User:James Chappell/ LuxR

Purification of LuxR

Aims:

Through our modeling it has become clear that in order to get the most effective Infecter detector system we will want the LuxR to be at steady state. This causes a fundamental problem with our construct 1 (pTet-LuxR-pLux-GFP) because it is unlikely that LuxR will reach steady state under control of the pTet promoter.
The Modeling Section for infecter detector shows how having LuxR at steady state changes the response.
We therefore are going to try to purify LuxR so we can add it to our reaction mixture.
We have based our pruification on several protocols to purify LuxR, both of which require plasmids overexpressing LuxR. We managed to obtain the following plasmids:

pMLU117 courtesy of Dr Everett Greenberg's Laboratory, contains LuxR with a His tag
pHK724 couretesy of Dr James Slock's Laboratory, contains LuxR

Status

We tried two seperate purification experiments with these two plasmids, in the end it was the pMLU117 that gave a positive results and is described below. The testing for pHK724 can be found on this link.......

Day 1 - Growing Cells

Inoculation of 100ml of media with BL21 E.coli containing the LuxR-His plasmid

Equipment

Plate containing BL21 (pMU117) colonies
Loop
Bunsen Burner
Incubator 37°C

Reagents

LB media containing 200ug of amp

Protocol

Collect equipment and set up bunsen burner
Pick 1 colony and using loop innoculate the 100ml of LB medium
Place flask in incubator at 37°C overnight

Day 2

Equipments

Stripettes
Bunsen Burner
Curvette
Spectrometer

Reagents

2 X 1Litres of LB media containing 200.amp
100ml of LB media inncolulated with BL21(pMU117) grown overnight

Protocol

Collect equipment and set up bunsen burner
Remove the 1ml of the 1 litre media and place in a curvette
Remove 50ml of the 100ml media grown overnight and put into 1 litre of LB meida. Repeat this process once again with the other litre of media.
Mix and place back in incubator at 37°C
After 3 hours check the O.D.₆₀₀. To do this first set the reference of the spectrometer by using a curvette containing 1ml, then place 1ml of our litre media and measure. If O.D.₆₀₀ is at 0.5 then remove cells from incubator.

Day 3

Purification

This protocol is based on the following protocol from the paper [1]
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

Making up Buffers
General Buffer
Total Volume: 1 litre

Tris-HCl: 0.05 x 131.4= 6.57g

Add 6.57g of Tris-HCl to a minimum amount of distilled water to dissolve it. Then add enough concentrated HCL to bring it to pH 7.0. Then top it up to 870ml with distilled water.

EDTA: 0.002 x 372.24= 0.744g

Add 0.744g of EDTA to the mixture.

DTT: 0002 x 154.3= 0.309g
10% glycerol

Add 100ml of 100% glycerol to the mixture.

0.5% of Tween-20

Add 5 ml of 100% Tween-20 to the mixture.

AHL stock 1mM

C1V1= C2V2 1 x V1= 0.025 x 1000ml Add 25ml of the stock solution to the mixture.

Give the mixture a good shake.

BUFFER A
Add NaCl and KCl to 1 liter of the general buffer.

NaCl: 0.05 x 58.442= 2.922g

Add 2.922g of NaCl to the mixture.

KCl: 0.1 x 74.55= 7.455g

Add 7.455g of KCl to the mixture.

Give it a good shake.

BUFFER A with 1M of NaCl
Add NaCl and KCl to 1 liter of the general buffer

NaCl: 1 x 58.442= 5.8442g

Add 5.8442g of NaCl to the mixture.

KCl: 0.1 x 74.55= 7.455g

Add 7.455g of KCl to the mixture.

Give it a good shake.

Buffer B
Add NaCl and KCl to 1 liter of the general buffer

NaCl: 0.025x 58.442= 1.4611g

Add 1.4611g of NaCl to the mixture.

KCl: 0.05 x 74.55= 3.7275g

Add 3.7275g KCl to the mixture.

Give it a good shake.

Prepare Cell Lysate

Colonies of BL21 with over expressing LuxR plasmid should be streaked onto LB-ampicillin plates and grown over night at 37 °C
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.
AHL 25uM 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
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:

The Cell pellet should be resuspended in 16ml of A buffer and 25uM AHL. This cell suspension should then be sonicated.
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

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.
The fractions collected need to see if they contain LuxR. This can be done using SDS-PAGE electrophoresis
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.
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.
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.
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.