Standardized GFP quantification

Contacts

Barry Canton, Caitlin Conboy, Jason Kelly, Ania Labno, Josh Michener

Introduction

This project falls into the larger category of standardizing biological technologies and processes (see also this open project). We think that this project would benefit a number of people in the Endy lab and others.

Standardization offers many benefits when designing and characterizing engineered biological systems. While standardization can be implemented at many levels with varying levels of difficulty, a fundamental standardization is to be able to reliably quantify the number of reporter proteins per cell in a culture sample. If this quantification can be done reliably and rapidly then experimental data can be compared across experiments, instruments and labs. Ideally, a calibration curve should exist for all instruments (e.g. plate readers, flow cytometers etc.) so that the relative units on the particular machine can be related back to absolute numbers of molecules per cell.

Specifically, we are proposing to calibrate the relative measurements from plate reader and flow cytometer to GFP molecule numbers per cell. In the longer term we would like to extend this to other fluorescent reporters and a wide range of instrument settings.

Prior Work

We can draw on a large body of work that has been done to quantify absolute numbers of molecules and to produce reproducible standards for instruments. We highlight a number of techniqes that can be leveraged to enable the standard quantification of molecule numbers.

• Protein purification techniqes
• Quantitative Western blotting and variants
• Fluorescence protein technologies
• Standardized fluorescent bead technologies
• Microscope image analysis techniques (a la Elowitz)

We have sufficient expertise in a number of these technologies to perform a standardization process on all the instruments we currently use.