Mining Environmental Plasmids for Synthetic Biology Parts and Devices

Martínez‐García, Esteban; Benedetti, Ilaria; Hueso, Angeles; Lorenzo, Vı́ctor de

doi:10.1128/9781555818982.ch32

Cited by 7 publications

(5 citation statements)

References 127 publications

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“…Successful attempts have been made in exploiting traits from environmental plasmids for standardized components in synthetic biology (Martínez-García et al, 2015). Environmental plasmids are exchangeable between different hosts and may express their genetic features in various genomic and metabolic backgrounds.…”

Section: Future Outlookmentioning

confidence: 99%

Comparative analysis reveals the modular functional structure of conjugative megaplasmid pTTS12 of Pseudomonas putida S12: A paradigm for transferable traits, plasmid stability, and inheritance?

et al. 2022

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Originating from various environmental niches, large numbers of bacterial plasmids have been found carrying heavy metal and antibiotic resistance genes, degradation pathways and specific transporter genes for organic solvents or aromatic compounds. Such genes may constitute promising candidates for novel synthetic biology applications. Our systematic analysis of gene clusters encoded on megaplasmid pTTS12 from Pseudomonas putida S12 underscores that a large portion of its genes is involved in stress response to increase survival under harsh conditions like the presence of heavy metal and organic solvent. We investigated putative roles of genes encoded on pTTS12 and further elaborated on their roles in the establishment and maintenance under several stress conditions, specifically focusing on solvent tolerance in P. putida strains. The backbone of pTTS12 was found to be closely related to that of the carbapenem-resistance plasmid pOZ176, member of the IncP-2 incompatibility group, although the carbapenem resistance cassette is absent from pTTS12. Megaplasmid pTTS12 contains multiple transposon-flanked cassettes mediating resistance to various heavy metals such as tellurite, chromate (Tn7), and mercury (Tn5053 and Tn5563). Additionally, pTTS12 also contains a P-type, Type IV secretion system (T4SS) supporting self-transfer to other P. putida strains. This study increases our understanding in the modular structure of pTTS12 as a member of IncP-2 plasmid family and several promising exchangeable gene clusters to construct robust microbial hosts for biotechnology applications.

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Section: Future Outlookmentioning

confidence: 99%

Comparative analysis reveals the modular functional structure of conjugative megaplasmid pTTS12 of Pseudomonas putida S12: A paradigm for transferable traits, plasmid stability, and inheritance?

et al. 2022

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“…In this work we have used the inducer dependent and AlkS-mediated activation of the PalkB promoter of the pOCT plasmid as an example of the roadmap that should be followed for reshaping a naturally-occurring regulatory node into an standardized device for engineering heterologous expression (40). As with any standard, there is a somewhat arbitrary but still reasonable and scientifically justifiable choice of a given composition rule (41).…”

Section: Discussionmentioning

confidence: 99%

Standardization of inducer-activated broad host range expression modules: Debugging and refactoring an alkane-responsive AlkS/P_alkBdevice

Arce-Rodríguez

Benedetti

Silva‐Rocha

2020

Preprint

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Although inducible heterologous expression systems have been available since the birth of recombinant DNA technology, the diversity of devices and genetic architectures of the corresponding vectors have often resulted in a lack of reproducibility and interoperability. In an effort to increase predictability of expression of genes of interest in a variety of possible bacterial hosts we propose a composition standard for debugging and reassembling all regulatory parts that participate in the performance of such devices. As a case study we address the n-octane and dicyclopropyl ketone (DCPK)-inducible PalkB promoter of the alkane biodegradation pOCT plasmid of Pseudomonas putida. The standardized expression module consisted of an edited alkS regulatory gene that is divergently expressed and separated of PalkB by a synthetic DNA buffer sequence. The native DNA sequence of the structural alkS gene was modified to alleviate the catabolite repression exerted by some carbon and nitrogen sources through the Crc/Hfq complex of some hosts. The PalkB promoter along with the alkS variants were then formatted as SEVA (Standard European Vector Architecture) cargoes and their activity parameters in P. putida determined with GFP and luminiscent reporters. The thereby refactored system showed improvements in various features desirable in conditional expression modules: inducibility, capacity, noise reduction and on/off ratio. When applied to other promoter/regulator pairs, the compositional standard thereby implemented in the AlkS/PalkB module will enable more complex genetic programming in non-model bacteria.

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“…This raises an interesting question on the design of universal expression systems for securing activity of the GoIs in any biological host—or using SynBio jargon, engineering orthogonal heterologous expression [155]. Transcription and translation signals of promiscuous mobile elements look like a good source of parts for engineering equally unrestrained expression devices [156]. Along the line, platforms based on viral promoters and polymerases have been proposed to this end [157].…”

Section: Engineering Horizontal Transfer Of Beneficial Traitsmentioning

confidence: 99%

Environmental Galenics: large-scale fortification of extant microbiomes with engineered bioremediation agents

de Lorenzo

2022

Phil. Trans. R. Soc. B

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Contemporary synthetic biology-based biotechnologies are generating tools and strategies for reprogramming genomes for specific purposes, including improvement and/or creation of microbial processes for tackling climate change. While such activities typically work well at a laboratory or bioreactor scale, the challenge of their extensive delivery to multiple spatio-temporal dimensions has hardly been tackled thus far. This state of affairs creates a research niche for what could be called Environmental Galenics (EG), i.e. the science and technology of releasing designed biological agents into deteriorated ecosystems for the sake of their safe and effective recovery. Such endeavour asks not just for an optimal performance of the biological activity at stake, but also the material form and formulation of the agents, their propagation and their interplay with the physico-chemical scenario where they are expected to perform. EG also encompasses adopting available physical carriers of microorganisms and channels of horizontal gene transfer as potential paths for spreading beneficial activities through environmental microbiomes. While some of these propositions may sound unsettling to anti-genetically modified organisms sensitivities, they may also fall under the tag of TINA (there is no alternative) technologies in the cases where a mere reduction of emissions will not help the revitalization of irreversibly lost ecosystems. This article is part of the theme issue ‘Ecological complexity and the biosphere: the next 30 years’.

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Mining Environmental Plasmids for Synthetic Biology Parts and Devices

Cited by 7 publications

References 127 publications

Comparative analysis reveals the modular functional structure of conjugative megaplasmid pTTS12 of Pseudomonas putida S12: A paradigm for transferable traits, plasmid stability, and inheritance?

Comparative analysis reveals the modular functional structure of conjugative megaplasmid pTTS12 of Pseudomonas putida S12: A paradigm for transferable traits, plasmid stability, and inheritance?

Standardization of inducer-activated broad host range expression modules: Debugging and refactoring an alkane-responsive AlkS/P_alkBdevice

Environmental Galenics: large-scale fortification of extant microbiomes with engineered bioremediation agents

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Mining Environmental Plasmids for Synthetic Biology Parts and Devices

Cited by 7 publications

References 127 publications

Comparative analysis reveals the modular functional structure of conjugative megaplasmid pTTS12 of Pseudomonas putida S12: A paradigm for transferable traits, plasmid stability, and inheritance?

Comparative analysis reveals the modular functional structure of conjugative megaplasmid pTTS12 of Pseudomonas putida S12: A paradigm for transferable traits, plasmid stability, and inheritance?

Standardization of inducer-activated broad host range expression modules: Debugging and refactoring an alkane-responsive AlkS/PalkBdevice

Environmental Galenics: large-scale fortification of extant microbiomes with engineered bioremediation agents

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Standardization of inducer-activated broad host range expression modules: Debugging and refactoring an alkane-responsive AlkS/P_alkBdevice