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 / medium

1.1 Fluorescence dependence on volume / medium
Constructs: None

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 equal dilutions in distilled water and in cell extract. We want to investigate whether FP has the same fluorescence in water and cell extract. Compare the differences (if any) of GFP fluorescence in the two solutions. ''Experiment 2: To maintain the constant volume of cell extract used for phase 2 and add a GFP solution. Our aim is to investigate whether the the total volume of sample has an affect on fluorescence given the moles of GFP are constant.

[+]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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<span class="_toggler_toggle-item1-2-0"> [+] 1.2 GFP Calibration Curve

1.2 GFP Calibration Curve
Constructs: None Aims: ''Experiment 1: ''Experiment 2: Status:
 * To create a series of dilutions of GFP using a purified sample of GFP
 * 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
 * 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

<span class="_toggler_toggle-item1-2-1">[+] 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

<span class="_toggler_toggle-item1-2-2">[+] Variables
 * [GFP] added to in vitro chassis varied

<span class="_toggler_toggle-item1-2-3">[+] Sampling Repetition:
 * Measure after addition of GFP to minimize degradation of GFP
 * 3 repeats

<span class="_toggler_toggle-item1-2-4">[+] Controls
 * Negative Control: In vitro systems only with no GFP added
 * Positive Control: DEFINE

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<span class="_toggler_toggle-item1-3-0"> [+] 1.3 Degradation Time of GFP

1.3 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: <span class="_toggler_toggle-item1-3-1">[+] Constant Conditions:
 * To determine the half life of GFP for a range of temperatures
 * 50μl in vitro chassis
 * [GFP] added

<span class="_toggler_toggle-item1-3-2">[+] Variables: <span class="_toggler_toggle-item1-3-3">[+] Sampling: Repetition: <span class="_toggler_toggle-item1-3-4">[+] Controls:
 * Temperature range: 10oC, 15oC, 20oC, 25oC, 30oC, 37oC, 45oC
 * Degradation of GFP
 * Every 30 minutes.
 * carried out over suitable time range - should be able to define after initial testing
 * 2 repeats
 * 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= <span class="_toggler_toggle-item2-1-0"> [+] 2.1 Cell by Date

<span class="_toggler_toggle-item2-1-1-0"> [+] 2.1.1 Operating Temperature Range

2.1.1 Operating Temperature Range
Constructs: pTet-GFP, pT7-GFP, 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: 8oC, 10oC, 20oC, 25oC, 30oC, 37oC, 45oC,
 * To determine specific life span at each temperature range.
 * To determine the maximum rate of GFP produced at each temperature range.

<span class="_toggler_toggle-item2-1-1-1">[+] Constant Conditions:
 * Defined volume of commercial cell extract
 * 2μg DNA concentration

<span class="_toggler_toggle-item2-1-1-2">[+] Variables:
 * Rate of GFP synthesis
 * Life span expression
 * Response time to get a fluorescence output
 * Temperature: 10oC, 15oC, 20oC, 25oC, 30oC, 37oC, 45oC,

<span class="_toggler_toggle-item2-1-1-3">[+] Sampling: Repetition:
 * Initially every 10 minutes for first hour then adjust to 30minutes
 * Also carrying out staggered readings, this is to narrow the gap of missed measurements overnight to 5 hours
 * 3 repeats

<span class="_toggler_toggle-item2-1-1-4">[+] 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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<span class="_toggler_toggle-item2-1-2-0"> [+] 2.1.2 Varying Temperature Changes: Gentle Gradient

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&deg;C to 37&deg;C and vice versa over different time periods.
 * Provide results for modelling.
 * Investigate k constant as a function of temperature and time.

