IGEM:IMPERIAL/2007/Projects/Experimental Design/Phase2: Difference between revisions
Line 289: | Line 289: | ||
*50μl in vitro chassis | *50μl in vitro chassis | ||
*DNA concentration | *DNA concentration | ||
</div> | |||
<span class="_toggler_toggle-item2-1- | <span class="_toggler_toggle-item2-1-3-2">'''[+] Variables:'''</span> | ||
<div class="toggle2-1- | <div class="toggle2-1-3 item2-1-3-2" style="display:none;"> | ||
<br> | <br> | ||
*Rate of GFP synthesis | *Rate of GFP synthesis | ||
Line 301: | Line 301: | ||
**Temperature change : from 4°C to 25<sup>o</sup>C, 37<sup>o</sup>C and vice versa | **Temperature change : from 4°C to 25<sup>o</sup>C, 37<sup>o</sup>C and vice versa | ||
**Time period before increment: 30 min, 1h, 2h, | **Time period before increment: 30 min, 1h, 2h, | ||
</div> | |||
<span class="_toggler_toggle-item2-1- | <span class="_toggler_toggle-item2-1-3-3">'''[+] Sampling:'''</span> | ||
<div class="toggle2-1- | <div class="toggle2-1-3 item2-1-3-3" style="display:none;"> | ||
<br> | <br> | ||
*Every 30 min interval. | *Every 30 min interval. | ||
Line 311: | Line 311: | ||
Repetition: | Repetition: | ||
*3 repeats | *3 repeats | ||
</div> | |||
<span class="_toggler_toggle-item2-1- | <span class="_toggler_toggle-item2-1-3-4">'''[+] Controls:'''</span> | ||
<div class="toggle2-1- | <div class="toggle2-1-3 item2-1-3-4" style="display:none;"> | ||
<br> | <br> | ||
*Negative Control: In vitro system only | *Negative Control: In vitro system only | ||
*Positive Control: In vitro system with purified GFP added | *Positive Control: In vitro system with purified GFP added | ||
</div> | |||
< | <div style="float:right; font-size: 150%;"><span class="_toggler_show-toggle2-1-3 _toggler-toggler2-1-3a _toggler-toggler2-1-3b toggler2-1-3a">Show All Details</span><span class="_toggler_hide-toggle2-1-3 _toggler-toggler2-1-3a _toggler-toggler2-1-3b toggler2-1-3b" style="display:none;">Hide All Details</span></div> | ||
</div> | </div> | ||
[<span class="_togglegroup _toggle _toggler">Infector Detector</span><span class="_toggle _toggler" style="display:none;">close level 1</span>] | [<span class="_togglegroup _toggle _toggler">Infector Detector</span><span class="_toggle _toggler" style="display:none;">close level 1</span>] | ||
<div class="_toggle" style="display:none;"> | <div class="_toggle" style="display:none;"> | ||
Line 350: | Line 351: | ||
<span class="_toggler_toggle-item2-1 | <span class="_toggler_toggle-item2-2-1-1">'''[+] Constant Conditions:'''</span> | ||
<div class="toggle2-1-2 | <div class="toggle2-2-1 item2-2-1-1" style="display:none;"> | ||
<br> | <br> | ||
*25 <sup>o</sup>C | *25 <sup>o</sup>C | ||
*50μl in vitro chassis | *50μl in vitro chassis | ||
*DNA added 2μg | *DNA added 2μg | ||
</div> | |||
<div style="float:right; font-size: 150%;"><span class="_toggler_show-toggle2-2-1 _toggler-toggler2-2-1a _toggler-toggler2-2-1b toggler2-2-1a">Show All Details</span><span class="_toggler_hide-toggle2-2-1 _toggler-toggler2-2-1a _toggler-toggler2-2-1b toggler2-2-1b" style="display:none;">Hide All Details</span></div> | |||
<br><br><br> | |||
<HR SIZE=2 WIDTH=90%><br> | |||
