HEARTBEAT Fuzzy Modeling

Introduction

In order to achieve defined protein levels in a cell, promoters of defined strength are an obvious requirement. Such promoters can only be valuable in synthetic biology if they are well characterized. For future eucaryotic devices that require [http://www.partsregistry.org/PoPS PoPS (Polymerase per second)] as an input, our promoters will be very suitable since they deliver PoPS as an output. PoPs is the standard unit of synthetic biology, but it is very difficult to measure directly. For bacteria, relative measurements (relative promoter units, RPU) are most commonly used and it has been shown how to convert them to PoPs [1]. We identified several challenges to achieve the same in mammalian cells, suggest solutions and provide easy-to use relative measures for application in mammalian cells - one based on RNA levels (Relative Mammalian Promoter Units, RMPU), the other based on folded protein levels (Relative Expression Units, REU). We apply those measurements to the characterization on CMV, an existing promoter from the registry. Finally, we discuss how to transform these units into PoPS.

Background / Motivation

The need for standardized measurements of promoter activity in vivo has been widely accepted across the synthetic biology community [1]. Only if a part is well characterized initially, function of an engineered device or system can be predicted reliably. Most work of synthetic biology has focused on bacteria, especially Escherichia Coli, as a model system. Novel tasks in synthetic biology, especially for medical applications, will require synthetic biology of mammalian cells. Mammalian systems are the most complex biological systems, and therefore, little work has been done in the field of mammalian synthetic biology, leaving a huge potential for future research.

[[Image:HD09_formula1.png|thumb|left|294px|Box 1: Calculation of PoPs, where γM is the mRNA degradation rate, a is the GFP maturation rate, γi is the degradation rate of immature GFP, ρ is the tranlation rate of immature GFP from mRNA and n is the number of copies of promoter per cell.

References

[1] Kelly, JR et al in Journal of Biological Engineering 3 (2009): "Measuring the activity of BioBrick promoters using an in vivo reference standard"
[2] http://www.promega.com/tbs/tm058/tm058.html