IGEM:IMPERIAL/2006/Protocols/S01656new

New Protocol to test aiiA Production

This can be easily adapted to measure AHL conc every hour so that you get a curve. However we will not need to do this for this aiiA part as the aiiA gene is untagged and may have different properties to the aiiA we will actually be using This test is solely to see if this gene works.

Controlls

-IPTG
Non aiia producing cells (gives idea of background breakdown of AHL in medium containing live cells)

Grow cultures of S01656 (4ml) and B0034 (2ml) overnight JS: What is B0034 used for? Isn't B0034 just an RBS?
Make Fresh Day Cultures (10ml) - Make 2 S01656 cultures and one B0034 culture they should have an optical density of 0.1 and you should use Pre-warmed media to prevent shock. Remember that the S01656 grows in kanomycin media.
You Should now have three cultures
Wait for 2 hours
Add AHL to eppindorf tubes (seven different concentrations) Use the AHL dilution series below Do we not need to dilute down to OD of 0.1 again to prevent going into the exponential phase?

Sample (ul)	Stock Concentration	AHL (ul)	Final AHL Concentration
990	1000uM	10	10uM
990	100uM P\|10	1uM
990	10uM	10	100nM
990	5uM	10	50nM
990	1uM	10	10nM
990	500nM	10	5nM
990	100nM	10	1nM

You should add 10ul of AHL to each eppindorf tube and 0.99ml of culture to each eppindorf in such a way that you have 21 tubes and each cell type has 7 tubes of different AHL concentrations
Add 50ul of IPTG to the + IPTG group of S01656 to induce aiiA production
Wait for 7 hours JS: what is the justification for keeping at 7 hours? I know that we were doing the experiment at 4 hours, but what is to say that 7 hours will work? Have we gotten any results from this experiment?
centrifuge down cells
Pipette off supernatant and freeze
Tomorrow use T9002 Protocol to measure amount of AHL

Any questions see JohnC