===2.2.2 Preliminary AHL Sensitivity Testing=== | ===2.2.2 Preliminary AHL Sensitivity Testing=== | ||
Line 381: | Line 387: | ||
<br> | <br> | ||
<span class="_toggler_toggle-item2- | <span class="_toggler_toggle-item2-2-2-1">'''[+] Constant Conditions:'''</span> | ||
<div class="toggle2- | <div class="toggle2-2-2 item2-2-2-1" style="display:none;"> | ||
<br> | <br> | ||
*25 <sup>o</sup>C | *25 <sup>o</sup>C | ||
*50μl in vitro chassis | *50μl in vitro chassis | ||
*DNA concentration ....... | *DNA concentration ....... | ||
</div> | |||
<span class="_toggler_toggle-item2- | <span class="_toggler_toggle-item2-2-2-2">'''[+] Variables:'''</span> | ||
<div class="toggle2- | <div class="toggle2-2-2 item2-2-2-2" style="display:none;"> | ||
<br> | <br> | ||
*Independent variables : | *Independent variables : | ||
Line 399: | Line 405: | ||
**Life span of chassis | **Life span of chassis | ||
**Response time | **Response time | ||
</div> | |||
<span class="_toggler_toggle-item2- | <span class="_toggler_toggle-item2-2-2-3">'''[+] Sampling:'''</span> | ||
<div class="toggle2- | <div class="toggle2-2-2 item2-2-2-3" style="display:none;"> | ||
<br> | <br> | ||
*Every 5minutes. | *Every 5minutes. | ||
Repetition: | Repetition: | ||
*3 repeats | *3 repeats | ||
</div> | |||
<span class="_toggler_toggle-item2- | <span class="_toggler_toggle-item2-2-2-4">'''[+] Controls:'''</span> | ||
<div class="toggle2- | <div class="toggle2-2-2 item2-2-2-4" style="display:none;"> | ||
<br> | <br> | ||
*Negative Control: In vitro system with no AHL added | *Negative Control: In vitro system with no AHL added | ||
*Positive Control: In vitro system with purified GFP added | *Positive Control: In vitro system with purified GFP added | ||
</div> | |||
<br> | <br> | ||
<div style="float:right; font-size: 150%;"><span class="_toggler_show-toggle2-2-2 _toggler-toggler2-2-2a _toggler-toggler2-2-2b toggler2-1-1a">Show All Details</span><span class="_toggler_hide-toggle2-2-2 _toggler-toggler2-2-2a _toggler-toggler2-2-2b toggler2-2-2b" style="display:none;">Hide All Details</span></div> | |||
<br><br><br> | |||
<HR SIZE=2 WIDTH=90%><br> | |||
===2.2.3 Test for AHL Sensitivity=== | ===2.2.3 Test for AHL Sensitivity=== | ||
Line 446: | Line 458: | ||
*To determine the response time of the system to AHL detection | *To determine the response time of the system to AHL detection | ||
<span class="_toggler_toggle-item2- | <span class="_toggler_toggle-item2-2-3-1">'''[+] Constant Conditions:'''</span> | ||
<div class="toggle2- | <div class="toggle2-2-3 item2-2-3-1" style="display:none;"> | ||
<br> | <br> | ||
*25 <sup>o</sup>C | *25 <sup>o</sup>C | ||
*50μl in vitro chassis | *50μl in vitro chassis | ||
*DNA added 2μg | *DNA added 2μg | ||
</div> | |||
<span class="_toggler_toggle-item2- | <span class="_toggler_toggle-item2-2-3-2">'''[+] Variables:'''</span> | ||
<div class="toggle2- | <div class="toggle2-2-3 item2-2-3-2" style="display:none;"> | ||
<br> | <br> | ||
*Independent variables : | *Independent variables : | ||
Line 464: | Line 476: | ||
**Life span of chassis | **Life span of chassis | ||
**Response time | **Response time | ||
</div> | |||
<span class="_toggler_toggle-item2- | <span class="_toggler_toggle-item2-2-3-3">'''[+] Sampling:'''</span> | ||
