Construction and Testing

From 2007.igem.org

Revision as of 12:26, 26 October 2007

Construction Tree

The diagram below outlines our parallel construction plan, from individual registry parts to the final half-adder. Expected fragment sizes are shown at every node.

Construction Chart

Diagram details:

• Vector resistances are shown, with shades of blue representing ampicillin and shades of red representing kanamycin

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
	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
	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
	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
	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
	The quorum-sensing promoters: Plas and Plux as above
	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