IGEM:IMPERIAL/2007/Projects/Experimental Design/Phase2: Difference between revisions
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<div class="tabs-blue"> | <div class="tabs-blue"> | ||
<ul> | <ul> | ||
<li>[[IGEM:IMPERIAL/2007/Experimental Design|Introduction]]</li> | <li>[[IGEM:IMPERIAL/2007/Projects/Experimental Design|Introduction]]</li> | ||
<li >[[IGEM:IMPERIAL/2007/Experimental Design/Phase1|Phase 1]]</li> | <li >[[IGEM:IMPERIAL/2007/Experimental Design/Phase1|Phase 1]]</li> | ||
<li id="current">[[IGEM:IMPERIAL/2007/Experimental Design/Phase2|Phase 2]]</li> | <li id="current">[[IGEM:IMPERIAL/2007/Experimental Design/Phase2|Phase 2]]</li> | ||
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| colspan="2" align="center" style="font-style:Bold; font-size:140%;" | Status | | colspan="2" align="center" style="font-style:Bold; font-size:140%;" | Status | ||
|- | |- | ||
| style="color:blue; font-style: | | colspan="2" align="center" style="color:#9933FF ; font-style:Bold; font-size:120%;" | '''Results uploaded !''' | ||
| style="color: | |- | ||
|align="center" style="font-size:100%;" | '''Experiment''' | |||
|align="center" style="font-size:100%;" | '''Status''' | |||
|- | |||
| style="color:blue; font-style:Bold; font-size:100%;" width="150" align="center" | '''GFP in water''' | |||
| align="center" style="color:blue; font-style:Bold; font-size:110%;" width="170" | [[IGEM:IMPERIAL/2007/Notebook/2007-8-21 | Completed - 21/8/07 ]] | |||
|- | |||
| style="color:blue; font-style:Bold; font-size:100%;" width="150" align="center" | '''GFP in cell extract''' | |||
| align="center" style="color:RED; font-style:Bold; font-size:110%;" width="170" | ABORTED | |||
|} | |} | ||
<br> | <br> | ||
'''Constructs:''' None | '''Constructs:''' None | ||
<br> | <br><br><br> | ||
<br> | <br><br> | ||
<br> | <br><br> | ||
{| border="0" align="right" style="font-style:Bold; font-size:140%; color:red;" | {| border="0" align="right" style="font-style:Bold; font-size:140%; color:red;" | ||
| align="left" width="170px" | | | align="left" width="170px" | | ||
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| colspan=2 align="right" style="font-style:Bold; font-size:110%;" | [[IGEM:IMPERIAL/2007/Experimental Design/Phase2/Results 1.1 |'''Results''']] | | colspan=2 align="right" style="font-style:Bold; font-size:110%;" | [[IGEM:IMPERIAL/2007/Experimental Design/Phase2/Results 1.1 |'''Results''']] | ||
|} | |} | ||
'''Aims:''' <br> | '''Aims:''' <br> | ||
*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 aim of the calibration curve is to determine a relationship between molecules of GFP in our cell extract and fluorescence. <br> 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:<br> | *The purified GFP that we wish to use for our calrbration curve is stored in a solution of buffer. <br>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. <br>The approaches are:<br> | ||
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| colspan="2" align="center" style="font-style:Bold; font-size:140%;" | Status | | colspan="2" align="center" style="font-style:Bold; font-size:140%;" | Status | ||
|- | |- | ||
| style="color:blue; font-style:outlined;" width="225" align="center" | [[IGEM:IMPERIAL/2007/Notebook/2007-8- | | style="color:blue; font-style:outlined;" width="225" align="center" | [[IGEM:IMPERIAL/2007/Notebook/2007-8-31 | '''Planned for 31/8/07''' ]] | ||
| style="color: | | style="color:blue; font-style:Bold; font-size:140%;" | First Test COMPLETED | ||
|- | |||
|style="color:blue; font-style:outlined;" width="225" align="center" |Attempt to purify GFPmut3b | |||
|style="color:blue; font-style:outlined;" width="225" align="center" | Week 11 | |||
|} | |} | ||
<br> | <br> | ||
'''Constructs:''' None | '''Constructs:''' None | ||
<br> | <br><br> | ||
<br> | <br> | ||
<br> | <br> | ||
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<div class="toggle1-3 item1-3-2" style="display:none;"> | <div class="toggle1-3 item1-3-2" style="display:none;"> | ||
<br> | <br> | ||
*Temperature range: | *Temperature range: 10<sup>o</sup>C, 15<sup>o</sup>C, 20<sup>o</sup>C, 25<sup>o</sup>C, 30<sup>o</sup>C, 37<sup>o</sup>C, 45<sup>o</sup>C | ||
*Degradation of GFP | *Degradation of GFP | ||
</div> | </div> | ||
