Building a pipeline to identify and engineer constitutive and repressible promoters

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Abstract

To support the increasingly complex circuits needed for plant synthetic biology applications, additional constitutive promoters are essential. Reusing promoter parts can lead to difficulty in cloning, increased heterogeneity between transformants, transgene silencing and trait instability. We have developed a pipeline to identify genes that have stable expression across a wide range of Arabidopsis tissues at different developmental stages and have identified a number of promoters that are well expressed in both transient ( Nicotiana benthamiana) and stable ( Arabidopsis) transformation assays. We have also introduced two genome-orthogonal gRNA target sites in a subset of the screened promoters, converting them into NOR logic gates. The work here establishes a pipeline to screen for additional constitutive promoters and can form the basis of constructing more complex information processing circuits in the future.

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Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2

Michael Love, Wolfgang Huber, Simon Anders (2014)

In comparative high-throughput sequencing assays, a fundamental task is the analysis of count data, such as read counts per gene in RNA-seq, for evidence of systematic changes across experimental conditions. Small replicate numbers, discreteness, large dynamic range and the presence of outliers require a suitable statistical approach. We present DESeq2, a method for differential analysis of count data, using shrinkage estimation for dispersions and fold changes to improve stability and interpretability of estimates. This enables a more quantitative analysis focused on the strength rather than the mere presence of differential expression. The DESeq2 package is available at http://www.bioconductor.org/packages/release/bioc/html/DESeq2.html. Electronic supplementary material The online version of this article (doi:10.1186/s13059-014-0550-8) contains supplementary material, which is available to authorized users.

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Fiji: an open-source platform for biological-image analysis.

Johannes Schindelin, Ignacio Arganda-Carreras, Erwin Frise … (2020)

Fiji is a distribution of the popular open-source software ImageJ focused on biological-image analysis. Fiji uses modern software engineering practices to combine powerful software libraries with a broad range of scripting languages to enable rapid prototyping of image-processing algorithms. Fiji facilitates the transformation of new algorithms into ImageJ plugins that can be shared with end users through an integrated update system. We propose Fiji as a platform for productive collaboration between computer science and biology research communities.

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A new mathematical model for relative quantification in real-time RT-PCR.

M. Pfaffl (2001)

Use of the real-time polymerase chain reaction (PCR) to amplify cDNA products reverse transcribed from mRNA is on the way to becoming a routine tool in molecular biology to study low abundance gene expression. Real-time PCR is easy to perform, provides the necessary accuracy and produces reliable as well as rapid quantification results. But accurate quantification of nucleic acids requires a reproducible methodology and an adequate mathematical model for data analysis. This study enters into the particular topics of the relative quantification in real-time RT-PCR of a target gene transcript in comparison to a reference gene transcript. Therefore, a new mathematical model is presented. The relative expression ratio is calculated only from the real-time PCR efficiencies and the crossing point deviation of an unknown sample versus a control. This model needs no calibration curve. Control levels were included in the model to standardise each reaction run with respect to RNA integrity, sample loading and inter-PCR variations. High accuracy and reproducibility (<2.5% variation) were reached in LightCycler PCR using the established mathematical model.

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Author and article information

Journal

Journal ID (nlm-ta): Quant Plant Biol

Journal ID (iso-abbrev): Quant Plant Biol

Journal ID (publisher-id): QPB

Title: Quantitative Plant Biology

Publisher: Cambridge University Press (Cambridge, UK )

ISSN (Electronic): 2632-8828

Publication date (Electronic, collection): 2023

Publication date (Electronic): 19 October 2023

Volume: 4

Electronic Location Identifier: e12

Affiliations

[1]Department of Biology, University of Washington , Seattle, WA, USA

Department of Biology, University of Washington , United States

Heinrich-Heine-Universität Düsseldorf , Germany

RDP, ENS de Lyon , France

Department of Biology, University of Washington , United States

Heinrich-Heine-Universität Düsseldorf , Germany

RDP, ENS de Lyon , France

Department of Biology, University of Washington , United States

Heinrich-Heine-Universität Düsseldorf , Germany

RDP, ENS de Lyon , France

Author notes

Corresponding author: Jennifer L. Nemhauser; Email: jn7@ 123456uw.edu

Author information

Eric J.Y. Yang https://orcid.org/0000-0001-5524-7471

Jennifer L. Nemhauser https://orcid.org/0000-0002-8909-735X

Article

Publisher Item ID: S2632882823000103

DOI: 10.1017/qpb.2023.10

PMC ID: 10600573

SO-VID: 65e7faa0-3073-4e2b-bd44-7eddd47fea51

License:

This is an Open Access article, distributed under the terms of the Creative Commons Attribution licence ( http://creativecommons.org/licenses/by/4.0), which permits unrestricted re-use, distribution and reproduction, provided the original article is properly cited.

History

Date received : 13 October 2022

Date revision received : 24 July 2023

Date accepted : 13 September 2023

Page count

Figures: 4, References: 54, Pages: 10

Funding

Funded by: Division of Integrative Organismal Systems, doi https://doi.org/10.13039/100000154;

Award ID: IOS-1546873

Funded by: Howard Hughes Medical Institute, doi https://doi.org/10.13039/100000011;

Funded by: National Institute of General Medical Sciences, doi https://doi.org/10.13039/100000057;

Building a pipeline to identify and engineer constitutive and repressible promoters

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Journal of Green Building

Most cited references 49

Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2

Fiji: an open-source platform for biological-image analysis.

A new mathematical model for relative quantification in real-time RT-PCR.

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