User:James Chappell/ LuxR

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Contents

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:
  1. pMLU117 courtesy of Dr Everett Greenberg's Laboratory, contains LuxR with a His tag
  2. 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

  1. Collect equipment and set up bunsen burner
  2. Pick 1 colony and using loop innoculate the 100ml of LB medium
  3. 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

  1. Collect equipment and set up bunsen burner
  2. Remove the 1ml of the 1 litre media and place in a curvette
  3. 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.
  4. Mix and place back in incubator at 37°C
  5. 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

  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 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
  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

http://www.pubmedcentral.nih.gov/picrender.fcgi?artid=299138&blobtype=pdf https://commerce.metapress.com/content/ueejdk2a2k86ha6f/resource-secured/?target=fulltext.pdf&sid=34whnizlntndbrb3clhmemah&sh=www.springerlink.com

http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=321501


  • Additives to increase protein folding:

http://wolfson.huji.ac.il/purification/Protocols/Additives_Folding.html

  • General overview on purification of proteins:

http://wolfson.huji.ac.il/purification/PDF/Literature/Middelberg2002.pdf
http://wolfson.huji.ac.il/purification/index.html

  • Solutes for refolding proteins:

http://www.proteinscience.org/cgi/reprint/15/2/304

  • Protocol for purification of inclusions bodies from e.coli

http://www.bms.ed.ac.uk/research/others/smaciver/Protocols/Inclusions.htm http://www.fhcrc.org/science/labs/strong/StrongLabRefoldingProtocol.pdf http://www.bio.com/protocolstools/protocol.jhtml?id=p495#overview

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