Construction and Testing

From 2007.igem.org

Construction Tree

The diagram below outlines our parallel construction plan, from individual registry parts to the final half-adder. Because many people were working on the project at once, it is designed to keep everyone organised.

Construction Chart

Diagram details:

• Expected fragment sizes are shown at every node so gel results can be analyzed quickly.
• Vector resistances for each part/construct are shown, with shades of blue representing ampicillin and shades of red representing kanamycin.
• Parts and developing constructs all have abbreviated names for ease of labeling. To make the subtle distinction between a coding sequence and a coding sequence with a ribosome binding site, we use a ' on the computer and an underline when writing. A double ' or double underline indicates a coding sequence with a ribosome binding site and a transcriptional terminator.
• Arrows show the direction of DNA cloning and specify which restriction enzymes to use for cutting. Hollow arrows are present when two parts are ligated from vectors of the same resistance, requiring additional methods such as the use of alkaline phosphatase, gel extraction, or a three-way ligation into a vector of difference resistance.
• The numbers 1-2-3-3.5-4 show the major protocols that need to be carried at each step in the assembly cycle.

Testing Constructs

We use the fluorescent proteins GFP and RFP as reporter genes to test the functionality of some of the components and constructs of our half-adder design. Where possible, we make use of the lac inducible promoter to control expression of the enhancing or repressing elements.

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

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