IGEM:IMPERIAL/2007/Projects/chassis/Notes

 Introduction Specifications Design Testing Notes   =Common Protocols:Comparison of Home-Made and Commercial Cell Extract= Commercial cell extracts are expensive, and since we are going to make a much larger quantity of cell extract, it would be sensible to use our home-made cell extracts for experimentations for both our applications.

Construct: pTet - GFP

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=Common Protocols:GFP=

1)Calibration Curve
Test to determine the relationship between fluorescence and in vitro concentration of GFP. To test this purified samples of known [GFP] are added to in vitro chassis and the fluorescence measured. From this a calibration curve of [GFP] vs Fluorescence can be made. This can be used for data analysis to convert fluorescence into a [GFP]. Aims: Constant Conditions:
 * To determine [GFP] vs Fluorescence

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2)Degradation Time
Test the half life of GFP protein in an in vitro chassis. To test this a purified sample of known [GFP] are added to an in vitro chassis, then fluorescence will be measured at regular time intervals. The fluorescence will be converted into GFP molecules using the calibration curve. This will give; degradation of GFP as a function of time, from this the half life of GFP can be obtained. In addition, temperature may affect the half life of GFP and so the half life will be measured for an appropriate temperature range. Aims: Constant Conditions:
 * To determine the half life of GFP for a range of temperatures

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=Protocol:Infector Detector=

1)Initial testing
Construct: pTet - LuxR - pLux - GFP Test to see if construct will express in vitro. Experiments carried out under room temperature 25oC and under inducer concentration that was shown to give a high induction in vivo. Aims: Constant Conditions:
 * To determine if construct expresses in vitro
 * To get approximations of: life span, response time and rate of GFP
 * To determine whether the constructs or the in vitro need to be optimised.

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1)Test for Steady State of LuxR Protein Expression
Construct: pTet - LuxR To determine the appropriate time for addition of AHL into the system for induction of pLux, we need to confirm that the amount of LuxR in the system is at a steady state. This is to ensure that the amount of LuxR does not affect the rate of production of reporter protein when the concentration of AHL is varied.

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2)Preliminary AHL Sensitivity Testing
Construct: pTet - LuxR - pLux - GFP To determine the sensitivity of the construct to AHL concentration. To do this, we induce in vitro chassis containing the construct, with known concentrations of AHL. We then record the change in GFP, such that we can calculate the rate of GFP production relative to concentration of AHL in solution. This preliminary experiment is to show an approximate range of concentrations that the construct is sensitive to. With the data from this test, AHL concentrations, sampling times and length of experiments can be optimised for more detailed characterisation. Aims: Constant Conditions:
 * To determine approximations of the threshold of response, time of response, life span and rate of GFP produced

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3)Test for AHL Sensitivity
Construct: pTet - LuxR - pLux - GFP Based upon previous preliminary AHL sensitivity tests, now define a new [AHL] to test and in addition optimise the sampling time and length of testing. Aims: For each AHL concentration: Constant Conditions:
 * To determine the transfer function of rate of GFP produced
 * To determine the maximum rate of GFP produced
 * To determine the lifespan of the chassis at varying AHL concentrations
 * To determine the lowest threshold of AHL detection
 * To determine the response time of the system to AHL detection

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4)Temperature sensitivity
Construct: pTet - LuxR - pLux - GFP To test the affect of temperature on the construct. Measure the sum of the temperature dependent variables by GFP output. Temperature dependent variables include degradation rates, diffusion rates and expression rates. Need to measure a suitable range of temperatures that cover the operating range. Aims: For each AHL concentration: Constant Conditions:
 * Find how temperature will change the output.

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