Reducing DNA context dependence in bacterial promoters
| Autor: | Swati Banerjee Carr, Douglas Densmore, Jacob Beal |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2017 |
| Předmět: |
0301 basic medicine
lcsh:Medicine Artificial Gene Amplification and Extension Insulator (genetics) Polymerase Chain Reaction Biochemistry chemistry.chemical_compound Plasmid Nucleic Acids Genomic library Promoter Regions Genetic lcsh:Science Insulator Element Multidisciplinary Synthetic Genetic Systems Research Design Synthetic Genetic Networks Physical Sciences Engineering and Technology Synthetic Biology Insulator Elements Genetic Engineering Research Article Biotechnology Plasmids DNA Bacterial Circuit performance Permutation Materials Science DNA transcription Computational biology Library Screening Research and Analysis Methods DNA sequencing 03 medical and health sciences Bacterial Proteins Genetics Escherichia coli Molecular Biology Techniques Molecular Biology Materials by Attribute Gene Library Molecular Biology Assays and Analysis Techniques Biology and life sciences Discrete Mathematics lcsh:R Promoter DNA Gene Expression Regulation Bacterial Insulators 030104 developmental biology chemistry Combinatorics lcsh:Q Gene expression Mathematics |
| Zdroj: | PLoS ONE, Vol 12, Iss 4, p e0176013 (2017) PLoS ONE |
| ISSN: | 1932-6203 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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