IGEM:IMPERIAL/2007/Projects/Experimental Design/Phase1

 Introduction Phase 1 Phase 2 Phase 3 Notes  

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

Test to see if construct will express in vivo. Experiments carried out at 37&deg;C in an incubator.

Aims:
 * To determine if construct expresses in vivo

[+] Constant Conditions
 * 50μl in vitro chassis
 * DNA concentration
 * Temperature stable at 37&deg;C

[+] Variables
 * Rate of GFP synthesis
 * Life span of chassis
 * Response time

[+] Sampling Repetition:
 * Every 5 minutes.
 * 3 repeats

[+] 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 Infector Detector
Construct: pTet-LuxR-pLux-GFP

Test to see if construct will express in vivo. Experiments carried out in incubator at 37oC at a constant inducer concentration. To do this, we induce E. coli cells transfected with the construct with a 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.

Aims:
 * To determine if construct expresses GFP in vivo

<span class="_toggler_toggle-item1-2-1">[+] Constant Conditions
 * Temperature stable at 37&deg;C
 * AHL concentration 1uM

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

<span class="_toggler_toggle-item1-2-3">[+] Sampling Repetition:
 * Every 30 minutes.
 * 3 repeats

<span class="_toggler_toggle-item1-2-4">[+] Controls
 * Negative Control: In vivo system with no AHL added
 * Positive Control: In vivo system GFP added. Ideal positive control is a construct known to work in this system, however none available at time of testing.

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2.1 Initial Testing for Cell by Date
Constructs: pTet-GFP, pT7-GFP, pcI-GFP Aims: Experiment 1 : Experiment 2:
 * 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.

<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. Ideal positive control is a construct known to work, however, we do not have one available at this stage

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2.2 Initial testing for Infector Detector
Construct: pTet-LuxR-pLux-GFP Aims: Test to see if construct will express in vitro. Experiments carried out at incubator at 37oC and under an inducer concentration that was shown to give a high induction in vivo.

Experiment 1 : Experiment 2:
 * Determine the DNA concentration and purity. From this determine volume of the DNA needed to add to the in vitro expression systems.
 * To determine if the infecter detector construct expresses in vitro
 * To Test the construct in home made and in commercial e.coli cell extract

<span class="_toggler_toggle-item2-2-1">[+] Constant Conditions
 * 37 oC
 * DNA added 2μg

<span class="_toggler_toggle-item2-2-2">[+] Variables
 * Type of cell extract used : Commercial and home made cell extract
 * AHL concentration added: 1uM

<span class="_toggler_toggle-item2-2-3">[+] Sampling Repetition:
 * Every 30 minutes initially and there after adjust sampling accordingly.
 * 3 repeats

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

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3.1 Investigate optimum counting time for fluorodetector
Construct: pTet-GFP

A test to see what is the optimum counting time to have during our experiments. The counting time is the time the fluorometer detector stays on top of each well. A small window time will only account for very discrete levels of fluorescence. These might include sudden spikes of radiation since fluorescence is not a uniform process. Hence we will get variation between samples of equal expression rates. A larger counting time results in a larger window size and hence a more average reading is taken from each sample smoothing out the variation due to the randomness of fluorescence emition. Care must be taken however because larger counting times will lead to faster fluorescence bleaching. A comprimise between the two must be found.

Aims: Determine the optimum counting time for the fluorometer. Avoid fluorescence bleaching.

<span class="_toggler_toggle-item3-1-1">[+] Constant Conditions
 * Temperature: 25 oC (Room temperature)
 * 200 µl aliquots from pTet culture in each well

<span class="_toggler_toggle-item3-1-2">[+] Variables
 * Vary the counting time of the fluorometer from 0.15 sec to 1.05 sec.
 * The counting times tested will be : 0.15, 0.30, 0.45, 0.60, 0.75, 0.90, 1.05 sec

<span class="_toggler_toggle-item3-1-3">[+] Sampling Repetition:
 * Take readings every 5 minutes for 1 hour.
 * 4 repeats

<span class="_toggler_toggle-item3-1-4">[+] Controls
 * Negative Control: E.Coli cultures without GFP expressing machinery
 * Positive Control: Diluted GFP in well

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3.2 Investigate the efficiency of a 1:1 mixture of commercial and homemade extract
Construct: pTet-GFP

This experiment is done to test if the commmercial cell extract and pre-incubation mix, if mixed in a ratio of 1:1 with the home made cell extract and reaction buffer, will produce sufficiently satisfying results to be used for carrying out the rest of the experiments for CELL BY DATE and INFECTOR DETECTOR.

Aims: To determine the working efficiency of the mixture of commercial and home-made cell extracts.

<span class="_toggler_toggle-item3-1-1">[+] Constant Conditions
 * Temperature: 25 oC (Room temperature)
 * 20 µl aliquots from the commercial cell extract mixture in each well
 * 20 µl aliquots from the home made cell extract mixture in each well
 * 20 µl of plasmi DNA in each sample

<span class="_toggler_toggle-item3-1-2">[+] Variables -

<span class="_toggler_toggle-item3-1-3">[+] Sampling Repetition:
 * Take readings at every 30 minutes interval.
 * 4 repeats

<span class="_toggler_toggle-item3-1-4">[+] Controls
 * Negative Control: E.Coli cultures without GFP expressing machinery (empty vector)
 * Positive Control: DNA in commercial cell extract

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