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<div class="toggle1-3 item1-3-3" style="display:none;"> | <div class="toggle1-3 item1-3-3" style="display:none;"> | ||
<br> | <br> | ||
*Every | *Every 30 minutes. | ||
*carried out over suitable time range - should be able to define after initial testing | |||
Repetition: | Repetition: | ||
* | *2 repeats | ||
</div> | </div> | ||
<span class="_toggler_toggle-item1-3-4">'''[+] Controls:'''</span> | <span class="_toggler_toggle-item1-3-4">'''[+] Controls:'''</span> | ||
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|align="center" style="font-size:100%;" | '''Status''' | |align="center" style="font-size:100%;" | '''Status''' | ||
|- | |- | ||
|style="font-style:Bold;" align="center" | [http://parts.mit.edu/registry/index.php/Part:BBa_I13522 pTet-GFP] | |style="font-style:Bold;" align="center"| [http://parts.mit.edu/registry/index.php/Part:BBa_I13522 pTet-GFP] | ||
|style="font-style:Bold; font-size:110%;" align="center" | 10 | |style="font-style:Bold; font-size:110%;" align="center" | 8°C, 15°C, 25°C ,30°C | ||
|style="font-style:Bold;" align="center" | Tested - [[IGEM:IMPERIAL/2007/Notebook/2007-8- | |style="font-style:Bold;" align="center" | Tested - [[IGEM:IMPERIAL/2007/Notebook/2007-8-30 | 14/09/2007]] | ||
|- | |||
|style="font-style:Bold;" align="center"| [http://parts.mit.edu/registry/index.php/Part:BBa_I13522 pTet-GFP] | |||
|style="font-style:Bold; font-size:110%;" align="center" | 10°C, 20°C, 37°C ,45°C | |||
|style="font-style:Bold;" align="center" | Tested - [[IGEM:IMPERIAL/2007/Notebook/2007-8-30 | 30/08/2007]] | |||
|- | |||
|} | |} | ||
<br=clearall> | |||
<br> | <br> | ||
'''Constructs:''' [http://parts.mit.edu/registry/index.php/Part:BBa_I13522 pTet-GFP], [http://parts.mit.edu/registry/index.php/Part:BBa_E7104 pT7-GFP] , [http://parts.mit.edu/registry/index.php/Part:BBa_I719022 pcI-GFP] | '''Constructs:''' [http://parts.mit.edu/registry/index.php/Part:BBa_I13522 pTet-GFP], [http://parts.mit.edu/registry/index.php/Part:BBa_E7104 pT7-GFP] , [http://parts.mit.edu/registry/index.php/Part:BBa_I719022 pcI-GFP] | ||
<br> | <br> | ||
Test to determine the operating range of the preferred construct ''in vitro''. Experiments carried out across various temperatures. | Test to determine the operating range of the preferred construct ''in vitro''. Experiments carried out across various temperatures. | ||
<br> | |||
<br> | |||
<br> | <br> | ||
<br> | <br> | ||
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'''Aims:''' | '''Aims:''' | ||
*To determine if construct expresses ''in vitro'' at temperatures of: | *To determine if construct expresses ''in vitro'' at temperatures of: 8<sup>o</sup>C, 10<sup>o</sup>C, 20<sup>o</sup>C, 25<sup>o</sup>C, 30<sup>o</sup>C, 37<sup>o</sup>C, 45<sup>o</sup>C, | ||
*To determine specific life span at each temperature range. | *To determine specific life span at each temperature range. | ||
*To determine the maximum rate of GFP produced at each temperature range. | *To determine the maximum rate of GFP produced at each temperature range. | ||
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<br> | <br> | ||
*Defined volume of commercial cell extract | *Defined volume of commercial cell extract | ||
*2μg DNA concentration | *2μg DNA concentration | ||
</div> | </div> | ||
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*Life span expression | *Life span expression | ||
*Response time to get a fluorescence output | *Response time to get a fluorescence output | ||
*Temperature: | *Temperature: 10<sup>o</sup>C, 15<sup>o</sup>C, 20<sup>o</sup>C, 25<sup>o</sup>C, 30<sup>o</sup>C, 37<sup>o</sup>C, 45<sup>o</sup>C, | ||
</div> | </div> | ||
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<div class="toggle2-1-1 item2-1-1-3" style="display:none;"> | <div class="toggle2-1-1 item2-1-1-3" style="display:none;"> | ||
<br> | <br> | ||
* | *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 | |||
Repetition: | Repetition: | ||
*3 repeats | *3 repeats | ||
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<div class="item2-2-0" style="display:none;"> | <div class="item2-2-0" style="display:none;"> | ||
