Precise and reliable gene expression via standard transcription and translation initiation elements

Nat Methods. 2013 Apr;10(4):354-60. doi: 10.1038/nmeth.2404. Epub 2013 Mar 10.

Abstract

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 ∼93% 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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Escherichia coli / genetics
  • Escherichia coli / metabolism*
  • Gene Expression Regulation, Bacterial* / physiology
  • Gene Library
  • Genetic Engineering
  • Genome, Bacterial
  • Prokaryotic Initiation Factors / genetics
  • Prokaryotic Initiation Factors / metabolism*
  • Promoter Regions, Genetic / genetics
  • RNA, Bacterial / genetics
  • RNA, Bacterial / metabolism
  • RNA, Messenger / genetics
  • RNA, Messenger / metabolism
  • Transcription, Genetic*

Substances

  • Prokaryotic Initiation Factors
  • RNA, Bacterial
  • RNA, Messenger