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

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*Several Problems have been identified, we have begun to address them. [[IGEM:IMPERIAL/2007/Projects/Experimental Design/Problems| Link here to problems.]]
*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]]
*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]]
*[[IGEM:IMPERIAL/2007/Projects/Experimental Design/Future Experiments| Planned experiments for future]]
*<font color=red> Problems with dsRED, it has failed quality control</font>
*Currently trying to purify LuxR and GFP.


==Time table==
==Time table==
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|- 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 || <font color=red>PROBLEM-Wrong GFP!!!!</font> || Purify GFP <br>DsRED arrives??<br>24hr experiment for mineral oil method
|| 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'''
||  ||  ||  Purify GFP <br> Prepare DsRED constructs
||  ||  ||   
|- style="background:#d4cae2"
|- style="background:#d4cae2"
| style="background:#aa99c5" | '''Friday'''
| style="background:#aa99c5" | '''Friday'''
||Prepare GFP solution-calibration <br> Construct Calibration Curve || ||  Purify GFP + Test Concentration <br> Prepare DsRED constructs
||Prepare GFP solution-calibration <br> Construct Calibration Curve || ||   
|}
|}


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| style="background:#aa99c5" | '''Wednesday'''
| style="background:#aa99c5" | '''Wednesday'''
|| || || 24 Hour Exp Room Temp
|| || ||
|- 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 ||  || Define the thresholds of temperature
|| 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 || ||
||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" |'''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|| Purified LuxR ready today
|| 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'''
||  || <br> Purify LuxR if plasmid arrives || Test Purified LuxR with construct 2
||  || <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'''
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|- style="background:#d4cae2"
|- style="background:#d4cae2"
| style="background:#aa99c5" | '''Friday'''
| style="background:#aa99c5" | '''Friday'''
|| Test construct with 5 AHL concentrations - 25 &deg;C ||  Begin purification of LuxR ||
|| Test construct with 5 AHL concentrations - 25 &deg;C ||  Begin purification of LuxR ||Test Purified LuxR with construct 2??
|}
|}


<br>
<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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