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Revision as of 03:58, 15 August 2007
1. Common GFP Experiments
1.1 GFP 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:
- To determine [GFP] vs Fluorescence
[+] Constant Conditions
- 100μl of commercial in vitro chassis
- ..... of our in vitro chassis
- carry out at room temperature
[+] Variables
[+] Sampling
- Measure after addition of GFP to minimize degradation of GFP
Repetition:
[+] Controls
- Negative Control: In vitro system only
- No GFP is added to an in vitro chassis
- Positive Control:-
Show All DetailsHide All Details
1.2 Degradation Time of GFP
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:
- To determine the half life of GFP for a range of temperatures
[+] Constant Conditions:
- 50μl in vitro chassis
- [GFP] added
[+] Variables:
- Temperature range: 4oC, 15oC, 25oC, 30oC, 37oC, 50oC
- Range may change based upon the initial testing; will only test ranges that in vitro is stable at.
- Degradation of GFP
[+] Sampling:
[+] Controls:
- Negative Control: In vitro system only
- Positive Control: In vitro chassis with high concentration of purified GFP added at high temperature
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2. Project Specific Experiments
[Cell by Dateclose level 1]
2.1 Cell By Date
2.1.1 Operating Temperature Range
Constructs: pTet-GFP, pT7-GFP or pcI-GFP
Test to determine the operating range of the preferred construct in vitro. Experiments carried out across various temperatures.
Aims:
- To determine if construct expresses in vitro at temperatures of: 4oC, 15oC, 25oC, 30oC, 37oC, 50oC
- To determine specific life span at each temperature range.
- To determine the maximum rate of GFP produced at each temperature range.
Conditions:
[show][hide]
- 50μl in vitro chassis
- DNA concentration
Variables:
[show][hide]
- Rate of GFP synthesis
- Life span of chassis
- Response time
- Temperature: 4oC, 15oC, 25oC, 30oC, 37oC, 50oC
Sampling:
[show][hide]
Controls:
[show][hide]
- Negative Control: In vitro system only
- Positive Control: In vitro system with purified GFP added
2.1.2 Varying Temperature Changes: Gentle Gradient
Constructs: pTet-GFP, pT7-GFP or pcI-GFP
Aims:
- To determine the effects of fluorescence with reference to a gentle change in temperature from 4°C to 37°C and vice versa over different time periods.
- Provide results for modelling.
- Investigate k constant as a function of temperature and time.
Conditions:
[show][hide]
- 50μl in vitro chassis
- DNA concentration
Variables:
[show][hide]
- Rate of GFP synthesis
- Life span of chassis
- Response time
- Temperature change 4oC, 25oC, 37oC
- Type of gradients: Gentle(1 hour)
- Temperature change : from 4°C to 25°C, 37°C and vice versa
- Time period before increment: 30 min, 1h, 2h (to be done in parallel)
Sampling:
[show][hide]
- Every 30 min interval.
- Every 15 minutes for 2 hours after change in temperature.
- Every 30 minutes thereafter.
Repetition:
Controls:
[show][hide]
- Negative Control: In vitro system only
- Positive Control: In vitro system with purified GFP added
2.1.3 Varying Temperature Changes: Steep Gradient
Constructs: pTet-GFP, pT7-GFP or pcI-GFP
Aims:
- To determine the effects of fluorescence with reference to a steep change in temperature from 4°C to 37°C and vice versa over different time periods.
- Provide results for modelling.
- Investigate k constant as a function of temperature and time.
Conditions:
[show][hide]
- 50μl in vitro chassis
- DNA concentration
Variables:
[show][hide]
- Rate of GFP synthesis
- Life span of chassis
- Response time
- Temperature change 4oC, 25oC, 37oC
- Type of gradients: Steep (5 min)
- Temperature change : from 4°C to 25oC, 37oC and vice versa
- Time period before increment: 30 min, 1h, 2h,
Sampling:
[show][hide]
- Every 30 min interval.
- Every 15 minutes for 2 hours after change in temperature.
- Every 30 minutes thereafter.
Repetition:
Controls:
[show][hide]
- Negative Control: In vitro system only
- Positive Control: In vitro system with purified GFP added
[Infector Detectorclose level 1]
2.2 Infector Detector
2.2.1 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.
Constant Conditions:
[show][hide]
- 25 oC
- 50μl in vitro chassis
- DNA added 2μg
2.2.2 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:
- To determine approximations of the threshold of response, time of response, life span and rate of GFP produced
Constant Conditions:
[show][hide]
- 25 oC
- 50μl in vitro chassis
- DNA concentration .......
Variables:
[show][hide]
- Independent variables :
- [AHL] concentration
- 0.1nM, 1nM, 10nM, 100nM, 1000nM
- Dependent variables:
- Rate of GFP produced and total GFP produced
- Life span of chassis
- Response time
Sampling:
[show][hide]
Controls:
[show][hide]
- Negative Control: In vitro system with no AHL added
- Positive Control: In vitro system with purified GFP added
2.2.3 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:
- 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
Constant Conditions:
[show][hide]
- 25 oC
- 50μl in vitro chassis
- DNA added 2μg
Variables:
[show][hide]
- Independent variables :
- [AHL] concentration
- Define after initial test
- Dependent variables:
- Rate of GFP produced and total GFP produced
- Life span of chassis
- Response time
Sampling:
[show][hide]
- Define after initial test
Repetition:
Controls:
[show][hide]
- Negative Control: In vitro system with no AHL added
- Positive Control: In vitro system with purified GFP added
2.2.4 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:
- Find how temperature will change the output.
Constant Conditions:
[show][hide]
- 50μl in vitro chassis
- DNA added 2μg
Variables:
[show][hide]
- Independent variables :
- [AHL] concentration
- Define after initial test
- Temperature
- Dependent variables:
- Rate of GFP produced and total GFP produced
Sampling:
[show][hide]
- Define after initial test
Repetition:
Controls:
[show][hide]
- Negative Control: In vitro system with no AHL added
- Positive Control: In vitro system with a construct gaurenteed to work
