IGEM:IMPERIAL/2007/Projects/Experimental Design/Phase2: Difference between revisions

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===2) Degradation Time of GFP===
===1.2 Degradation Time of GFP===
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Revision as of 07:58, 14 August 2007



1. Common GFP Protocols

1.1 GFP Calibration Curve


Status
Planned for 20/8/07 INCOMPLETE

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].



Protocol
Results

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:

Dependent

  • [GFP] added

Independent

  • Fluorescence

Sampling:

  • Measure after addition of GFP to minimize degradation of GFP

Repetition:

  • 3 repeats

Controls:

  • Negative Control: In vitro system only
    • No GFP is added to an in vitro chassis
  • Positive Control:-


1.2 Degradation Time of GFP


Status
Planned for 20/8/07 INCOMPLETE

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.

Protocol
Results

Aims:

  • To determine the half life of GFP for a range of temperatures

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:

  • Every 15 minutes.

Repetition:

  • 3 repeats

Controls:

  • Negative Control: In vitro system only
  • Positive Control: In vitro chassis with high concentration of purified GFP added at high temperature


Infecter 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:

  • 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.

Constant Conditions:

  • 25 oC
  • 50μl in vitro chassis
  • DNA added 2μg
  • AHL concentration 1000nM

Variables:

  • Independent variables :
  • Dependent variables:
    • Rate of GFP synthesis
    • Life span of chassis
    • Response time

Sampling:

  • Every 5 minutes.

Repetition:

  • 3 repeats

Controls:

  • Negative Control: In vitro system with no AHL added
  • Positive Control: In vitro system with purified GFP added


Phase 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:

  • 25 oC
  • 50μl in vitro chassis
  • DNA added 2μg

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:

  • 25 oC
  • 50μl in vitro chassis
  • DNA concentration .......

Variables:

  • 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:

  • Every 5minutes.

Repetition:

  • 3 repeats

Controls:

  • Negative Control: In vitro system with no AHL added
  • Positive Control: In vitro system with purified GFP added


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:

  • 25 oC
  • 50μl in vitro chassis
  • DNA added 2μg

Variables:

  • 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:

  • Define after initial test

Repetition:

  • 3 repeats

Controls:

  • Negative Control: In vitro system with no AHL added
  • Positive Control: In vitro system with purified GFP added

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:

  • 50μl in vitro chassis
  • DNA added 2μg

Variables:

  • Independent variables :
  • [AHL] concentration
    • Define after initial test
  • Temperature
    • 30oC and 37oC
  • Dependent variables:
    • Rate of GFP produced and total GFP produced

Sampling:

  • Define after initial test

Repetition:

  • 3 repeats

Controls:

  • Negative Control: In vitro system with no AHL added
  • Positive Control: In vitro system with a construct gaurenteed to work

Phase 2: Characterizing Specific Construct

1) Calibration Curve for GFP

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

Conditions:

  • 50μl in vitro chassis
  • DNA added
    • Do we need DNA, as it may absorb some of the fluorescence
  • 25oC

Variables:

  • [GFP] added
  • Fluorescence

Sampling:

  • Measure after addition of GFP to minimize degradation of GFP

Repetition:

  • 3 repeats

Controls:

  • Negative Control: In vitro system only
    • No GFP is added to an in vitro chassis


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

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:

  • Every 15 minutes.

Repetition:

  • 3 repeats

Controls:

  • Negative Control: In vitro system only
  • Positive Control: In vitro chassis with high concentration of purified GFP added at high temperature


3) 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:

  • 50μl in vitro chassis
  • DNA concentration

Variables:

  • Rate of GFP synthesis
  • Life span of chassis
  • Response time
  • Temperature: 4oC, 15oC, 25oC, 30oC, 37oC, 50oC

Sampling:

  • Every 15 minutes.

Repetition:

  • 3 repeats

Controls:

  • Negative Control: In vitro system only
  • Positive Control: In vitro system with purified GFP added


4) 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:

  • 50μl in vitro chassis
  • DNA concentration

Variables:

  • 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:

  • Every 30 min interval.
  • Every 15 minutes for 2 hours after change in temperature.
  • Every 30 minutes thereafter.

Repetition:

  • 3 repeats

Controls:

  • Negative Control: In vitro system only
  • Positive Control: In vitro system with purified GFP added


5) 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:

  • 50μl in vitro chassis
  • DNA concentration

Variables:

  • 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:

  • Every 30 min interval.
  • Every 15 minutes for 2 hours after change in temperature.
  • Every 30 minutes thereafter.

Repetition:

  • 3 repeats

Controls:

  • Negative Control: In vitro system only
  • Positive Control: In vitro system with purified GFP added


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