External control of transgene expression in tobacco plastids using the bacterial <i>lac</i> repressor

Mühlbauer, Stefan Klaus; Koop, Hans‐Ulrich

doi:10.1111/j.1365-313x.2005.02495.x

Cited by 60 publications

(29 citation statements)

References 20 publications

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“…32, 33). Solutions to these problems have long been sought (24)(25)(26), but progress has been hampered so far by the lack of tightly regulated inducible gene expression systems for plastids that are independent of additional nuclear transgenes. The theophylline riboswitch offers a "plastid-only" solution to inducible gene expression from the chloroplast genome that does not require additional (nuclear or plastid) transgenes and thus should be widely applicable.…”

Section: Discussionmentioning

confidence: 99%

“…Their complexity has also hampered the development of tightly controlled inducible gene expression systems for plastids. Although recent progress has made it possible to genetically engineer the chloroplast genomes of an increasing number of higher plants (23), transgene expression from the plastid genome is usually constitutive and attempts to construct inducible gene expression systems have been afflicted with the requirement for additional nuclear transgenes and/or a substantial leakiness of gene expression in the uninduced state (24)(25)(26). This is highly unfortunate, because tightly controllable expression systems represent versatile tools in both functional genomics and biotechnology.…”

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confidence: 99%

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Inducible gene expression from the plastid genome by a synthetic riboswitch

Verhounig

Karcher

Bock

2010

Proc. Natl. Acad. Sci. U.S.A.

120

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Riboswitches are natural RNA sensors that regulate gene expression in response to ligand binding. Riboswitches have been identified in prokaryotes and eukaryotes but are unknown in organelles (mitochondria and plastids). Here we have tested the possibility to engineer riboswitches for plastids (chloroplasts), a genetic system that largely relies on translational control of gene expression. To this end, we have used bacterial riboswitches and modified them in silico to meet the requirements of translational regulation in plastids. These engineered switches were then tested for functionality in vivo by stable transformation of the tobacco chloroplast genome. We report the identification of a synthetic riboswitch that functions as an efficient translational regulator of gene expression in plastids in response to its exogenously applied ligand theophylline. This riboswitch provides a novel tool for plastid genome engineering that facilitates the tightly regulated inducible expression of chloroplast genes and transgenes and thus has wide applications in functional genomics and biotechnology.chloroplast | plastid transformation | translational regulation R iboswitches are natural RNA sensors that mediate control of gene expression via their capacity to bind small molecules (metabolites). They fold into RNA secondary structures whose conformation switches between an "on" state and an "off" state in response to ligand binding (reviewed, e.g., in refs. 1-4). Riboswitches can be divided into two distinct structural domains: an aptamer and an expression platform. The aptamer domain binds the metabolite and this triggers conformational changes in the expression platform, which either permit or prevent gene expression. Depending on the nature of the response to metabolite binding, "on" switches and "off" switches are distinguished. In bacteria, riboswitches reside mainly in the 5 0 untranslated regions (UTRs) of mRNAs and an emerging theme is that anabolic genes are mainly controlled by "off" switches (5-7), whereas catabolic genes are controlled by "on" switches (8), thus providing an efficient mechanism of feedback control of metabolic pathways. In bacteria, most riboswitches act at the transcriptional level [e.g., by resolution of rho-independent transcription termination hairpins (8, 9)], but a few translational switches have also been identified (5, 7). Translational riboswitches usually function via sequestration of the ribosome-binding site (Shine-Dalgarno (SD) sequence), thereby blocking translation initiation in a metabolitedependent manner. The increasing understanding of the functional principles of bacterial riboswitches and the possibility to produce novel aptamers by in vitro evolution of RNA molecules has facilitated attempts to design synthetic switches (10-12), which potentially can provide versatile tools for genetic engineering and synthetic biology applications.While, over the past years, numerous riboswitches have been discovered in prokaryotes (13) and also a few in eukaryotes including plants (14,15), ...

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Inducible gene expression from the plastid genome by a synthetic riboswitch

Verhounig

Karcher

Bock

2010

Proc. Natl. Acad. Sci. U.S.A.

