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From 2010.igem.org

generation of subparts, linkers and vectors

subparts

	Protein	primers	PCR product	library of transformants	single colonies	storage vector	insert
che proteins	CheY-A	recieved	purified on stock	on agar plate	not available	purified on stock	on stock
	CheY-C	recieved	purified in stock	on agar plate	not available	purified on stock	on stock
	CheB-A	recieved	purified in stock	on agar plate	3 on agar plate	purified on stock	on stock
	CheB-C	recieved	purified in stock	on agar plate	3 on agar plate	purified on stock	on stock
	CheR-A	recieved	purified in stock	on agar plate	3 on agar plate	digest has to be redone
	CheR-C	recieved	purified in stock	on agar plate	3 on agar plate	digest has to be redone
localizers	TrigF-A	recieved	purified in stock	on agar plate	3 on agar plate	purified on stock
	MreB-C	recieved	purified in stock	in process
	tetR-A	recieved	purified in stock	on agar plate	3 on agar plate	purified on stock	on stock
couplers	PIF3-A	recieved	purified in stock	on agar plate	3 on agar plate	purified on stock
	PIF3-C	recieved	purified in stock	on agar plate	3 on agar plate	purified on stock
	PhyB-C	recieved	purified in stock	on agar plate	3 on agar plate	purified on stock
reporters	cyFP-A	recieved	purified in stock	on agar plate	3 on agar plate	purified on stock	on stock
	cyFP-C	recieved	purified in stock	on agar plate	3 on agar plate	purified on stock

Linker oligos are recieved and don't need to be treated further. They can be used directly for the generation of the BioBricks.
storage and acceptor vectors are available.
PIF3 and PhyB were additionally ordered for synthesis (codon optimized). Not yet recieved.

generation of biobricks

We'll talk to Fabian. Apparently he has a nice protocol to generate acceptor vectors and transform E. coli sequentially in a 96 well plate.

testing of biobricks

testing of che-fusions

We're currently trying to establish an assay to test for the chemotactic behavour of E. coli. A first try with a "syringe test" failed. We'll redo the test and are looking for other possibilites. Maybe a swarm test?

• tested biobricks: che-fusions
• negative control: E. coli mutated for the accoding che-protein
• positive control: wild type E. coli

testing of localicer-fusions

We are planning to use a fluorescence microscope in order to visualize the distribution of localizer-cyFP fusions within a single E. coli.

testing of couplers

We're planning to use the setup as for the testing of the localizers.

• tested biobricks: localizer-PhyB and PIF3-cyFP, localizer-PIF3 and PhyB-cyFP

Images should look as in testing of localizer-fusions. Preliminary clarificaiton of the suitability of the approach are to be done.

bringing E. lemming alive

timetable