Jan 07, 2025
96 well E.coli fluorescence assay
This protocol is a draft, published without a DOI.
- 1Harvard University
Protocol Citation: Simon d'Oelsnitz 2025. 96 well E.coli fluorescence assay. protocols.io https://protocols.io/view/96-well-e-coli-fluorescence-assay-dxag7ibw
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: In development
We are still developing and optimizing this protocol
Created: January 07, 2025
Last Modified: January 07, 2025
Protocol Integer ID: 117800
Keywords: Genetic biosensor, Synthetic biology
Abstract
This method is used to measure the fluorescence of E. coli cells exposed to a small molecule. It's primary purpose is to characterize the performance of a genetic sensor (such as TetR or LacI) that is used to regulate the expression of a fluorescent protein. From this protocol, the following metrics are obtained:
1. G0 (background signal)
if a saturation in the fluorescence response is observed, the following additional metrics can be obtained:
2. EC50 (sensitivity)
3. Ginf (maximum signal)
4. Dynamic range (Ginf/G0)
5. Hill coefficient
If multiple ligands are being tested with the same genetic sensor circuit, additional information is collected:
6. Selectivity
Materials
Starting cultures:
- Glycerol stocks of cells
Reagents:
- LB media
Create the overnight culture
Create the overnight culture
Prepare 3 mL of LB media with appropriate antibiotics in a 15 mL tube for each strain you will be testing
Scrap glycerol stocks of frozen strains and mix them in the prepared tubes.
Incubate shaking at 275 rpm, 37oC for 12 - 24 hours (to reach stationary phase).
Step up the assay plate
Step up the assay plate
Prepare LB media with appropriate antibiotic, mix, and dispense 880 uL of this media in each well of a 96 deep well plate
Add cells
Incubate
Induce cultures with small molecule
Induce cultures with small molecule
Prepare inducer molecule
Dilute inducer molecule
Add inducer to cultures
Return to incubator
Measure cell fluorescence
Measure cell fluorescence
Centrifuge plate
Discard supernatant into hazardous waste
Resuspend cell pellets in PBS
Transfer 100 uL of cell resuspension into a black, clear bottom assay plate
Measure optical density and fluorescence