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From 2012.igem.org

Implementation

Contents 1 Implementation 1.1 Plasmid 1 1.2 Plasmid 2 1.3 Plasmid 3 1.4 Measurement

The following plasmid constructs are needed to implement the measurement system. Using two receptors, two signalling systems and two reporter genes the implementation contains redundancy to select for a sensitive system.

Plasmid 1

Task: Expression of membrane receptor.

backbone pRS#	part #1	part #2	part #3
313	Padh1	mPR Danio rerio	Tadh1
313	Padh1	mPR Xenopus laevis	Tadh1

Plasmid 2

Task: Signalling, inhibitor controlled by membrane receptor through Pfet3.

backbone pRS#	part #1	part #2	part #3
315	Pfet3	rox1	Tadh1
targets Plasmid 3 with Panb1
315	Pfet3	mig1	Tadh1
targets Plasmid 3 with Psuc2

Plasmid 3

Task: Expression of reporter gene, controlled by inhibitor (plasmid 2).

backbone pRS#	part #1	part #2	part #3
316	Panb1	luciferase	Tadh1
316	Panb1	lacZ	Tadh1
316	Psuc2	luciferase	Tadh1
316	Psuc2	lacZ	Tadh1

Measurement

The measurement itself should be limited to an optical one. With this idea in mind, we decided for reporter genes like lacZ and luciferase, because these produce signals which can simply be quantified by optical measurement methods.