Transformation of the Plastid Genome in Tobacco: The Model System for Chloroplast Genome Engineering

Maliga, Pál; Tungsuchat-Huang, Tarinee; Lutz, Kerry Ann

doi:10.1007/978-1-0716-1472-3_6

Cited by 6 publications

(4 citation statements)

References 39 publications

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“…Plastid transformation was carried out as described (Lutz et al, 2006; Maliga et al, 2021). Plasmid DNA for plastid transformation was prepared using the QIAGEN Plasmid Maxi Kit (Cat.…”

Section: Methodsmentioning

confidence: 99%

Posttranscriptional tuning of gene expression over a large dynamic range in synthetic tobacco chloroplast operons

Yu,

Tungsuchat-Huang,

Ioannou

et al. 2024

Preprint

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Achieving balanced gene expression within synthetic operons requires a spectrum of expression levels. Here we investigate the expression of gfp reporter gene in tobacco chloroplasts, guided by variants of the plastid atpH 5' UTR, which harbors a binding site for PPR10, a protein that activates atpH at the post-transcriptional level. Our findings reveal that endogenous tobacco PPR10 confers distinct levels of reporter activation when coupled with the tobacco and maize atpH 5' UTRs in different design contexts. Notably, high GFP expression was not coupled to stabilization of monocistronic gfp transcripts in dicistronic reporter lines, adding to the evidence that PPR10 activates translation via a mechanism that is independent of its stabilization of monocistronic transcripts. Furthermore, the incorporation of a tRNA upstream of the UTR nearly abolishes gfp mRNA (and GFP protein), resulting in a substantial reduction in GFP accumulation. When combined with a mutant atpH 5' UTR, the tRNA leads to an exceptionally low level of transgene expression. Collectively, this approach allows for tuning reporter gene expression across a wide range, spanning from 0.02% to 25% of the total soluble cellular protein (TSP). These findings highlight the toolbox available for plastid synthetic biology applications requiring multigene expression at varying levels.

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“…Plastid transformation was carried out as described (Lutz et al, 2006; Maliga et al, 2021). Plasmid DNA for plastid transformation was prepared using the QIAGEN Plasmid Maxi Kit (Cat.…”

Section: Methodsmentioning

confidence: 99%

Posttranscriptional tuning of gene expression over a large dynamic range in synthetic tobacco chloroplast operons

Yu,

Tungsuchat-Huang,

Ioannou

et al. 2024

Preprint

Self Cite

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show abstract

“…In contrast, obtaining homoplasmic plastid transformants and testing parts in vivo requires several months of selection, or in case of poplar trees even up to an entire year. [18][19][20] In order to overcome these limitations, rapid prototyping platforms that facilitate an accelerated Design-Build-Test-Learn cycle (DBTL) need to be developed. 21 Such a platform would enable rapid iterations of genetic designs, testing, and modifications, significantly reducing the time and resources required for part characterization in plastids.…”

Section: Introductionmentioning

confidence: 99%

Chloroplast Cell-Free Systems from Different Plant Species as a Rapid Prototyping Platform

Boehm,

Inckemann,

Burgis

et al. 2024

Preprint

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Climate change poses a significant threat to global agriculture, necessitating innovative solutions. Plant synthetic biology, particularly chloroplast engineering, holds promise as a viable approach to this challenge. Chloroplasts present a variety of advantageous traits for genetic engineering, but the development of genetic tools and genetic part characterization in these organelles is hindered by the lengthy timescales required to generate transplastomic organisms. To address these challenges, we have established a versatile protocol for generating chloroplast-based cell-free gene expression (CFE) systems derived from a diverse range of plant species, including wheat (monocot), spinach, and poplar trees (dicots). We show that these systems work with conventionally used T7 RNA polymerase, as well as the endogenous chloroplast polymerases, allowing for detailed characterization and prototyping of regulatory sequences at both transcription and translation levels. To demonstrate the platform for characterization of promoters and 5' and 3' untranslated regions (UTRs) in higher plant chloroplast gene expression, we analyze a collection of 23 5'UTRs, 10 3'UTRs, and 6 chloroplast promoters, assessed their expression in spinach and wheat extracts, and found consistency in expression patterns, suggesting cross-species compatibility. Looking forward, our chloroplast CFE systems open new avenues for plant synthetic biology, offering prototyping tools for both understanding gene expression and developing engineered plants, which could help meet the demands of a changing global climate.

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“…Extensive and efficient genetic tools enable rapid engineering within days in microbial chassis. In contrast, obtaining homoplasmic plastid transformants and testing parts in vivo requires several months of selection, or in the case of poplar trees even up to an entire year. − In order to overcome these limitations, rapid prototyping platforms that facilitate an accelerated Design-Build-Test-Learn cycle (DBTL) need to be developed. , Such a platform would enable rapid iterations of genetic designs, testing, and modifications, significantly reducing the time and resources required for part characterization in plastids.…”

Section: Introductionmentioning

confidence: 99%

Chloroplast Cell-Free Systems from Different Plant Species as a Rapid Prototyping Platform

Böhm,

Inckemann,

Burgis

et al. 2024

ACS Synth. Biol.

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Climate change poses a significant threat to global agriculture, necessitating innovative solutions. Plant synthetic biology, particularly chloroplast engineering, holds promise as a viable approach to this challenge. Chloroplasts present a variety of advantageous traits for genetic engineering, but the development of genetic tools and genetic part characterization in these organelles is hindered by the lengthy time scales required to generate transplastomic organisms. To address these challenges, we have established a versatile protocol for generating highly active chloroplast-based cell-free gene expression (CFE) systems derived from a diverse range of plant species, including wheat (monocot), spinach, and poplar trees (dicots). We show that these systems work with conventionally used T7 RNA polymerase as well as the endogenous chloroplast polymerases, allowing for detailed characterization and prototyping of regulatory sequences at both transcription and translation levels. To demonstrate the platform for characterization of promoters and 5′ and 3′ untranslated regions (UTRs) in higher plant chloroplast gene expression, we analyze a collection of 23 5′UTRs, 10 3′UTRs, and 6 chloroplast promoters, assessed their expression in spinach and wheat extracts, and found consistency in expression patterns, suggesting crossspecies compatibility. Looking forward, our chloroplast CFE systems open new avenues for plant synthetic biology, offering prototyping tools for both understanding gene expression and developing engineered plants, which could help meet the demands of a changing global climate.

show abstract

Transformation of the Plastid Genome in Tobacco: The Model System for Chloroplast Genome Engineering

Cited by 6 publications

References 39 publications

Posttranscriptional tuning of gene expression over a large dynamic range in synthetic tobacco chloroplast operons

Posttranscriptional tuning of gene expression over a large dynamic range in synthetic tobacco chloroplast operons

Chloroplast Cell-Free Systems from Different Plant Species as a Rapid Prototyping Platform

Chloroplast Cell-Free Systems from Different Plant Species as a Rapid Prototyping Platform

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