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

From OpenWetWare
Jump to navigationJump to search
Line 108: Line 108:
#*pLux Construct 2 - Wait for intial testing
#*pLux Construct 2 - Wait for intial testing
#*Negative Control - Constuct rbs:GFP:Terminator, No promotoer
#*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

Revision as of 07:13, 10 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.


Time table

Testing of constructs and cell extracts

Week 8 Week 9 Week 10
Monday - Refine the wiki Planning the week
DsRED arrives
Tuesday Prepare GFP solution-calibration
Construct Calibration Curve
Refine the wiki		 Prepare DsRED constructs (Ask Ben)
Wednesday Test Home made cell extract
Test varying levels of cell extract		 PROBLEM-Wrong GFP!!!! Purify GFP
Prepare DsRED constructs
Thursday Purify GFP
Prepare DsRED constructs
Friday Prepare GFP solution-calibration
Construct Calibration Curve		 Purify GFP + Test Concentration
Prepare DsRED constructs

CBD

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

ID

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


Future Tests

(CE:Number of Cell extracts needed)

  1. Calibration curve GFP (CE:10)
  2. Calibration curve dsRED (CE:10)
  3. ID - Final Testing of AHL for dsRED (CE:20)
  4. 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

  • Week 10: 20
  • Future testing: 60
  • In Stock: 72 Reactions

Dependencies

  1. DsRed construct ( Tuesday)
  2. Maxi Preps:
    • pLux Construct 2 - Wait for intial testing
    • Negative Control - Constuct rbs:GFP:Terminator, No promotoer
Retrieved from "https://openwetware.org/mediawiki/index.php?title=IGEM:IMPERIAL/2007/Projects/Experimental_Design&oldid=149296"