Reducing DNA context dependence in bacterial promoters

PLoS One. 2017 Apr 19;12(4):e0176013. doi: 10.1371/journal.pone.0176013. eCollection 2017.

Abstract

Variation in the DNA sequence upstream of bacterial promoters is known to affect the expression levels of the products they regulate, sometimes dramatically. While neutral synthetic insulator sequences have been found to buffer promoters from upstream DNA context, there are no established methods for designing effective insulator sequences with predictable effects on expression levels. We address this problem with Degenerate Insulation Screening (DIS), a novel method based on a randomized 36-nucleotide insulator library and a simple, high-throughput, flow-cytometry-based screen that randomly samples from a library of 436 potential insulated promoters. The results of this screen can then be compared against a reference uninsulated device to select a set of insulated promoters providing a precise level of expression. We verify this method by insulating the constitutive, inducible, and repressible promotors of a four transcriptional-unit inverter (NOT-gate) circuit, finding both that order dependence is largely eliminated by insulation and that circuit performance is also significantly improved, with a 5.8-fold mean improvement in on/off ratio.

MeSH terms

  • Bacterial Proteins / genetics*
  • DNA, Bacterial / genetics*
  • Escherichia coli / genetics*
  • Gene Expression Regulation, Bacterial*
  • Gene Library
  • Genetic Engineering
  • Insulator Elements*
  • Plasmids / chemistry
  • Plasmids / metabolism
  • Promoter Regions, Genetic*
  • Research Design

Substances

  • Bacterial Proteins
  • DNA, Bacterial

Grants and funding

Funding for this work comes from the National Science Foundation (NSF; nsf.gov) Expeditions in Computing Program Award #1522074 as part of the Living Computing Project (JB, DMD), as well as from National Science Foundation (NSF; nsf.gov) Award #1253856 (SBC, DMD) and from the Office of Naval Research (ONR; www.onr.navy.mil) under award N000141110725 (SBC, DMD). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Raytheon BBN Technologies provided support in the form of salary for author JB, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of this author are articulated in the “author contributions” section.