<span class="_toggler_toggle-item2-1-2-1">[+] Constant Conditions:
 * 50μl in vitro chassis
 * DNA concentration

<span class="_toggler_toggle-item2-1-2-2">[+] 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)

<span class="_toggler_toggle-item2-1-2-3">[+] Sampling: Repetition:
 * Every 30 min interval.
 * Every 15 minutes for 2 hours after change in temperature.
 * Every 30 minutes thereafter.
 * 3 repeats

<span class="_toggler_toggle-item2-1-2-4">[+] Controls:
 * Negative Control: In vitro system only
 * Positive Control: In vitro system with purified GFP added

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<span class="_toggler_toggle-item2-1-3-0"> [+] 2.1.3 Varying Temperature Changes: Steep Gradient

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&deg;C to 37&deg;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

<span class="_toggler_toggle-item2-1-3-1">[+] Constant Conditions:
 * 50μl in vitro chassis
 * DNA concentration

<span class="_toggler_toggle-item2-1-3-2">[+] 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,

<span class="_toggler_toggle-item2-1-3-3">[+] Sampling: Repetition:
 * Every 30 min interval.
 * Every 15 minutes for 2 hours after change in temperature.
 * Every 30 minutes thereafter.
 * 3 repeats

<span class="_toggler_toggle-item2-1-3-4">[+] Controls:
 * Negative Control: In vitro system only
 * Positive Control: In vitro system with purified GFP added

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<span class="_toggler_toggle-item2-2-0"> [+] 2.2 Infector Detector

<span class="_toggler_toggle-item2-2-1-0"> [+] 2.2.1 Preliminary AHL Sensitivity Testing

2.2.1 Preliminary AHL Sensitivity Testing
Construct: pTet - LuxR - pLux - GFP Aims Experiment 1: Experiment 2:
 * Prepare suitable dilutions of AHL
 * To determine the sensitivity of the construct to AHL concentration.
 * This preliminary experiment is to show an approximate range of concentrations that the construct is sensitive to. From this choose more AHL concentrations to test.

Aims:
 * To determine approximations of the threshold of response, time of response, life span and rate of GFP produced

<span class="_toggler_toggle-item2-2-1-1">[+] Constant Conditions:
 * 25 oC
 * 50μl in vitro chassis
 * DNA concentration 2ug

<span class="_toggler_toggle-item2-2-1-2">[+] Variables:
 * Independent variables :
 * [AHL] concentration
 * 10nM, 50nM, 100nM,
 * Dependent variables:
 * Rate of GFP produced and total GFP produced
 * Life span of chassis
 * Response time

<span class="_toggler_toggle-item2-2-1-3">[+] Sampling: Repetition:
 * Every 10 minutes for first hour and then adjust to 30 minutes
 * 3 repeats

<span class="_toggler_toggle-item2-2-1-4">[+] Controls:
 * Negative Control: In vitro system with no AHL added
 * Positive Control: In vitro system with purified GFP added

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<span class="_toggler_toggle-item2-2-3-0"> [+] 2.2.3 Test for AHL Sensitivity

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

<span class="_toggler_toggle-item2-2-3-1">[+] Constant Conditions:
 * 25 oC
 * 50μl in vitro chassis
 * DNA added 2μg

<span class="_toggler_toggle-item2-2-3-2">[+] 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

<span class="_toggler_toggle-item2-2-3-3">[+] Sampling: Repetition:
 * Now defined our range of interest as 1-10nM, first test:
 * 3nM, 5nM, 7nM
 * 3 repeats

<span class="_toggler_toggle-item2-2-3-4">[+] Controls:
 * Negative Control: In vitro system with no AHL added
 * Positive Control: In vitro system with purified GFP added

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<span class="_toggler_toggle-item2-2-4-0"> [+] 2.2.4 Temperature sensitivity

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 three suitable AHL concentration covering a low, intermediate and high induction range test at 37&deg;C
 * This is to show that the infecter detector can work at a suitable range, however, we are modeling our system at 25&deg;C

<span class="_toggler_toggle-item2-2-4-1">[+] Constant Conditions:
 * 50μl in vitro chassis
 * DNA added 2μg

<span class="_toggler_toggle-item2-2-4-2">[+] Variables:


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

<span class="_toggler_toggle-item2-2-4-3">[+] Sampling: Repetition:
 * Define after initial test
 * 3 repeats

<span class="_toggler_toggle-item2-2-4-4">[+] 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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