Building the Plant SynBio Toolbox through Combinatorial Analysis of DNA Regulatory Elements

Alexander C Pfotenhauer; Alessandro Occhialini; Mary-Anne Nguyen; Helen Scott; Lezlee T Dice; Stacee A Harbison; Li Li; D Nikki Reuter; Tayler M Schimel; C Neal Stewart Jr; Jacob Beal; Scott C Lenaghan

doi:10.1021/acssynbio.2c00147

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Building the Plant SynBio Toolbox through Combinatorial Analysis of DNA Regulatory Elements

Journal article

Open access

Peer reviewed

Building the Plant SynBio Toolbox through Combinatorial Analysis of DNA Regulatory Elements

Alexander C Pfotenhauer, Alessandro Occhialini, Mary-Anne Nguyen, Helen Scott, Lezlee T Dice, Stacee A Harbison, Li Li, D Nikki Reuter, Tayler M Schimel, C Neal Stewart Jr, …

ACS synthetic biology, Vol.11(8), pp.2741-2755

08/19/2022

DOI: 10.1021/acssynbio.2c00147

PMCID: PMC9396662

PMID: 35901078

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https://doi.org/10.1021/acssynbio.2c00147View

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Abstract

While the installation of complex genetic circuits in microorganisms is relatively routine, the synthetic biology toolbox is severely limited in plants. Of particular concern is the absence of combinatorial analysis of regulatory elements, the long design-build-test cycles associated with transgenic plant analysis, and a lack of naming standardization for cloning parts. Here, we use previously described plant regulatory elements to design, build, and test 91 transgene cassettes for relative expression strength. Constructs were transiently transfected into leaves and expression of a fluorescent reporter was measured from plant canopies, leaves, and protoplasts isolated from transfected plants. As anticipated, a dynamic level of expression was achieved from the library, ranging from near undetectable for the weakest cassette to a ∼200-fold increase for the strongest. Analysis of expression levels in plant canopies, individual leaves, and protoplasts were correlated, indicating that any of the methods could be used to evaluate regulatory elements in plants. Through this effort, a well-curated 37-member part library of plant regulatory elements was characterized, providing the necessary data to standardize construct design for precision metabolic engineering in plants.

DNA - metabolism

Nicotiana - genetics

Plant Leaves - genetics

Plant Leaves - metabolism

Plants, Genetically Modified - genetics

Synthetic Biology - methods

Details

Title: Subtitle: Building the Plant SynBio Toolbox through Combinatorial Analysis of DNA Regulatory Elements
Creators: Alexander C Pfotenhauer - University of Tennessee at Knoxville
Alessandro Occhialini - University of Tennessee at Knoxville
Mary-Anne Nguyen - University of Tennessee at Knoxville
Helen Scott - RTX (United States)
Lezlee T Dice - University of Tennessee at Knoxville
Stacee A Harbison - University of Tennessee at Knoxville
Li Li - University of Tennessee at Knoxville
D Nikki Reuter - University of Tennessee Institute of Agriculture
Tayler M Schimel - University of Tennessee at Knoxville
C Neal Stewart Jr - University of Tennessee at Knoxville
Jacob Beal - RTX (United States)
Scott C Lenaghan - University of Tennessee at Knoxville
Resource Type: Journal article
Publication Details: ACS synthetic biology, Vol.11(8), pp.2741-2755
DOI: 10.1021/acssynbio.2c00147
PMID: 35901078
PMCID: PMC9396662
NLM abbreviation: ACS Synth Biol
ISSN: 2161-5063
eISSN: 2161-5063
Language: English
Date published: 08/19/2022
Academic Unit: Electrical and Computer Engineering
Record Identifier: 9984627232002771

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