Chromosome-free bacterial cells are safe and programmable platforms for synthetic biology

Fan, Catherine; Davison, Paul A.; Habgood, Robert; Zeng, Hong; Decker, Christoph M.; Salazar, Manuela Gesell; Lueangwattanapong, Khemmathin; Townley, Helen E.; Yang, Aidong; Thompson, Ian P.; Ye, Hua; Cui, Zhanfeng; Schmidt, Frank; Hunter, C. Neil; Huang, Wei E.

doi:10.1073/pnas.1918859117

Cited by 36 publications

(45 citation statements)

References 74 publications

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“…Moreover, the chromosome-degraded cells maintain ATP levels and can produce plasmid-encoded proteins for several hours, enabling targeted expression of exogenous genes without interference from chromosomal gene expression. Removing all endogenous gene circuitry from E. coli cells but maintaining the transcription machinery provides customizable non-viable containers for a range of applications, including expression of synthetic gene circuits, biosensing, and drug delivery ( Caliando and Voigt, 2015 ; Fan et al., 2020 ; MacDiarmid et al., 2007 ; Rampley et al., 2017 ).…”

Section: Discussionmentioning

confidence: 99%

Transient non-specific DNA binding dominates the target search of bacterial DNA-binding proteins

et al. 2021

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Summary Despite their diverse biochemical characteristics and functions, all DNA-binding proteins share the ability to accurately locate their target sites among the vast excess of non-target DNA. Toward identifying universal mechanisms of the target search, we used single-molecule tracking of 11 diverse DNA-binding proteins in living Escherichia coli . The mobility of these proteins during the target search was dictated by DNA interactions rather than by their molecular weights. By generating cells devoid of all chromosomal DNA, we discovered that the nucleoid is not a physical barrier for protein diffusion but significantly slows the motion of DNA-binding proteins through frequent short-lived DNA interactions. The representative DNA-binding proteins (irrespective of their size, concentration, or function) spend the majority (58%–99%) of their search time bound to DNA and occupy as much as ∼30% of the chromosomal DNA at any time. Chromosome crowding likely has important implications for the function of all DNA-binding proteins.

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Section: Discussionmentioning

confidence: 99%

Transient non-specific DNA binding dominates the target search of bacterial DNA-binding proteins

et al. 2021

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“…coli cells maintain ATP levels and continue to produce plasmid-encoded proteins for several hours, enabling targeted expression of exogenous genes without interference from chromosomal gene expression. Removing all endogenous gene circuitry from E. coli cells but maintaining the transcription machinery provides customizable non-viable containers for a range of applications, including expression of synthetic gene circuits, biosensing, and drug delivery (Caliando and Voigt, 2015;Fan et al, 2020;MacDiarmid et al, 2007;Rampley et al, 2017).…”

Section: Discussionmentioning

confidence: 99%

Transient non-specific DNA binding dominates the target search of bacterial DNA-binding proteins

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et al. 2020

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Despite their diverse biochemical characteristics and functions, all DNA-binding proteins share the ability to accurately locate their target sites among the vast excess of non-target DNA. Towards identifying universal mechanisms of the target search, we used single-molecule tracking of 11 diverse DNA-binding proteins in living Escherichia coli. The mobility of these proteins during the target search was dictated by DNA interactions, rather than by their molecular weights. By generating cells devoid of all chromosomal DNA, we discovered that the nucleoid does not pose a physical barrier for protein diffusion, but significantly slows the motion of DNA-binding proteins through frequent short-lived DNA interactions. The representative DNA-binding proteins (irrespective of their size, concentration, or function) spend the majority (58-99%) of their search time bound to DNA and occupy as much as ~30% of the chromosomal DNA at any time. Chromosome-crowding likely has important implications for the function of all DNA-binding proteins.

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“…By virtue of genetic engineering approaches, several bacterial features, such as targeting, adhesion capability, secretion, and synthesis of specific metabolites, could be altered to treat a broad array of diseases, including tumors, hyperammonemia, inflammation, and chronic wounds. [ 8,53 ] Moreover, engineered bacteria with a synthetic biology design can function as an in situ “therapeutic factory” to produce desired biotherapeutics, including enzymes and protein drugs, specifically at the disease site for enhanced treatment efficacy and decreased side effects.…”

Section: Design Principles For Bacteria‐based Delivery Systemsmentioning

confidence: 99%

“…dislodged the chromosome in several types of bacteria with heterologous I‐CeuI endonuclease and other nucleases and introduced genetic circuits into bacteria with different plasmids. [ 8 ] SimCells without native chromosome for replication still maintained functionalities for up to 10 days, providing adequate time for engineering with desired genetic circuits and eliminated uncontrollable colonization. The SimCells demonstrated the advantages of utilizing genetic engineering tools to transform bacteria as multifunctional delivery platforms for disease treatment.…”

Section: Design Principles For Bacteria‐based Delivery Systemsmentioning

confidence: 99%

“…For example, bacteria can be genetically engineered to display a certain bio‐functionality with the incorporation of genetic circuits, such as synthesis of immune regulatory proteins or expression of disease‐specific therapeutics. [ 8 ] Furthermore, different chemical engineering strategies can be applied to bacteria to form chemistry‐oriented bacteria‐based delivery systems, such as endowing bacteria with external/internal stimuli‐responsive capability. [ 9 ] Despite growing interest in this field and remarkable progress in both preclinical and clinical studies, there are still significant challenges for the clinical translation of leveraging engineered bacteria as delivery systems or therapeutics, including the scaled‐up manufacturing, the viability of bacteria during delivery, precise control of bacteria colonization, dosage determination, and potential biosafety.…”

Section: Introductionmentioning

confidence: 99%

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Chemically and Biologically Engineered Bacteria‐Based Delivery Systems for Emerging Diagnosis and Advanced Therapy

et al. 2021

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Bacteria are one of the main groups of organisms, which dynamically and closely participate in human health and disease development. With the integration of chemical biotechnology, bacteria have been utilized as an emerging delivery system for various biomedical applications. Given the unique features of bacteria such as their intrinsic biocompatibility and motility, bacteria‐based delivery systems have drawn wide interest in the diagnosis and treatment of various diseases, including cancer, infectious diseases, kidney failure, and hyperammonemia. Notably, at the interface of chemical biotechnology and bacteria, many research opportunities have been initiated, opening a promising frontier in biomedical application. Herein, the current synergy of chemical biotechnology and bacteria, the design principles for bacteria‐based delivery systems, the microbial modulation, and the clinical translation are reviewed, with a special focus on the emerging advances in diagnosis and therapy.

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Chromosome-free bacterial cells are safe and programmable platforms for synthetic biology

Cited by 36 publications

References 74 publications

Transient non-specific DNA binding dominates the target search of bacterial DNA-binding proteins

Transient non-specific DNA binding dominates the target search of bacterial DNA-binding proteins

Transient non-specific DNA binding dominates the target search of bacterial DNA-binding proteins

Chemically and Biologically Engineered Bacteria‐Based Delivery Systems for Emerging Diagnosis and Advanced Therapy

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