IGEM:IMPERIAL/2007/Projects/Experimental Design/Phase2

• Introduction
• Phase 1
• Phase 2
• Phase 3
• Notes

1. Common GFP Experiments

[+] 1.1 Fluorescence dependence on volume

1.1 Fluorescence dependence

Status
Planned for 20/8/07	INCOMPLETE

Constructs: None

	Protocol
Results

Aims:

• The aim of the calibration curve is to determine a relationship between molecules of GFP in our cell extract and fluorescence. It should be noted that throughout our phase 2 experiments we will be maintaining the volume of cell extract used.
• The purified GFP that we wish to use for our calrbration curve is stored in a solution of buffer. Using this GFP there are several approachs to construct a calubration curve, and the purpose of this experiment is to validate one of these method. The approaches are:
  

Experiment 1:
To use the [GFP] solution and create dilutions using distilled water and using cell extract. Assumes that [GFP] has the same fluorescence in water and Cell extract.
Experiment 2:
To maintain the constant volume of cell extract used for phase 2 and add a [GFP] solution. Assumes that the the total volume of sample has no affect on fluorescence as long as moles of GFP are constant.
Experiment 3:
To use less volume than the constant cell extract used for phase 2 and add a [GFP] solution, this will bring the total volume to that used for phase 2 experiments. Assumes that varing volumes of cell extract volume has no affect on fluorescence i.e. minimal flourescence absorbance from cell extract.

• We need to test these three methods to see which is the best for constructing a calibration curve

Status:

• Planned for 20/8/2007

[+]Constant Conditions and Variables

• Exp 1. : Varying Total volume of sample, volume of cell extract constant
• Exp 2. : Varying volume of cell extract added , total volume constant
• Carry out at room temperature

[+] Sampling

• Carry out several readings to average out
• Vary the flourometer reading window
• 2 Repeats

[+] Controls

• Negative Controls :
  • Cell extract and no GFP
  • Blank wells

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[+] 1.2 GFP Calibration Curve

1.2 GFP Calibration Curve

Status
Planned for 23/8/07	INCOMPLETE

Constructs: None

	Protocol
Results

Aims:
Experiment 1:

• To create a series of dilutions of GFP using a purified sample of GFP

Experiment 2:

• To create a calibration curve to determine: [GFP] in in vitro chassis vs Fluorescence
• This is essential for our data analysis to convert the fluorescence into GFP molecules

Status:

• Purified GFP is to be ordered from clone tech at the moment waiting for a quote from clone tech. Phone Friday 17th August to hurry along.
• Need to finalise which in vitro chassis we will be using for our experiments, will be determined in inital in vitro testing of phase 1

[+] Constant Conditions

• Use a defined in vitro chassis, this will be determined after initial testing. It is most likely we will carry out a calibration curve 100ul commercial extract and a defined amount of home made cell extract
• carry out at room temperature

[+] Variables

• [GFP] added to in vitro chassis varied

[+] Sampling

• Measure after addition of GFP to minimize degradation of GFP

Repetition:

• 3 repeats

[+] Controls

• Negative Control: In vitro systems only with no GFP added
• Positive Control: DEFINE

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[+] 1.3 Degradation Time of GFP

1.3 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

[+] Constant Conditions:

• 50μl in vitro chassis
• [GFP] added

[+] Variables:

• Temperature range: 4^oC, 15^oC, 25^oC, 30^oC, 37^oC, 50^oC
• 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

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2. Project Specific Experiments

[+] 2.1 Cell by Date

[+] 2.1.1 Operating Temperature Range

2.1.1 Operating Temperature Range

Status
Planned for 20/8/07	INCOMPLETE

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.

	Protocol
Results

Aims:

• To determine if construct expresses in vitro at temperatures of: 4^oC, 15^oC, 25^oC, 30^oC, 37^oC, 50^oC
• To determine specific life span at each temperature range.
• To determine the maximum rate of GFP produced at each temperature range.

[+] Constant Conditions:

• Defined volume of commercial cell extract
  • Determine later if we can use home made cell extract
• 2μg DNA concentration

[+] Variables:

• Rate of GFP synthesis
• Life span expression
• Response time to get a fluorescence output
• Temperature: 4^oC, 15^oC, 25^oC, 30^oC, 37^oC, 50^oC

[+] Sampling:

• Every 30 minutes

Repetition:

• 3 repeats

[+] Controls:

• Negative Control: In vitro system and empty vector
• Positive Control:
  • In vitro system with a vector containing a construct that has been proven to work in vitro
  • In vitro system with purified GFP

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[+] 2.1.2 Varying Temperature Changes: Gentle Gradient

2.1.2 Varying Temperature Changes: Gentle Gradient

Status
Planned for 20/8/07	INCOMPLETE

Constructs: pTet-GFP, pT7-GFP or pcI-GFP

	Protocol
Results

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.

[+] Constant Conditions:

• 50μl in vitro chassis
• DNA concentration

[+] Variables:

• Rate of GFP synthesis
• Life span of chassis
• Response time
• Temperature change 4^oC, 25^oC, 37^oC
  • 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

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[+] 2.1.3 Varying Temperature Changes: Steep Gradient

2.1.3 Varying Temperature Changes: Steep Gradient

Status
Planned for 20/8/07	INCOMPLETE

Constructs: pTet-GFP, pT7-GFP or pcI-GFP

	Protocol
Results

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.
• Investigate how temperature change affects the rate of expression of GFP
• How the temperature change affects the life span of the system
• How the age of the in vitro system affects the response to temperature in terms of rate of GFP expressed

[+] Constant Conditions:

• 50μl in vitro chassis
• DNA concentration

[+] Variables:

• Rate of GFP synthesis
• Life span of chassis
• Response time
• Temperature change 4^oC, 25^oC, 37^oC
  • Type of gradients: Steep (5 min)
  • Temperature change : from 4°C to 25^oC, 37^oC 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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[+] 2.2 Infector Detector

[+] 2.2.1 Test for Steady State of LuxR Protein Expression

2.2.1 Test for Steady State of LuxR Protein Expression

Status
Planned for 20/8/07	INCOMPLETE

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.

	Protocol
Results

[+] Constant Conditions:

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

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[+] 2.2.2 Preliminary AHL Sensitivity Testing

2.2.2 Preliminary AHL Sensitivity Testing

Status
Planned for 20/8/07	INCOMPLETE

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.

	Protocol
Results

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

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[+] 2.2.3 Test for AHL Sensitivity

2.2.3 Test for AHL Sensitivity

Status
Planned for 20/8/07	INCOMPLETE

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.

	Protocol
Results

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

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[+] 2.2.4 Temperature sensitivity

2.2.4 Temperature sensitivity

Status
Planned for 20/8/07	INCOMPLETE

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.

	Protocol
Results

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
  • 30^oC and 37^oC
• 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

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