Predator Transfer Function Characterisation

This experiment should give us a value for the rate of LuxR and aiiA production as a function of the total amount of HSL in the medium. It should also allow us to work out the Km and Vm for the binding of LuxR-AHL with luxpR. 

Total amount of HSL = Amount of HSL we add to the medium = Free HSL + HSL bound to LuxR 

This should aid us in putting realistic values into our models

Modelling

For each value of HSL conc (assuming HSL is constant) a steady state of GFP (blue line) should be reached.

•	Green = AHL
•	Blue = GFP
•	Light Blue = LuxR
•	Red = Gene
•	Yellow = LuxR+AHL

As transcription at lux pR is proportional to the amount of LuxR+HSL which is dependent on the amount of HSL added the rate of GFP production will be proportional to the amount of HSL added.

If we know the Rate of GFP degradation and the equilibrium conc of GFP then we can work out the rate of GFP synthesis for that amount of HSL as degradation = synthesis at equilibrium.

If we know the rates and the substrate conc for those rates we can make a lineweaver burke plot. Plot 1/v against 1/[s].

1 / V = (Km / Vm)(1 / [S]) + (1 / Vm)
Y   =         M         X    +      C

This will allow us to work out the real values of Km and Vm for LuxR+AHL when it binds LuxPr, 

Notes:
The model is very sensitive to the Km of luxR ie it should be as high as possible (therefore as low an affinity). It may be possible to increase the apparent Km by adding a competitive inhibitor plasmid or a competing but non-functional activator (such as a synthetic zinc finger, (I don’t know how hard/expensive they are to make))

Experiment

Part J37016: Characterization
1.	Inoculate eight different cultures from a single bacterial colony in growth medium containing appropriate antibiotic and aTc (at at least 50ng/ml). 
2.	Incubate at 37oC for 15 hours in a shaker. 
3.	Remove cultures from shaker and dilute by a factor of 10 into fresh medium. 
4.	Incubate at 37oC for 2 hours in a shaker. 
5.	While incubating, make up a solution of 200uL 100uM AHL (stock). 
6.	Serial dilute to give final stock concentrations of 100uM, 10uM, 1uM, 100nM, 10nM, 1nM, 0.1nM, 0nM AHL. 
7.	Add the following to 8 separate flat bottom wells. Repeat for each of the bacterial colonies (total 64 wells) 
8.	Add the aTc to the plates

Stock Concentration 	AHL Added (ul) 	Water Added(ul) 	Final AHL Concentration 	
100uM 	20 	0 	10uM 	
10uM 	20 	0 	1uM 	
1uM 	20 	0 	100nM 	
100nM 	20 	0 	10nM 	
10nM 	20 	0 	1nM 	
1nM 	20 	0 	0.1nM 	
0.1nM 	20 	0 	0.01nM 	
N/A 	0 	20 	0nM 	

1.	Add 200uL of growth medium to a well to act as a control. 
2.	Transfer 8 180ul samples of one of the cultures into flat-bottom wells into the 8 different AHL concentrations. 
3.	Repeat for the remaining 7 cultures. 
4.	Grow the cultures in a Wallac Victor 3 multi-well fluorimeter (or alternative apparatus) at 37 oC. 
5.	Assay with an automatically repeating protocol of absorbance measurements (600nm filter, 0.1 second absorbance through approximately 0.5cm of fluid), fluorescence readings (488nm excitation filter, 525nm emission filter, 0.5 second, CW lamp energy 12902 units), and shaking (1 mm, linear, normal speed). 
6.	Take measurements every 3 minutes (if automatic) or 30 minutes (if manual). 
7.	Continue until no more change is observed. 

This experiment should give you the GFP concentration at steady state for each concentration of HSL
Depending on the rates of degradation of HSL the output may look like this

X-axis – time
Y-axis – GFP expression
Different lines = different AHL conc yellow is the lowest, red highest
Here the concentration of HSL is not constant ie. It can degrade and be used by the bacteria. You get a peak where the max hight of the peak is proportional to the amount of HSL added then the peak falls as HSL gets degraded. The measurement should be taken at the peak.

John Chattaway