Journal article
Reducing DNA context dependence in bacterial promoters
PloS one, Vol.12(4), pp.e0176013-e0176013
04/19/2017
DOI: 10.1371/journal.pone.0176013
PMCID: PMC5396932
PMID: 28422998
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.
Details
- Title: Subtitle
- Reducing DNA context dependence in bacterial promoters
- Creators
- Swati B Carr - Boston UniversityJacob Beal - RTXDouglas M Densmore - Boston University
- Resource Type
- Journal article
- Publication Details
- PloS one, Vol.12(4), pp.e0176013-e0176013
- DOI
- 10.1371/journal.pone.0176013
- PMID
- 28422998
- PMCID
- PMC5396932
- ISSN
- 1932-6203
- eISSN
- 1932-6203
- Language
- English
- Date published
- 04/19/2017
- Academic Unit
- Electrical and Computer Engineering
- Record Identifier
- 9984627183302771
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