<div class="toggle2- | <div class="toggle2-2-3 item2-2-3-3" style="display:none;"> | ||
<br> | <br> | ||
*Define after initial test | *Define after initial test | ||
Repetition: | Repetition: | ||
*3 repeats | *3 repeats | ||
</div> | |||
<span class="_toggler_toggle-item2- | <span class="_toggler_toggle-item2-2-3-4">'''[+] Controls:'''</span> | ||
<div class="toggle2- | <div class="toggle2-2-3 item2-2-3-4" style="display:none;"> | ||
<br> | <br> | ||
*Negative Control: In vitro system with no AHL added | *Negative Control: In vitro system with no AHL added | ||
*Positive Control: In vitro system with purified GFP added | *Positive Control: In vitro system with purified GFP added | ||
</div> | |||
<div style="float:right; font-size: 150%;"><span class="_toggler_show-toggle2-2-3 _toggler-toggler2-2-3a _toggler-toggler2-2-3b toggler2-2-3a">Show All Details</span><span class="_toggler_hide-toggle2-2-3 _toggler-toggler2-2-3a _toggler-toggler2-2-3b toggler2-2-3b" style="display:none;">Hide All Details</span></div> | |||
<br><br><br> | |||
<HR SIZE=2 WIDTH=90%><br> | |||
===2.2.4 Temperature sensitivity=== | ===2.2.4 Temperature sensitivity=== | ||
Line 506: | Line 523: | ||
<br> | <br> | ||
<span class="_toggler_toggle-item2- | <span class="_toggler_toggle-item2-2-4-1">'''[+] Constant Conditions:'''</span> | ||
<div class="toggle2- | <div class="toggle2-2-4 item2-1-2-4" style="display:none;"> | ||
<br> | <br> | ||
*50μl in vitro chassis | *50μl in vitro chassis | ||
*DNA added 2μg | *DNA added 2μg | ||
</div> | |||
<span class="_toggler_toggle-item2- | <span class="_toggler_toggle-item2-2-4-2">'''[+] Variables:'''</span> | ||
<div class="toggle2- | <div class="toggle2-2-4 item2-2-4-2" style="display:none;"> | ||
<br> | <br> | ||
Line 524: | Line 541: | ||
*Dependent variables: | *Dependent variables: | ||
**Rate of GFP produced and total GFP produced | **Rate of GFP produced and total GFP produced | ||
</div> | |||
<span class="_toggler_toggle-item2- | <span class="_toggler_toggle-item2-2-4-3">'''[+] Sampling:'''</span> | ||
<div class="toggle2- | <div class="toggle2-2-4 item2-2-4-3" style="display:none;"> | ||
<br> | <br> | ||
*Define after initial test | *Define after initial test | ||
Repetition: | Repetition: | ||
*3 repeats | *3 repeats | ||
</div> | |||
<span class="_toggler_toggle-item2- | <span class="_toggler_toggle-item2-2-4-4">'''[+] Controls:'''</span> | ||
<div class="toggle2- | <div class="toggle2-2-4 item2-2-4-4" style="display:none;"> | ||
<br> | <br> | ||
*Negative Control: In vitro system with no AHL added | *Negative Control: In vitro system with no AHL added | ||
*Positive Control: In vitro system with a construct gaurenteed to work | *Positive Control: In vitro system with a construct gaurenteed to work | ||
</div> | |||
</div> | </div> |
Revision as of 06:50, 15 August 2007
1. Common GFP Experiments
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
[+] Variables
[+] Sampling
[+] Controls
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
[+] Constant Conditions:
[+] Variables:
[+] Sampling:
[+] Controls:
2. Project Specific Experiments
[Cell by Date ]
[Infector Detector ]