<span class="_toggler_toggle-item2-2-1-0"><span style="font-style:Bold; font-size:130%; text-indent: 10%;"> [+] 2.2.1 | <span class="_toggler_toggle-item2-2-1-0"><span style="font-style:Bold; font-size:130%; text-indent: 10%;"> [+] 2.2.1 Preliminary AHL Sensitivity Testing</span></span> | ||
<div class="item2-2-1-0" style="display:none;"> | <div class="item2-2-1-0" style="display:none;"> | ||
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| colspan="2" align="center" style="font-style:Bold; font-size:140%;" | Status | | colspan="2" align="center" style="font-style:Bold; font-size:140%;" | Status | ||
|- | |- | ||
| style="color:blue; font-style:outlined;" width="225" align="center" | [[IGEM:IMPERIAL/2007/Notebook/2007-8-20 | ''' | | style="color:blue; font-style:outlined;" width="225" align="center" | [[IGEM:IMPERIAL/2007/Notebook/2007-8-20 | '''Completed 30/8/07''' ]] | ||
| style="color: | | style="color:blue; font-style:Bold; font-size:140%;" | COMPLETED | ||
|} | |} | ||
'''Construct:''' pTet - LuxR - pLux - GFP <br> | '''Construct:''' pTet - LuxR - pLux - GFP <br> | ||
To determine the sensitivity of the construct to AHL concentration. | '''Aims'''<br> | ||
''Experiment 1:'' | |||
*Prepare suitable dilutions of AHL | |||
''Experiment 2:'' | |||
*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. | |||
<br><br> | <br><br> | ||
{| border="0" align="right" style="font-style:Bold; font-size:140%; color:red;" | {| border="0" align="right" style="font-style:Bold; font-size:140%; color:red;" | ||
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*Independent variables : | *Independent variables : | ||
*[AHL] concentration | *[AHL] concentration | ||
** | **10nM, 50nM, 100nM, | ||
*Dependent variables: | *Dependent variables: | ||
**Rate of GFP produced and total GFP produced | **Rate of GFP produced and total GFP produced | ||
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<div class="toggle2-2-2 item2-2-1-3" style="display:none;"> | <div class="toggle2-2-2 item2-2-1-3" style="display:none;"> | ||
<br> | <br> | ||
*Every | *Every 10 minutes for first hour and then adjust to 30 minutes | ||
Repetition: | Repetition: | ||
*3 repeats | *3 repeats | ||
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<span class="_toggler_toggle-item2-2-3-0"><span style="font-style:Bold; font-size:130%; text-indent: 10%;"> [+] 2.2.3 Test for AHL Sensitivity</span></span> | <span class="_toggler_toggle-item2-2-3-0"><span style="font-style:Bold; font-size:130%; text-indent: 10%;"> [+] 2.2.3 Test for AHL Sensitivity</span></span> | ||
<div class="item2-2-3-0" style="display:none;"> | <div class="item2-2-3-0" style="display:none;"> | ||
==2.2.3 Test for AHL Sensitivity== | ==2.2.3 Test for AHL Sensitivity== | ||
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| align="right" style="font-style:Bold; font-size:110%;" | [[IGEM:IMPERIAL/2007/Experimental Design/Phase2/Protocol 2.2.3 |'''Protocol''']] | | align="right" style="font-style:Bold; font-size:110%;" | [[IGEM:IMPERIAL/2007/Experimental Design/Phase2/Protocol 2.2.3 |'''Protocol''']] | ||
|- | |- | ||
| colspan=2 align="right" style="font-style:Bold; font-size:110%;" | [[IGEM:IMPERIAL/2007/Experimental Design/Phase2/Results | | colspan=2 align="right" style="font-style:Bold; font-size:110%;" | [[IGEM:IMPERIAL/2007/Experimental Design/Phase2/Results 2.2.3 |'''Results''']] | ||
|}</div> | |}</div> | ||
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<div class="toggle2-2-3 item2-2-3-3" style="display:none;"> | <div class="toggle2-2-3 item2-2-3-3" style="display:none;"> | ||
<br> | <br> | ||
* | *Now defined our range of interest as 1-10nM, first test: | ||
**3nM, 5nM, 7nM | |||
Repetition: | Repetition: | ||
*3 repeats | *3 repeats | ||
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'''Aims:''' | '''Aims:''' | ||
For | For three suitable AHL concentration covering a low, intermediate and high induction range test at 37°C | ||
* | *This is to show that the infecter detector can work at a suitable range, however, we are modeling our system at 25°C | ||
<br> | <br> | ||
Latest revision as of 02:12, 18 September 2007
1. Common GFP Experiments
[+] 1.1 Fluorescence dependence on volume / medium
[+] 1.2 GFP Calibration Curve
[+] 1.3 Degradation Time of GFP
2. Project Specific Experiments
[+] 2.1 Cell by Date
[+] 2.2 Infector Detector