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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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 || Planning the week <br> DsRED arrives
|| - || 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 DsRED constructs (Ask Ben)
||  || 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 || <font color=red>PROBLEM-Wrong GFP!!!!</font> || Purify GFP <br> Prepare DsRED constructs
|| 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" |'''Tuesday'''
| style="background:#aa99c5" |'''Tuesday'''
|| Phase 1 - test pT7 || || 24 Hour Exp Room Temp
|| Phase 1 - test pT7 || ||  
|- style="background:#d4cae2"
|- style="background:#d4cae2"


| style="background:#aa99c5" | '''Wednesday'''
| style="background:#aa99c5" | '''Wednesday'''
|| || || Define the thresholds of temperature
|| || ||
|- 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 ||  ||  
|| 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" | '''Monday'''
| style="background:#aa99c5" | '''Monday'''
|| - ||  || Purified LuxR ready today   
|| - ||  || 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 Purified LuxR with construct 2
|| 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 ||  
||  || <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>
==Future Tests==
(CE:Number of Cell extracts needed)
#Calibration curve GFP (CE:10)
#Calibration curve dsRED (CE:10)
#ID - Final Testing of AHL for dsRED (CE:20)
#CBD - Steep Gradient dsRED- ( Ask Alex about final app)
#*Slow Gradient dsRED
#*Final Test Temp range for DsREd(CE:20)
'''Total Cell Extracts Reactions Required For Project'''<br>
*Week 10: 20
*Future testing: 60
*In Stock: 72 Reactions
===Dependencies===
#DsRed construct ( Tuesday)
#Maxi Preps:
#*pLux Construct 2 - Wait for intial testing
#*Negative Control - Constuct rbs:GFP:Terminator, No promotoer
===Meeting to sort the GFP crisis!===
====Options====
#'''GFP'''
#*AcGFP -
#*GFP mut3b - need to get the purified form
#**Ask around!
#'''RFP'''
#*DsRed - wild type
#*DsRed2 - can be visualied but has a long response time ~ 24hrs
#*DsRed express - can be visualised with UV, has response time of ~ 7-8hrs
====Proposed plan of action====
#'''DsRed express''' - prioritise cloning and testing the constructs with DsRed Express
#*Currently testing DsRed Express in-vitro (Calibration curve)
#'''GFP mut 3b''' - carry on looking for purified form so calibration and degradation curves can be derived
#*Do research to find degradation and calibration data in literature
#DesRed 2 will be cloned just in case the other options don't work out
====Flurometer====
*Might get access to 24hr fluorometer - programmed data collection (biochem lab, level 6)
**Can carry out only the experiments at room temperature and degradation experiments

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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