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

Abstract

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.

Genetically stable CRISPR-based kill switches for engineered microbes

“…In contrast, omission of any antibiotic treatment resulted in undetectable levels of either EcN strain (Supplementary Fig. 5c ), indicating a failure to colonize, while treatment with carbenicillin 45 48 hours before EcN gavage resulted in low and variable titers of both EcN strains (Supplementary Fig. 5d ).…”

“…24 or 72 hours after streptomycin administration, mice were orally gavaged with 10 8 CFU EcN in 100 μL phosphate buffered saline (PBS). To test alternative microbiome ablation strategies, mice were instead orally gavaged with 6 mg carbenicillin disodium salt (C1389, Sigma-Aldrich) in 100 μL H 2 O 48 hours prior to EcN administration 45 , or left untreated prior to EcN administration.…”

Microcin MccI47 selectively inhibits enteric bacteria and reduces carbapenem-resistant Klebsiella pneumoniae colonization in vivo when administered via an engineered live biotherapeutic

“…While recent studies relied on chromosomal integration [33,34], we sought to maximize MccI47 output using a multicopy plasmid [21,35,36] and developed an expression system using the native, self-retaining EcN plasmid pMut2 as backbone for constitutive heterologous protein expression [36].…”

“…EcN is used for the construction of genetically engineered bacteriotherapeutic agents which are being tested in multiple ongoing clinical trials[32]. While recent studies relied on chromosomal integration[33, 34], we sought to maximize MccI47 output using a multicopy plasmid[21, 35, 36] and developed an expression system using the native, self-retaining EcN plasmid pMut2 as backbone for constitutive heterologous protein expression[36]. We first cured EcN from pMut2 by utilizing MccI47 ( mciA ) in a counterselection approach ( Figure 2A ).…”

Potential of Escherichia coli Probiotics for Improved Health and Disease Management

“…Effects of colonization and survival of the plasmid-cured strain with decreased DNA content as compared to the wild-type strain need to be investigated to determine the impact of metabolic load. Alternatively, both the cryptic plasmids of EcN have been engineered for stable maintenance and expression of recombinant proteins [53].…”