IGEM:IMPERIAL/2007/Projects/Experimental Design: Difference between revisions

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<div class="tabs-blue">
<div class="tabs-blue">
<ul>
<ul>
<li id="current">[[IGEM:IMPERIAL/2007/Experimental Design|Introduction]]</li>
<li id="current">[[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>[[IGEM:IMPERIAL/2007/Experimental Design/Phase2|Phase 2]]</li>
<li>[[IGEM:IMPERIAL/2007/Experimental Design/Phase2|Phase 2]]</li>
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*Phase 3 - Testing/Validation of final constructs
*Phase 3 - Testing/Validation of final constructs
In addition any overlap between the experiments of the projects has been identified and considered when making the experimental design.
In addition any overlap between the experiments of the projects has been identified and considered when making the experimental design.
==Status==
*Several Problems have been identified, we have begun to address them. [[IGEM:IMPERIAL/2007/Projects/Experimental Design/Problems| Link here to problems.]]
*We are currently trying to improve our methodology to get better results. [[IGEM:IMPERIAL/2007/Projects/Experimental Design/Improve Methodology| Link to improving our Methodology]]
*<font color=red> Problems with dsRED, it has failed quality control</font>
*Currently trying to purify LuxR and GFP.


==Time table==
==Time table==
===Testing of constructs and cell extracts===
===General timetable===
{| style="background:#FAF5FF; border: 1px solid #aa99c5; color:Black"
{| style="background:#FAF5FF; border: 1px solid #aa99c5; color:Black"
|- style="background:#aa99c5; color:Black"
|- style="background:#aa99c5; color:Black"
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|- style="background:#d4cae2"
|- style="background:#d4cae2"
| style="background:#aa99c5" | '''Monday'''
| style="background:#aa99c5" | '''Monday'''
|| - || Refine the wiki ||  
|| - || Refine the wiki || Planning the week
|- style="background:#d4cae2"
|- style="background:#d4cae2"
| style="background:#aa99c5" |'''Tuesday'''
| style="background:#aa99c5" |'''Tuesday'''
||  || Prepare GFP solution-calibration <br> Construct Calibration Curve <br>Refine the wiki ||  
||  || Prepare GFP solution-calibration <br> Construct Calibration Curve <br>Refine the wiki || Testing to reduce evaporation
|- style="background:#d4cae2"
|- style="background:#d4cae2"
| style="background:#aa99c5" | '''Wednesday'''
| style="background:#aa99c5" | '''Wednesday'''
|| Test Home made cell extract <br>Test varying levels of cell extract || ||  
|| Test Home made cell extract <br>Test varying levels of cell extract || <font color=red>PROBLEM-Wrong GFP!!!!</font> || 24hr experiment with pTet using paraffin oil
|- style="background:#d4cae2"
|- style="background:#d4cae2"
| style="background:#aa99c5" | '''Thursday'''
| style="background:#aa99c5" | '''Thursday'''
||  ||  ||  
||  ||  ||
|- style="background:#d4cae2"
|- style="background:#d4cae2"
| style="background:#aa99c5" | '''Friday'''
| style="background:#aa99c5" | '''Friday'''
||Prepare GFP solution-calibration <br> Construct Calibration Curve || ||  
||Prepare GFP solution-calibration <br> Construct Calibration Curve || ||
|}
|}


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|- style="background:#aa99c5; color:Black"
|- style="background:#aa99c5; color:Black"
|  || Week 8 || Week 9 || Week 10  
|  || Week 8 || Week 9 || Week 10  
|- style="background:#d4cae2"
|- style="background:#d4cae2"
| style="background:#aa99c5" | '''Monday'''
| style="background:#aa99c5" | '''Monday'''
|| - ||  || Test suitable gradient of temperature,<br> minimum working temp - maximum working temp 
|| - ||  ||
|- style="background:#d4cae2"
|- style="background:#d4cae2"
| style="background:#aa99c5" |'''Tuesday'''
| style="background:#aa99c5" |'''Tuesday'''
|| Phase 1 - test pT7 || || Test suitable step increases 
|| Phase 1 - test pT7 || ||  
|- style="background:#d4cae2"
|- style="background:#d4cae2"
| style="background:#aa99c5" | '''Wednesday'''
| style="background:#aa99c5" | '''Wednesday'''
|| || Exp with 15 and 25 &deg;C ||  
|| || ||
|- style="background:#d4cae2"
|- style="background:#d4cae2"
| style="background:#aa99c5" | '''Thursday'''
| style="background:#aa99c5" | '''Thursday'''
|| Test 20 and 37 &deg;C,<br>Test GFP degradation at 20 and 37 &deg;C || Exp with 30 and 40 &deg;C, <br> continue measuring 15 and 25 &deg;C ||  
|| Test 20 and 37 &deg;C,<br>Test GFP degradation at 20 and 37 &deg;C || || 24 Hour Exp Room Temp
 
