IGEM:IMPERIAL/2007/Projects/Hrp System/Testing/Protocol Hrp

Ben Yi Tew 09:39, 3 August 2007 (EDT): I was looking at your protocol and I realized that you're measuring flourescence directly from the cells in the medium, is it really recommended? What happened to your protocols to lyse cells? And are you sure that you want to test it at 10 mM AHL? That is very high concentration, and I doubt it is in the detection range of the lux promoter. And secondly, we might not have that much AHL... also, our ampicillin stock is at 50 mg/ml, and we're using 50 μg/ml working solution for simplicity of measurement. Please delete after reading, thank you.

Equipment

• Twinkle Plate Reader Fluorometer
• 96 well plate
• Eppendorf Tubes
• Gilson Pippettes
• Plastic Curvetts
• Incubator 37^o shaker
• Vortex and Centrifuge
• Spectrometer

Materials and Reagents

• M9 minimal media 40μg/ml Ampicillin
• Inducer of promoter at varying concentrations
• Recombinant acGFP of known concentration
• Agar Plates
• E.coli strain containing vector

Protocols

Promoter Test Construct

The promoter pLux is to be tested at 8 varying ranges of inducer concentration.

• pLux will have a range of 0 to 10mM AHL added at intervals of 1.25mM Protocol

Device Test Construct

For this test construct we initially need to run a preliminary test in order to find out the time intervals and input ranges we need. We will start the preliminary test using the range of concentrations used initially for promoters. This device uses a pLac promoter, the proposed range of concentrations is going to be defined from the promoter experiment, this is use a range of concentrations to cover an appropriate range of promoter. From looking at literature an appropriate range is 0-100μM.

• Preliminary experiment Protocol
• Device experiment Protocol