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

Making up of Buffers
R Buffer
Total Volume: 1 litre

1. Tris: 50 x 10-3 x 131.4= 6.57g

Add 6.57g to just enough dH2O so that it dissolves, equilibrate it to pH 7.8 with concentrated HCl, then top it up to 950ul with dH2O.

1. NaCl: 0.2 x 58.442= 11.6884g

Add 11.6884g of NaCl to the existing solution.

1. EDTA: 0.1 x 10-3 x 372.24= 0.037224g

Add 0.037224g of EDTA to the existing mixture.

1. DTT: 0.1 x 10-3 x 154.3= 0.01543g

Add 0.01543g of DTT to to the existing mixture.

1. 5% glycerol

Add 50ml to the mixture.

1. Give it a good mix.

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

• This protocol is based on the following protocol from the paper [2]
• It requires the plasmid pMLU117

Reagents

• A Buffer - 50mM Tris-HCL [pH 7.0 at 22°C] 100mM KCL, 50mM NaCl, 2mM EDTA, 2mM dithiothreitol, 10% glycerol, 0.5% Tween 20
• B Buffer - 50mM Tris-HCL [pH 7.0 at 22°C] 50mM KCL, 25mM NaCl, 2mM EDTA, 2mM dithiothreitol, 10% glycerol, 0.5% Tween 20
• 25uM solution AHL
• 1 Litre LB-amp
• NaCl gradients

Equipment

• Temp controlled Centrifuge
• SP column
• LB amp plates

Protocol

Prepare Cell Lysate

1. Colonies of BL21 with over expressing LuxR plasmid should be streaked onto LB-ampicillin plates and grown over night at 37 °C
2. Colonies were used to inoculate a liter of LB-ampicillin prewarmed at 37 °C, this is grown until an OD₆₀₀ of 0.5 is reached, this is then chilled to 28 °C.
3. AHL and IPTG to final concentration of 1mM is then added with additional ampicillin (200ug/ml) and the culture continued to grown at 28 °C for 4 hours, this step is when we want to express the LuxR protein and so we need to first induce with IPTG, then add AHL for correct LuxR protein folding and finally a lower temperature to try to prevent inclusion body formation
4. Cells were harvested by centrifugation at 8000 x g for 10mins at 4 °C and were frozen overnight at -80°C

• The following steps were performed at 4 °C:

1. The Cell pellet should be resuspended in 16ml of A buffer and 25uM AHL. This cell suspension should then be sonicated.
2. After sonication the lysate was centrifuged at 20,000 x g for 10 min. After this, remove the supernatant and centrifuge again at 100,000 x g for 1.5h.

Chromatography

1. The cell extract from 100,000 x g is passed through a 5ml HiTrap herparin column and equilibrated with A buffer and 25uM AHL. Bound proteins are then eluted with a 25ml NaCL step gradient from 0.15 to 1M.
2. The fractions collected need to see if they contain LuxR. This can be done using SDS-PAGE electrophoresis
3. Now we need to remove the high levels of NaCl from our cell extract. Fractions containing LuxR are then pooled and dialyzed against 500ml of A buffer containing 25uM AHL and 20% (wt/vol) polyethylene glycerol(mw 8000)for four hours.
4. After dialysis the sample was diluted 1:1 with a KCL and NaCL free version of A buffer plus 25uM AHL, this gives a final salt concentration of 75mM.
5. Now we need to prepare a SP column, this needs to be equilibrated with buffer B and 25uM AHL.Then the sample can be run through and washed with B buffer.
6. Finally, to remove the bound protein the SP column can be eluted with 100ml linear NaCl gradients of 0.075 to 0.5M. To test fraction content and purity of LuxR, SDS-PAGE can be carried out.

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.