CRISPR‐based curing and analysis of metabolic burden of cryptic plasmids in <i>Escherichia coli</i> Nissle 1917

Zainuddin, Halimatun S.; Bai, Yanhong; Mansell, Thomas J.

doi:10.1002/elsc.201900003

Cited by 20 publications

(17 citation statements)

References 33 publications

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“…30 E. coli Nissle harbors a native cryptic plasmid pMUT1, 38 which has been shown to be highly stable. 39 Therefore, E. coli Nissle was cured from its native pMUT1, to leverage pMUT1 for the strain engineering. The kanR kanamycin resistance gene was added to the plasmid for cloning purposes along the aadK streptomycin resistance gene that was included to enable the strain to resist streptomycin pre-treatment used in the in vivo experiments.…”

Section: ■ Resultsmentioning

confidence: 99%

Escherichia coli Promoters with Consistent Expression throughout the Murine Gut

et al. 2021

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Advanced microbial therapeutics have great potential as a novel modality to diagnose and treat a wide range of diseases. Yet, to realize this potential, robust parts for regulating gene expression and consequent therapeutic activity in situ are needed. In this study, we characterized the expression level of more than 8000 variants of the Escherichia coli sigma factor 70 (σ70) promoter in a range of different environmental conditions and growth states using fluorescence-activated cell sorting and deep sequencing. Sampled conditions include aerobic and anaerobic culture in the laboratory as well as growth in several locations of the murine gastrointestinal tract. We found that σ70 promoters in E. coli generally maintain consistent expression levels across the murine gut (R 2: 0.55–0.85, p value < 1 × 10–5), suggesting a limited environmental influence but a higher variability between in vitro and in vivo expression levels, highlighting the challenges of translating in vitro promoter activity to in vivo applications. Based on these data, we design the Schantzetta library, composed of eight promoters spanning a wide expression range and displaying a high degree of robustness in both laboratory and in vivo conditions (R 2 = 0.98, p = 0.000827). This study provides a systematic assessment of the σ70 promoter activity in E. coli as it transits the murine gut leading to the definition of robust expression cassettes that could be a valuable tool for reliable engineering and development of advanced microbial therapeutics.

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Section: ■ Resultsmentioning

confidence: 99%

Escherichia coli Promoters with Consistent Expression throughout the Murine Gut

et al. 2021

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“…The pMUT plasmids have no known function, but are stable within EcN during passage through the gut, and can thus act as vectors for recombinant DNA. While previous studies have used the pMUT plasmids, 3 and shown their high plasmid retention in vitro , 24 their in vivo efficacy had never been systematically characterized. Through our attempts to cure the native pMUT plasmids, our results suggest that pMUT2 stability in EcN is improved by the RelB/RelE toxin-antitoxin system, as we could not cure pMUT2 without expressing the antitoxin gene from our pCryptDel4.8 plasmid.…”

Section: Discussionmentioning

confidence: 99%

Plasmid Vectors for in Vivo Selection-Free Use with the Probiotic E. coli Nissle 1917

et al. 2020

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Escherichia coli Nissle 1917 (EcN) is a probiotic bacterium, commonly employed to treat certain gastrointestinal disorders. It is fast emerging as an important target for the development of therapeutic engineered bacteria, benefiting from the wealth of knowledge of E. coli biology and ease of manipulation. Bacterial synthetic biology projects commonly utilize engineered plasmid vectors, which are simple to engineer and can reliably achieve high levels of protein expression. However, plasmids typically require antibiotics for maintenance, and the administration of an antibiotic is often incompatible with in vivo experimentation or treatment. EcN natively contains plasmids pMUT1 and pMUT2, which have no known function but are stable within the bacteria. Here, we describe the development of the pMUT plasmids into a robust platform for engineering EcN for in vivo experimentation, alongside a CRISPR-Cas9 system to remove the native plasmids. We systematically engineered both pMUT plasmids to contain selection markers, fluorescent markers, temperature sensitive expression, and curli secretion systems to export a customizable functional material into the extracellular space. We then demonstrate that the engineered plasmids were maintained in bacteria as the engineered bacteria pass through the mouse GI tract without selection, and that the secretion system remains functional, exporting functionalized curli proteins into the gut. Our plasmid system presents a platform for the rapid development of therapeutic EcN bacteria.

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“…EcN was detected in the fecal matter at 45 days after oral inoculation. EcN contains two cryptic plasmids MUT1 and MUT2, and these plasmids were cured using CRISP-Cas9-assisted double-strand breaks [47]. EcN strain cured of these plasmids had similar growth under Luria broth conditions despite differences in the DNA content.…”

Section: Genetic Modifications Of Probiotic E Colimentioning

confidence: 99%

Potential of Escherichia coli Probiotics for Improved Health and Disease Management

Archana¹

2023

Escherichia Coli - Old and New Insights

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Although natural gut microbiota containsEscherichia coli as a commensal, this bacterium, along with other members of the Enterobacteriaceae family, are usually known for their pathogenic potential. Interestingly, E. coli colonizes first and remains all through life, and in fact, some strains possess beneficial properties such as antibacterial colicin secretion. Among the beneficial strains, E. coli Nissle, isolated in 1917, has been the most extensively explored strain. Adaptability to survive under diverse conditions coupled with facile genetic manipulations enabled the design of E. coli strains with properties to deliver antioxidant, anti-inflammatory, and antitumor molecules. Moreover, genetically modified E. coli strains secreting enzymes for converting sucrose and fructose into insulin and mannitol, respectively, were very effective in preventing the onset of metabolic disease by acting as synbiotics. Thus, E. coli is emerging as a very potent probiotic platform for developing strains with the potential of controlling many metabolic and multifactorial diseases, including cancer.

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CRISPR‐based curing and analysis of metabolic burden of cryptic plasmids in Escherichia coli Nissle 1917

Cited by 20 publications

References 33 publications

Escherichia coli Promoters with Consistent Expression throughout the Murine Gut

Escherichia coli Promoters with Consistent Expression throughout the Murine Gut

Plasmid Vectors for in Vivo Selection-Free Use with the Probiotic E. coli Nissle 1917

Potential of Escherichia coli Probiotics for Improved Health and Disease Management

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