IGEM:IMPERIAL/2007/Projects/Cell by date/TestingValidation

=Cell by Date: Testing/Validation=

 Introduction Specifications Design Modelling Implementation Testing/Validation Notes References </ul> <br style="clear:both">

Lab Notebook

 * Week 5 (6 Aug): Building Constructs
 * Week 6 (13 Aug): Building Constructs / Phase 1
 * Week 7 (20 Aug): Phase 1 / Phase 2
 * Week 8 (27 Aug):
 * Week 9 (3 Sep):
 * Week 10 (10 Sep):

name=iGEM:IMPERIAL/2007/Notebook date=2007/09/15 view=threemonths format=%name/%year-%month-%day weekstart=7

1.1 Initial testing for Cell by Date in vivo
Constructs: pTet-GFP, pT7-GFP ,pcI-GFP

Test to see if construct will express in vivo. Experiments carried out at 30&deg;C in an incubator.

Aims:
 * To determine if construct expresses in vivo

<span class="_toggler_toggle-item1-1-1">[+] Constant Conditions
 * 50μl in vitro chassis
 * DNA concentration
 * Temperature stable at 30&deg;C

<span class="_toggler_toggle-item1-1-2">[+] Variables
 * Rate of GFP synthesis
 * Life span of chassis
 * Response time

<span class="_toggler_toggle-item1-1-3">[+] Sampling Repetition:
 * Every 5 minutes.
 * 3 repeats

<span class="_toggler_toggle-item1-1-4">[+] Controls
 * Negative Control: E.Coli cultures without GFP expressing machinery
 * Positive Control: Diluted GFP in well

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1.2 Initial testing for Cell by Date in vitro
Constructs: pTet-GFP, pT7-GFP, pcI-GFP Aims: Experiment 1 : Experiment 2: Status:
 * Determine the DNA concentration and purity. From this determine volume of the DNA needed to add to the in vitro expression systems.
 * To determine which constructs for cell by date expresses in vitro
 * To test the pTet and pcI constructs in commercial S30 E.coli cell extract and home made S30 extract
 * To test the pT7 construct in commercial S30 T7 cell extract and home made S30 extract.
 * To investigate optimum volume of home made S30 extract
 * To investigate the differences between home made and commercially bought S30 extract. This is in terms of rates of expression, length of expression and total output.
 * Home made extract on hold until next week, this is because of problem that there was no amino acid or tRNA supplements added. We are carrying on with the commercial extracts for our testing
 * DNA Constructs: Purified samples of pTet+pT7 available Friday, pcI available next week.
 * T7 Commercial extract arrived
 * E.coli commercial extract to arrive Friday

<span class="_toggler_toggle-item2-1-1">[+] Constant Conditions
 * 2ug DNA added
 * 37&deg;C

<span class="_toggler_toggle-item2-1-2">[+] Variables
 * Type of cell extract used : Commercial or home made
 * For home made extract we will vary the volumes used

<span class="_toggler_toggle-item2-1-3">[+] Sampling Repetition:
 * 30minutes
 * 2 repeats

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

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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: Conditions:
 * To determine [GFP] vs Fluorescence

Variables:

Sampling:

Controls:

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: Conditions:
 * To determine the half life of GFP for a range of temperatures

Variables:

Sampling:

Controls:

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: Conditions:
 * 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.

Variables:

Sampling:

Controls:

4) Varying Temperature Changes: Gentle Gradient
Constructs: pTet-GFP, pT7-GFP or pcI-GFP Aims: Conditions:
 * 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.

Variables:

Sampling:

Controls:

5) Varying Temperature Changes: Steep Gradient
Constructs: pTet-GFP, pT7-GFP or pcI-GFP Aims: Conditions:
 * 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.
 * Provide results for modelling.
 * Investigate k constant as a function of temperature and time.

Variables:

Sampling:

Controls:

1) Testing/Validation of Device
Constructs: pTet-GFP, pT7-GFP or pcI-GFP Aims: Conditions:
 * To validate the predictions of modelling analysis.
 * To fine-tune the in vitro system

Variables:

Sampling:

Controls: