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

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

Purification of LuxR

• Through our modeling it has become clear that in order to get the most effective Infecter detector system we will need LuxR to be at steady state. This is a fundamental problem with our initial construct because it is unlikely that LuxR will reach steady state under control of the pTet promoter. We are therefore are pursuing an alternative where by we will add purified LuxR to the in vitro system.
• The Modeling Section on the infecter detector describes the initial modeling of both infecter detector constructs.
• Through literature a protocol that we can use to purify LuxR. We are planning to use this as a basis and modify it to suit our needs.
• For purification of LuxR we require a plasmid that can over express LuxR, we have identified two suitable plasmids and we are currently trying to obtain both. Depending on which one we choose we will have to vary the protocol.

Purification 1

• This protocol is based on the following protocol from the paper [1]
• It is dependent on the plasmid phk724 kindly provided by James Slock

Reagents and Equipment needed

• 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 The principle of the experiment is to over express Lux R and isolate this from the E.coli Cells. When LuxR is over expressed in E.coli it forms inclusion bodies, which are aggregates of insoluble misfolded proteins. Our aim is to isolate the LuxR inclusion bodies, and to re-solubalise the inclusion body and then to cause the refolding of the LuxR, to end up with highly concentrated and active sample of LuxR.

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 supernatant is carefully removed and discarded to leave the pellet. We then resuspended the pellet in 2.4ml R buffer.
3. Then add the following to the supernatant: 0.24ml of 50mM EDTA in 10% X-100 and a fresh solution of lysozyme (to give a final concentration of 150jg/ml). This solution should be incubated for 45 mins. The lysozymes will cause lysis of the Cells and the X-100 is to solubalise the membrane proteins, which would otherwise form inclusion bodies.
4. After incubation the sample should be sonicated and centrifuged at 100,000 x g for 30 min. This second sonication step is to lyse any unlysed cell.
5. From the sample the supernatant is again carefully removed and discarded, leaving just the pellet. This pellet is composed of insoluble proteins from the cell
6. The pellet should then be suspended in 2.4 ml R buffer containing 6 M guanidine hydrochloride. This solution should then be incubated for 20 min. This solution of 6 M guanidine hydrochloride will cause certain proteins such as LuxR to become soluble.
7. 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.
8. The supernatant is again removed into a new tube, this supernatant contains the soluble luxR proteins.
9. The supernatant needs to then be diluted back to 1M guanidine hydrochloride by dropwise addition of RS buffer. By slowly neutralizing the guanidine hydrochloride the LuxR is brought out of solubility and refolds back into a functional protein.
10. Finally the dilution of LuxR sample in 1M guanidine hydrochloride is dialyzed first against RS buffer containing 0.5M guanidine hydrochloride and then dialyzed against an RS buffer containing 0.25M guanidine hydrochloride
11. To check for purity of LuxR a SDS-Polyacrylamide gel should be run out. We should see a strong band at 27kDa

Purification 2

• • his protocol is based on the following protocol from the paper [2]
• It is dependent on the plasmid pMLU117
• The basis of the protocol is to over express a LuxR protein that contains an His tag. The over expressed protein is then isolated and denatured. This denatured LuxR-His is run through an affinity chromatography.
• Once LuxR has been isolated the protein is then refolded.

Experiment 1 - Affinity Chromatography

References

3. Kaplan HB and Greenberg EP. Overproduction and purification of the luxR gene product: Transcriptional activator of the Vibrio fischeri luminescence system. Proc Natl Acad Sci U S A. 1987 Oct;84(19):6639-43. DOI:10.1073/pnas.84.19.6639 |
4. Urbanowski ML, Lostroh CP, and Greenberg EP. Reversible acyl-homoserine lactone binding to purified Vibrio fischeri LuxR protein. J Bacteriol. 2004 Feb;186(3):631-7. DOI:10.1128/JB.186.3.631-637.2004 |
5. Bruist MF and Simon MI. Phase variation and the Hin protein: in vivo activity measurements, protein overproduction, and purification. J Bacteriol. 1984 Jul;159(1):71-9. DOI:10.1128/jb.159.1.71-79.1984 |
6. Georgiou G, Telford JN, Shuler ML, and Wilson DB. Localization of inclusion bodies in Escherichia coli overproducing beta-lactamase or alkaline phosphatase. Appl Environ Microbiol. 1986 Nov;52(5):1157-61. DOI:10.1128/aem.52.5.1157-1161.1986 |

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.