Composability of regulatory sequences controlling transcription and translation in Escherichia coli
Open accessAbstract
The inability to predict heterologous gene expression levels precisely hinders our ability to engineer biological systems. Using well-characterized regulatory elements offers a potential solution only if such elements behave predictably when combined. We synthesized 12,563 combinations of common promoters and ribosome binding sites and simultaneously measured DNA, RNA, and protein levels from the entire library. Using a simple model, we found that RNA and protein expression were within twofold of expected levels 80% and 64% of the time, respectively. The large dataset allowed quantitation of global effects, such as translation rate on mRNA stability and mRNA secondary structure on translation rate. However, the worst 5% of constructs deviated from prediction by 13-fold on average, which could hinder large-scale genetic engineering projects. The ease and scale this of approach indicates that rather than relying on prediction or standardization, we can screen synthetic libraries for desired behavior.
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FACS- and growth-selection-based MPRAs require expression of a single library variant per cell that must be transcribed within the cell from a DNA template, making it difficult to distinguish between the effects of transcriptional and translational control.
“These techniques require the expression of a single library variant per cell that must be transcribed within the cell from a DNA template, making it difficult to distinguish between the effects of transcriptional and translational control.”
From Human 5′ UTR design and variant effect prediction from a massively parallel translation assay (2019) · cites this paper as [11]
Earlier MPRAs designed to learn aspects of 5′ UTR cis-regulation relied on FACS or growth selection to stratify libraries by activity.
“Earlier MPRAs designed to learn aspects of 5′ UTR cis-regulation relied on FACS10,11 or growth selection12 to stratify libraries by activity.”
From Human 5′ UTR design and variant effect prediction from a massively parallel translation assay (2019) · cites this paper as [11]
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- Authors
- Kosuri, S., Goodman, D. B., Cambray, G., Mutalik, V. K., Gao, Y., Arkin, A. P., Endy, D., & Church, G. M.
- Affiliations
- Harvard University (US)
- Lawrence Berkeley National Laboratory (US)
- Johns Hopkins University (US)
- Stanford University (US)
- ORCID
- Venue
- Proceedings of the National Academy of Sciences
- Publisher
- National Academy of Sciences
- Published
- 2013-08-07
- Volume
- 110
- Issue
- 34
- Pages
- 14024-14029
- ISSN
- 0027-8424 (ISSN-L)
- 1091-6490
- PMC
- PMC3752251
- OpenAlex
- W2018645639
- Cited by
- 392
- References
- 5
- Citations by year
- 2026: 20 · 2025: 23 · 2024: 23 · 2023: 26 · 2022: 30 · 2021: 28 · 2020: 35 · 2019: 35 · 2018: 38 · 2017: 38 · 2016: 34 · 2015: 28 · 2014: 27 · 2013: 6
- FWCI
- 11.93
- Type
- journal article
- Language
- en
- Open access
- green
- europepmc.org
- Locations
- Proceedings of the National Academy of Sciences (publishedVersion)
- Europe PMC (PubMed Central) (publishedVersion)
- PubMed Central (publishedVersion)
- PubMed (publishedVersion)
- Digital Access to Scholarship at Harvard (DASH) (Harvard University) (submittedVersion)
- HAL (submittedVersion, other-oa)
- DSpace@MIT (Massachusetts Institute of Technology) (submittedVersion, cc-by-nc)
- eScholarship (California Digital Library) (submittedVersion)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Microbial Metabolic Engineering and Bioproduction
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Gene Regulatory Network Analysis
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA and protein synthesis mechanisms
- Keywords
- synthetic gene circuits, promoter library, ribosome binding site, heterologous expression, transcriptional regulation, translational regulation, mRNA stability, mRNA secondary structure, translation rate, Escherichia coli, synthetic biology, high-throughput screening
- MeSH terms
- Cloning, Molecular, Escherichia coli, Flow Cytometry, Genetic Engineering, Models, Genetic, Promoter Regions, Genetic, Ribosomes, RNA, Messenger, Gene Library, Gene Expression Regulation, Bacterial, DNA Primers, Reverse Transcriptase Polymerase Chain Reaction, Systems Biology, Regulatory Elements, Transcriptional, High-Throughput Nucleotide Sequencing
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