120

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“…Previous studies have also highlighted the activity of chloroplast promoters in bacteria (Brixey et al 1997) and of bacterial promoters in the chloroplast (Newell et al 2003). Another bacterial gene expression element, the widely used lac repressor from E. coli, has been adapted for IPTG-inducible chloroplast transgene expression (Mühlbauer and Koop 2005). Alternatively, chloroplast-based translational regulaChlorophyll 610-700 nm CFP 466-495 nm Merge Figure 3.…”

Section: Cr Boehm Et Almentioning

confidence: 99%

Synthetic Botany

Boehm

Pollak

Purswani³

et al. 2017

Cold Spring Harb Perspect Biol

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Plants are attractive platforms for synthetic biology and metabolic engineering. Plants' modular and plastic body plans, capacity for photosynthesis, extensive secondary metabolism, and agronomic systems for large-scale production make them ideal targets for genetic reprogramming. However, efforts in this area have been constrained by slow growth, long life cycles, the requirement for specialized facilities, a paucity of efficient tools for genetic manipulation, and the complexity of multicellularity. There is a need for better experimental and theoretical frameworks to understand the way genetic networks, cellular populations, and tissue-wide physical processes interact at different scales. We highlight new approaches to the DNA-based manipulation of plants and the use of advanced quantitative imaging techniques in simple plant models such as Marchantia polymorpha. These offer the prospects of improved understanding of plant dynamics and new approaches to rational engineering of plant traits.

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“…We therefore suspected that expression of the genes from the (prokaryotic-type) plastid expression signals results in high levels of Pal and Cpl-1 accumulation in bacteria, which is lethal, most likely caused by the cell wall-hydrolyzing properties of the enzymes. As currently-available inducible expression systems for plastids suffer from either low efficiency (11) or problems with undesired side effects on plastid gene expression (12), we sought an alternative solution by developing a strategy that would prevent expression of the potentially toxic proteins in E. coli without impairing expression in plastids. One of the few differences in gene expression between plastids and eubacteria lies in the mechanism of transcription termination.…”

Section: Toxicity Of Pal and Cpl-1 And Design Of A Toxin Shuttle Vectormentioning

confidence: 99%

Plastid production of protein antibiotics against pneumonia via a new strategy for high-level expression of antimicrobial proteins

Oey

Lohse

Scharff

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

101

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Plastid transformation has become an attractive tool in biotechnology. Because of the prokaryotic nature of the plastid's gene expression machinery, expression elements (promoters and untranslated regions) that trigger high-level foreign protein accumulation in plastids usually also confer high expression in bacterial cloning hosts. This can cause problems, for example, when production of antimicrobial compounds is attempted. Their bactericidal activity can make the cloning of the corresponding genes in plastid transformation vectors impossible. Here, we report a general solution to this problem. We have designed a strategy (referred to as toxin shuttle) that allows the expression in plastids of proteins that are toxic to Escherichia coli. The strategy is based on blocking transcription in E. coli by bacterial transcription terminators upstream of the gene of interest, which subsequently are excised in planta by site-specific recombination. We demonstrate the applicability of the strategy by the high-level expression in plastids (to up to 30% of the plant's total soluble protein) of 2 phage-derived protein antibiotics that are toxic to E. coli. We also show that the plastid-produced antibiotics efficiently kill pathogenic strains of Streptococcus pneumoniae, the causative agent of pneumonia, thus providing a promising strategy for the production of next-generation antibiotics in plants.chloroplast ͉ molecular farming ͉ plastid transformation ͉ phage lysin ͉ site-specific recombination

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External control of transgene expression in tobacco plastids using the bacterial lac repressor

Cited by 60 publications

References 20 publications

Inducible gene expression from the plastid genome by a synthetic riboswitch

Inducible gene expression from the plastid genome by a synthetic riboswitch

Synthetic Botany

Plastid production of protein antibiotics against pneumonia via a new strategy for high-level expression of antimicrobial proteins

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