Multiplexed characterization of rationally designed promoter architectures deconstructs combinatorial logic for IPTG-inducible systems

Timothy C Yu; Winnie L Liu; Marcia S Brinck; Jessica E Davis; Jeremy Shek; Grace Bower; Tal Einav; Kimberly D Insigne; Rob Phillips; Sriram Kosuri; Guillaume Urtecho

doi:10.1038/s41467-020-20094-3

Multiplexed characterization of rationally designed promoter architectures deconstructs combinatorial logic for IPTG-inducible systems

Nat Commun. 2021 Jan 12;12(1):325. doi: 10.1038/s41467-020-20094-3.

Authors

Timothy C Yu¹, Winnie L Liu², Marcia S Brinck³, Jessica E Davis⁴, Jeremy Shek⁴, Grace Bower², Tal Einav⁵, Kimberly D Insigne⁶, Rob Phillips^{5

7

8}, Sriram Kosuri^{9

10

11

12

13

14}, Guillaume Urtecho¹⁵

Affiliations

¹ Department of Bioengineering, University of California, Los Angeles, CA, 90095, USA.
² Department of Molecular, Cell, and Developmental Biology, University of California, Los Angeles, CA, 90095, USA.
³ Department of Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles, CA, 90095, USA.
⁴ Department of Chemistry and Biochemistry, University of California, Los Angeles, CA, 90095, USA.
⁵ Department of Physics, California Institute of Technology, Pasadena, CA, 91125, USA.
⁶ Bioinformatics Interdepartmental Graduate Program, University of California, Los Angeles, CA, 90095, USA.
⁷ Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA, 91125, USA.
⁸ Department of Applied Physics, California Institute of Technology, Pasadena, CA, 91125, USA.
⁹ Department of Chemistry and Biochemistry, University of California, Los Angeles, CA, 90095, USA. sri@ucla.edu.
¹⁰ UCLA-DOE Institute for Genomics and Proteomics, Los Angeles, CA, 90095, USA. sri@ucla.edu.
¹¹ Institute for Quantitative and Computational Biosciences (QCB), University of California, Los Angeles, Los Angeles, CA, 90095, USA. sri@ucla.edu.
¹² Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, University of California, Los Angeles, Los Angeles, CA, 90095, USA. sri@ucla.edu.
¹³ Jonsson Comprehensive Cancer Center, University of California, Los Angeles, CA, 90095, USA. sri@ucla.edu.
¹⁴ Molecular Biology Interdepartmental Doctoral Program, University of California, Los Angeles, CA, 90095, USA. sri@ucla.edu.
¹⁵ Molecular Biology Interdepartmental Doctoral Program, University of California, Los Angeles, CA, 90095, USA. gurtecho@ucla.edu.

Abstract

A crucial step towards engineering biological systems is the ability to precisely tune the genetic response to environmental stimuli. In the case of Escherichia coli inducible promoters, our incomplete understanding of the relationship between sequence composition and gene expression hinders our ability to predictably control transcriptional responses. Here, we profile the expression dynamics of 8269 rationally designed, IPTG-inducible promoters that collectively explore the individual and combinatorial effects of RNA polymerase and LacI repressor binding site strengths. We then fit a statistical mechanics model to measured expression that accurately models gene expression and reveals properties of theoretically optimal inducible promoters. Furthermore, we characterize three alternative promoter architectures and show that repositioning binding sites within promoters influences the types of combinatorial effects observed between promoter elements. In total, this approach enables us to deconstruct relationships between inducible promoter elements and discover practical insights for engineering inducible promoters with desirable characteristics.

Publication types

Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

Binding Sites
Biophysical Phenomena
DNA-Directed RNA Polymerases / metabolism
Escherichia coli / drug effects
Escherichia coli / metabolism
Fluorescence
Genes, Reporter
Isopropyl Thiogalactoside / pharmacology*
Logic*
Mutation / genetics
Operator Regions, Genetic / genetics
Promoter Regions, Genetic*
Protein Binding
Reproducibility of Results
Thermodynamics
Transcription Factors / metabolism

Substances

Transcription Factors
Isopropyl Thiogalactoside
DNA-Directed RNA Polymerases

Abstract

Publication types

MeSH terms

Substances

Grants and funding