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.

Initial Testing for LuxR solubility

 * Add 0.5 litre of cells (grown to OD 0.5 and induced with AHL) to centrifuge tubes and spin them down at 8000g.
 * Remove the supernatant and dissolve the pellet in 10ml of Buffer A
 * Sonicate the mixture
 * Centrifuge them again at 100 000g and separate the supernatant from the pellet. Keep the supernatant (Tube A).
 * Resuspend the pellet in water (Tube B)to make up approximately the same volume as in Tube A.
 * Remove 100ul of each solution into another two tubes and add SDS page gel loading buffer to them.
 * Add 2ul of each of the solution to the SDS PAGE gell and run for 2 hours.

Purification 2
Reagents Equipment
 * This protocol is based on the following protocol from the paper Synbio2
 * It requires the plasmid pMLU117
 * A Buffer - 50mM Tris-HCL [pH 7.0 at 22&deg;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&deg;C] 50mM KCL, 25mM NaCl, 2mM EDTA, 2mM dithiothreitol, 10% glycerol, 0.5% Tween 20
 * 25uM solution AHL
 * 1 Litre LB-amp
 * NaCl gradients
 * Temp controlled Centrifuge
 * SP column
 * LB amp plates

Protocol
Making up Buffers General Buffer Total Volume: 1 litre 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. Add 0.744g of EDTA to the mixture. Add 100ml of 100% glycerol to the mixture. Add 5 ml of 100% Tween-20 to the mixture. C1V1= C2V2 1 x V1= 0.025 x 1000ml Add 25ml of the stock solution to the mixture.
 * Tris-HCl: 0.05 x 131.4= 6.57g
 * EDTA: 0.002 x 372.24= 0.744g
 * DTT: 0002 x 154.3= 0.309g
 * 10% glycerol
 * 0.5% of Tween-20
 * AHL stock 1mM
 * Give the mixture a good shake.

BUFFER A Add NaCl and KCl to 1 liter of the general buffer. Add 2.922g of NaCl to the mixture. Add 7.455g of KCl to the mixture.
 * NaCl: 0.05 x 58.442= 2.922g
 * KCl: 0.1 x 74.55= 7.455g
 * Give it a good shake.

BUFFER A with 1M of NaCl Add NaCl and KCl to 1 liter of the general buffer Add 5.8442g of NaCl to the mixture. Add 7.455g of KCl to the mixture.
 * NaCl: 1 x 58.442= 5.8442g
 * KCl: 0.1 x 74.55= 7.455g
 * Give it a good shake.

Buffer B Add NaCl and KCl to 1 liter of the general buffer Add 1.4611g of NaCl to the mixture. Add 3.7275g KCl to the mixture.
 * NaCl: 0.025x 58.442= 1.4611g
 * KCl: 0.05 x 74.55= 3.7275g
 * 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 &deg;C
 * 2) Colonies were used to inoculate a liter of LB-ampicillin prewarmed at 37 &deg;C, this is grown until an OD600 of 0.5 is reached, this is then chilled to 28 &deg;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 &deg;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 &deg;C and were frozen overnight at -80&deg;C
 * The following steps were performed at 4 &deg;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.

Comparison of Batch mode and Plate mode
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.

Options

 * 1) GFP
 * 2) *AcGFP -
 * 3) *GFP mut3b - need to get the purified form
 * 4) **Ask around!
 * 5) RFP
 * 6) *DsRed - wild type
 * 7) *DsRed2 - can be visualied but has a long response time ~ 24hrs
 * 8) *DsRed express - can be visualised with UV, has response time of ~ 7-8hrs

Proposed plan of action

 * 1) DsRed express - prioritise cloning and testing the constructs with DsRed Express
 * 2) *Currently testing DsRed Express in-vitro (Calibration curve)
 * 3) GFP mut 3b - carry on looking for purified form so calibration and degradation curves can be derived
 * 4) *Do research to find degradation and calibration data in literature
 * 5) DesRed 2 will be cloned just in case the other options don't work out

Flurometer

 * Might get access to 24hr fluorometer - programmed data collection (biochem lab, level 6)
 * Can carry out only the experiments at room temperature and degradation experiments