IGEM:IMPERIAL/2007/Projects/Experimental Design/Notes

• Introduction
• Phase 1
• Phase 2
• Phase 3
• Notes

Purification of LuxR

Reagents and Equipment needed

• An over producing vector in LuxR
  • pHK724?
• R buffer (50 mM Tris, pH 7.8 at

5°C/200 mM NaCl/0.1 mM EDTA/0.1 mM dithiothreitol/5% glycerol)

• o.24mlx 50mM EDTA in 10% X-100
• 6M Guanidine Hydrochloride
• Fresh solution of lysozyme
• Dialysis tube
• Polyacrylamide electrophoresis

Protocol

1. Cells are grown and induced to produce LuxR. These cells are then isolated from the media by centrifugation to give a cell pellet weight of 0.6g
2. The pellet is removed and we resuspended in the 2.4ml R buffer. Ensure that the pellet has been completely resuspended.
3. Then add 0.24ml of 50mM EDTA in 10% X-100 and add a fresh solution of lysozyme for a final concentration of 150jg/ml. This solution should be incubated for 45 mins temp? .
4. After incubation the sample should be sonicated and centrifuged at 100,000 x g for 30 min

The suspension was incubated for 45 min, sonicated, and

1. The supernatant fluid should be removed and discarded. The pellet should then be suspended

in 2.4 ml R buffer that contains 6 M guanidine hydrochloride. This solution should then be incubated for 20 min.

1. After incubation remove the supernatant and place in a new tube. The supernatant should then be centrifuged for 30 min at 100,000 x g.
2. The supernatant is again removed into a new tube. The supernatant needs to then be diluted back to 1M guanidine hydrochloride by dropwise addition of RS buffer.
3. After the dilution to 1M guanidine hydrochloride, the solution is dialyzed first against RS buffer containing 0.5M guanidine hydrochloride and then dialyzed against an RS buffer containing 0.25M guanidine hydrochloride

Comparison of Batch mode and Plate mode

	Batch Mode	Plates
Pros	Minimises evaporation	Less samples used
	No temperature fluctuations	No pipetting involved from stock solution
Cons	A lot of cell extract is needed	Problem with evaporation
	Home-made cell extracts (by virtue of its large quantity) must be used	Fluctuation of temperature
	Home made cell extracts might not be reliable	Only 50µL is used per well, and this amount is less than the optimum amount for measurement by the fluorometer

Evaporation Problem: With volumes of 0.5 ~1.0 or less and incubation times longer than 30 minutes, each sample is covered with a 3 to 5 mm column of redistilled hexane. link The amount of samples we are looking at is about 100µl, so evaporation would not be a problem.