct since operator fusion may affect promoter activity