Rational Design of Evolutionarily Stable Microbial Kill Switches

Stirling, Finn; Bitzan, Lisa; O’Keefe, Samuel; Redfield, Elizabeth; Oliver, John W. K.; Way, Jeffrey C.; Silver, Pamela A.

doi:10.1016/j.molcel.2018.10.002

Cited by 8 publications

(4 citation statements)

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“…Antibiotic addition quickly becomes expensive when scaling for industrial scale manufacturing of nonreplication chassis, but integrating other killing switches (toxin−antitoxin, auxotrophy-based, growth-sensing) can provide excellent alternatives to antibiotics. 52,53 Our goal was to build an easy-to-use and simple chassis to express user-designed genetic circuits, such as reporters, and value-added products; therefore, we confirmed that the produced chassis (with fragmented chromosomes) are biosynthetically active and can express user-designed genetic units. We coupled our sgRNA-C with a tetracycline-inducible green fluorescent protein (GFP) reporter system and transformed the plasmid into cells expressing Cas12 under the arabinose-inducible promoter (Figure S7A).…”

Section: ■ Results and Discussionsupporting

confidence: 57%

“…Our results demonstrated that ceftriaxone in conjunction with the CRISRP-Cas12 provides a reliable method to purify nonreplicative CS-cells without apparent changes in CS-cells morphology. Antibiotic addition quickly becomes expensive when scaling for industrial scale manufacturing of nonreplication chassis, but integrating other killing switches (toxin–antitoxin, auxotrophy-based, growth-sensing) can provide excellent alternatives to antibiotics. , …”

Section: Resultsmentioning

confidence: 99%

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CS-Cells: A CRISPR-Cas12 DNA Device to Generate Chromosome-Shredded Cells for Efficient and Safe Molecular Biomanufacturing

2022

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Synthetic biology holds great promise for translating ideas into products to address the grand challenges facing humanity. Molecular biomanufacturing is an emerging technology that facilitates the production of key products of value, including therapeutics and select chemical compounds. Current biomanufacturing technologies require improvements to overcome limiting factors, including efficient production, cost, and safe release; therefore, developing optimum chassis for biomolecular manufacturing is of great interest for enabling diverse synthetic biology applications. Here, we harnessed the power of the CRISPR-Cas12 system to design, build, and test a DNA device for genome shredding, which fragments the native genome to enable the conversion of bacterial cells into nonreplicative, biosynthetically active, and programmable molecular biomanufacturing chassis. As a proof of concept, we demonstrated the efficient production of green fluorescent protein and violacein, an antimicrobial and antitumorigenic compound. Our CRISPR-Cas12-based chromosome-shredder DNA device has built-in biocontainment features providing a roadmap for the conversion of any bacterial cell into a chromosome-shredded chassis amenable to high-efficiency molecular biomanufacturing, thereby enabling exciting and diverse biotechnological applications.

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Section: ■ Results and Discussionsupporting

confidence: 57%

Section: Resultsmentioning

confidence: 99%

CS-Cells: A CRISPR-Cas12 DNA Device to Generate Chromosome-Shredded Cells for Efficient and Safe Molecular Biomanufacturing

2022

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“…This strategy proposes a novel ultrasound-responsive bacterial drug delivery approach. Interestingly, the temperature-responsive element is also used to prevent environmental pollution caused by the excretion of microbial drugs in the body 121 , 122 .…”

Section: The Condition-responsive Elementmentioning

confidence: 99%

Recent advances in bacterial therapeutics based on sense and response

Feng

Wang

et al. 2023

Acta Pharmaceutica Sinica B

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“…Recently reported organisms with a reduced genetic code may be less likely to acquire natural genetic material that could confer new phenotypes and contribute synthetic genetic material to the natural gene pool (Lau et al 2017;Robertson et al 2021). Novel kill switches and synthetic auxotrophy may also restrict the activity and replication of engineered organisms to controlled environments (Stirling et al 2018Whitford et al 2018;Stirling and Silver 2020). In 2022, the Engineering Biology Research Consortium released a road map for the application of synthetic biology to climate and sustainability that urges the identification, assessment, and open communication of benefits and risks associated with research efforts (Chen et al 2022).…”

Section: The Upper Limit Of Microbially Mediated Dissolution and Bios...mentioning

confidence: 99%

Microbial Catalysis for CO₂Sequestration: A Geobiological Approach

Van Den Berghe,

Walworth,

Dalvie

et al. 2023

Cold Spring Harb Perspect Biol

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One of the greatest threats facing the planet is the continued increase in excess greenhouse gasses, with CO 2 being the primary driver due to its rapid increase in only a century. Excess CO 2 is exacerbating known climate tipping points that will have cascading local and global effects including loss of biodiversity, global warming, and climate migration. However, global reduction of CO 2 emissions is not enough. Carbon dioxide removal (CDR) will also be needed to avoid the catastrophic effects of global warming. Although the drawdown and storage of CO 2 occur naturally via the coupling of the silicate and carbonate cycles, they operate over geological timescales (thousands of years). Here, we suggest that microbes can be used to accelerate this process, perhaps by orders of magnitude, while simultaneously producing potentially valuable by-products. This could provide both a sustainable pathway for global drawdown of CO 2 and an environmentally benign biosynthesis of materials. We discuss several different approaches, all of which involve enhancing the rate of silicate weathering. We use the silicate mineral olivine as a case study because of its favorable weathering properties, global abundance, and growing interest in CDR applications. Extensive research is needed to determine both the upper limit of the rate of silicate dissolution and its potential to economically scale to draw down significant amounts (Mt/Gt) of CO 2 . Other industrial processes have successfully cultivated microbial consortia to provide valuable services at scale (e.g., wastewater treatment, anaerobic digestion, fermentation), and we argue that similar economies of scale could be achieved from this research.

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Rational Design of Evolutionarily Stable Microbial Kill Switches

Cited by 8 publications

References 0 publications

CS-Cells: A CRISPR-Cas12 DNA Device to Generate Chromosome-Shredded Cells for Efficient and Safe Molecular Biomanufacturing

CS-Cells: A CRISPR-Cas12 DNA Device to Generate Chromosome-Shredded Cells for Efficient and Safe Molecular Biomanufacturing

Recent advances in bacterial therapeutics based on sense and response

Microbial Catalysis for CO₂Sequestration: A Geobiological Approach

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Rational Design of Evolutionarily Stable Microbial Kill Switches

Cited by 8 publications

References 0 publications

CS-Cells: A CRISPR-Cas12 DNA Device to Generate Chromosome-Shredded Cells for Efficient and Safe Molecular Biomanufacturing

CS-Cells: A CRISPR-Cas12 DNA Device to Generate Chromosome-Shredded Cells for Efficient and Safe Molecular Biomanufacturing

Recent advances in bacterial therapeutics based on sense and response

Microbial Catalysis for CO2Sequestration: A Geobiological Approach

Contact Info

Product

Resources

About

Microbial Catalysis for CO₂Sequestration: A Geobiological Approach