RESUMO
An inability to reliably predict quantitative behaviors for novel combinations of genetic elements limits the rational engineering of biological systems. We developed an expression cassette architecture for genetic elements controlling transcription and translation initiation in Escherichia coli: transcription elements encode a common mRNA start, and translation elements use an overlapping genetic motif found in many natural systems. We engineered libraries of constitutive and repressor-regulated promoters along with translation initiation elements following these definitions. We measured activity distributions for each library and selected elements that collectively resulted in expression across a 1,000-fold observed dynamic range. We studied all combinations of curated elements, demonstrating that arbitrary genes are reliably expressed to within twofold relative target expression windows with â¼93% reliability. We expect the genetic element definitions validated here can be collectively expanded to create collections of public-domain standard biological parts that support reliable forward engineering of gene expression at genome scales.
Assuntos
Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Fatores de Iniciação em Procariotos/metabolismo , Transcrição Gênica , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica/fisiologia , Biblioteca Gênica , Engenharia Genética , Genoma Bacteriano , Fatores de Iniciação em Procariotos/genética , Regiões Promotoras Genéticas/genética , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
The practice of engineering biology now depends on the ad hoc reuse of genetic elements whose precise activities vary across changing contexts. Methods are lacking for researchers to affordably coordinate the quantification and analysis of part performance across varied environments, as needed to identify, evaluate and improve problematic part types. We developed an easy-to-use analysis of variance (ANOVA) framework for quantifying the performance of genetic elements. For proof of concept, we assembled and analyzed combinations of prokaryotic transcription and translation initiation elements in Escherichia coli. We determined how estimation of part activity relates to the number of unique element combinations tested, and we show how to estimate expected ensemble-wide part activity from just one or two measurements. We propose a new statistic, biomolecular part 'quality', for tracking quantitative variation in part performance across changing contexts.