|- style="background:#d4cae2"  
|- style="background:#d4cae2"  
| style="background:#aa99c5" | '''Friday'''
| style="background:#aa99c5" | '''Friday'''
||Second day testing 20 and 37 &deg;C ||Exp with 10 and 45 &deg;C, <br> continue measuring 30 and 40 &deg;C  ||  
||Second day testing 20 and 37 &deg;C || ||Define the thresholds of temperature
 
|}
|}


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|- style="background:#d4cae2"
|- style="background:#d4cae2"
| style="background:#aa99c5" | '''Monday'''
| style="background:#aa99c5" | '''Monday'''
|| - ||  ||  
|| - ||  || Purify LuxR 
|- style="background:#d4cae2"
|- style="background:#d4cae2"
| style="background:#aa99c5" |'''Tuesday'''
| style="background:#aa99c5" |'''Tuesday'''
|| test pLux in-vitro - 1nM AHL|| Refine AHL concentrations range 25&deg;C<br> <font color=red> Purify LuxR if plasmid arrives||  
|| test pLux in-vitro - 1nM AHL|| Refine AHL concentrations range 25&deg;C<br> <font color=red> Purify LuxR if plasmid arrives|| Purification of LuxR - <font color=red> problems <font color=red>
|- style="background:#d4cae2"
|- style="background:#d4cae2"
| style="background:#aa99c5" | '''Wednesday'''
| style="background:#aa99c5" | '''Wednesday'''
||  || Choose a range of AHL concentrations range 37&deg;C<font color=red> <br> Purify LuxR if plasmid arrives ||  
||  || <br> Purify LuxR if plasmid arrives || <font color=red> problems with LuxR pufification<font color=red>
|- style="background:#d4cae2"
|- style="background:#d4cae2"
| style="background:#aa99c5" | '''Thursday'''
| style="background:#aa99c5" | '''Thursday'''
||  || Test purified LuxR ||  
||  || Clone in plasmid ||  
|- style="background:#d4cae2"
|- style="background:#d4cae2"
| style="background:#aa99c5" | '''Friday'''
| style="background:#aa99c5" | '''Friday'''
|| Test construct with 5 AHL concentrations - 25 &deg;C || Test purified LuxR ||  
|| Test construct with 5 AHL concentrations - 25 &deg;C || Begin purification of LuxR ||Test Purified LuxR with construct 2??
|}
|}
<br>

Latest revision as of 02:35, 21 September 2007



We have identified three phases of experiments for each of the project:

  • Phase 1 - Intial testing of the promoters and test constructs in vitro
  • Phase 2 - Testing/Validation of the test constructs
  • Phase 3 - Testing/Validation of final constructs

In addition any overlap between the experiments of the projects has been identified and considered when making the experimental design.

Status

  • Several Problems have been identified, we have begun to address them. Link here to problems.
  • We are currently trying to improve our methodology to get better results. Link to improving our Methodology
  • Problems with dsRED, it has failed quality control
  • Currently trying to purify LuxR and GFP.

Time table

General timetable

Week 8 Week 9 Week 10
Monday - Refine the wiki Planning the week
Tuesday Prepare GFP solution-calibration
Construct Calibration Curve
Refine the wiki		 Testing to reduce evaporation
Wednesday Test Home made cell extract
Test varying levels of cell extract		 PROBLEM-Wrong GFP!!!! 24hr experiment with pTet using paraffin oil
Thursday
Friday Prepare GFP solution-calibration
Construct Calibration Curve

CBD

Week 8 Week 9 Week 10
Monday -
Tuesday Phase 1 - test pT7
Wednesday
Thursday Test 20 and 37 °C,
Test GFP degradation at 20 and 37 °C		 24 Hour Exp Room Temp
Friday Second day testing 20 and 37 °C Define the thresholds of temperature

ID

Week 8 Week 9 Week 10
Monday - Purify LuxR
Tuesday test pLux in-vitro - 1nM AHL Refine AHL concentrations range 25°C
Purify LuxR if plasmid arrives		 Purification of LuxR - problems
Wednesday
Purify LuxR if plasmid arrives		 problems with LuxR pufification
Thursday Clone in plasmid
Friday Test construct with 5 AHL concentrations - 25 °C Begin purification of LuxR Test Purified LuxR with construct 2??


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