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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<span class="_toggler_toggle-item1-1-0"><div style="font-style:Bold; font-size:130%; text-indent: 5%;"> [+] 1.1 Fluorescence dependence on volume</div></span> | <span class="_toggler_toggle-item1-1-0"><div style="font-style:Bold; font-size:130%; text-indent: 5%;"> [+] 1.1 Fluorescence dependence on volume / medium</div></span> | ||
<div class="item1-1-0" style="display:none;"> | <div class="item1-1-0" style="display:none;"> | ||
==1.1 Fluorescence dependence== | ==1.1 Fluorescence dependence on volume / medium== | ||
<br> | <br> | ||
{| border="2" style="background-color:#ABCDEF;" align="right" | {| border="2" style="background-color:#ABCDEF;" align="right" | ||
| 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" | | ||
| align="right" style="font-style:Bold; font-size:110%;" | [[IGEM:IMPERIAL/2007/Experimental Design/Phase2/Protocol 1. | | align="right" style="font-style:Bold; font-size:110%;" | [[IGEM:IMPERIAL/2007/Experimental Design/Phase2/Protocol 1.1 |'''Protocol''']] | ||
|- | |- | ||
| colspan=2 align="right" style="font-style:Bold; font-size:110%;" | [[IGEM:IMPERIAL/2007/Experimental Design/Phase2/Results 1. | | 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> | ||
''Experiment 1: | ''Experiment 1: | ||
<br> | <br> | ||
To use the | 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. | ||
<br> | <br><br> | ||
''Experiment 2: | ''Experiment 2: | ||
<br> | <br> | ||
To maintain the constant volume of cell extract used for phase 2 and add a | 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. | ||
<br> | <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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==2.1.1 Operating Temperature Range== | ==2.1.1 Operating Temperature Range== | ||
{| width= 30% border="2" style="background-color:#ABCDEF;" align="right" | |||
| colspan="3" align="center" style="font-style:Bold; font-size:140%;" | Overall Status | |||
|- | |||
| colspan="3" align="center" style="color:#9933FF ; font-style:Bold; font-size:140%;" | Results uploaded ! | |||
| | |- | ||
|align="center" style="font-size:100%;" | '''Construct''' | |||
|align="center" style="font-size:100%;" | '''Temperature''' | |||
|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; font-size:110%;" align="center" | 8°C, 15°C, 25°C ,30°C | |||
|style="font-style:Bold;" align="center" | Tested - [[IGEM:IMPERIAL/2007/Notebook/2007-8-30 | 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> | |||
'''Constructs:''' pTet-GFP, pT7-GFP | <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] | |||
<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> | |||
<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" | | ||
| align="right" style="font-style:Bold; font-size:110%;" | [[IGEM:IMPERIAL/2007/Experimental Design/Phase2/Protocol 2.1.1 |'''Protocol''']] | | align="right" style="font-style:Bold; font-size:110%;" | [[IGEM:IMPERIAL/2007/Experimental Design/Phase2/Protocol 2.1.1 |'''Protocol''']] | ||
|- | |- | ||
| 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 2.1.1 |'''Results''']] | ||
|} | |} | ||
'''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;"> | ||
==2.2. | ==2.2.1 Preliminary AHL Sensitivity Testing== | ||
<br clear="all"> | <br clear="all"> | ||
<div style="position: relative; top: 5px;"> | <div style="position: relative; top: 5px;"> | ||
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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;" | ||
| align="left" width="170px" | | | align="left" width="170px" | | ||
| align="right" style="font-style:Bold; font-size:110%;" | [[IGEM:IMPERIAL/2007/Experimental Design/Phase2/Protocol 2.2. | | align="right" style="font-style:Bold; font-size:110%;" | [[IGEM:IMPERIAL/2007/Experimental Design/Phase2/Protocol 2.2.1 |'''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 2.2.1 |'''Results''']] | ||
|}</div> | |}</div> | ||
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<br> | <br> | ||
<span class="_toggler_toggle-item2-2- | <span class="_toggler_toggle-item2-2-1-1">'''[+] Constant Conditions:'''</span> | ||
<div class="toggle2-2- | <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 concentration | *DNA concentration 2ug | ||
</div> | </div> | ||
<span class="_toggler_toggle-item2-2- | <span class="_toggler_toggle-item2-2-1-2">'''[+] Variables:'''</span> | ||
<div class="toggle2-2- | <div class="toggle2-2-1 item2-2-1-2" style="display:none;"> | ||
<br> | <br> | ||
*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> | </div> | ||
<span class="_toggler_toggle-item2-2- | <span class="_toggler_toggle-item2-2-1-3">'''[+] Sampling:'''</span> | ||
<div class="toggle2-2-2 item2-2- | <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 | ||
</div> | </div> | ||
<span class="_toggler_toggle-item2-2- | <span class="_toggler_toggle-item2-2-1-4">'''[+] Controls:'''</span> | ||
<div class="toggle2-2- | <div class="toggle2-2-1 item2-2-1-4" style="display:none;"> | ||
<br> | <br> | ||
*Negative Control: In vitro system with no AHL added | *Negative Control: In vitro system with no AHL added | ||
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<div style="float:right; font-size: 150%;"><span class="_toggler_show-toggle2-2- | <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> | <br><br><br> | ||
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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