RESUMEN
Controlled gene expression is fundamental for the study of gene function and our ability to engineer bacteria. However, there is currently no easy-to-use genetics toolbox that enables controlled gene expression in a wide range of diverse species. To facilitate the development of genetics systems in a fast, easy, and standardized manner, we constructed and tested a plasmid assembly toolbox that will enable the identification of well-regulated promoters in many Proteobacteria and potentially beyond. Each plasmid is composed of four categories of genetic parts (i) the origin of replication, (ii) resistance marker, (iii) promoter-regulator and (iv) reporter. The plasmids can be efficiently assembled using ligation-independent cloning, and any gene of interest can be easily inserted in place of the reporter. We tested this toolbox in nine different Proteobacteria and identified regulated promoters with over fifty-fold induction range in eight of these bacteria. We also constructed variant libraries that enabled the identification of promoter-regulators with varied expression levels and increased inducible fold change relative to the original promoter. A selection of over 50 plasmids, which contain all of the toolbox's genetic parts, are available for community use and will enable easy construction and testing of genetics systems in both model and non-model bacteria.
Asunto(s)
Regulación de la Expresión Génica , Plásmidos/genética , Proteobacteria/genética , Bioingeniería , Regiones Promotoras GenéticasRESUMEN
Controlled gene expression is crucial for engineering bacteria for basic and applied research. Inducible systems enable tight regulation of expression, wherein a small-molecule inducer causes the transcription factor to activate or repress transcriptional initiation. The T7 expression system is one of the most widely used inducible systems, particularly for high overexpression of proteins. However, it is well known that the highly active T7 RNA polymerase (RNAP) has several drawbacks, including toxicity to the host and substantial leaky expression in the absence of an inducer. Much work has been done to address these issues; current solutions require special strains or additional plasmids, making the system more complicated and less accessible. Here, we challenge the assumption that the T7 expression system is the best choice for obtaining high protein titers. We hypothesized that expression from strong inducible promoters expressed from high-copy plasmids could compete with expression levels obtained from T7 RNAP but that such promoters would possess improved control of transcription. Employing inducible systems from a toolbox we developed previously, we demonstrate that our plasmids consistently give higher outputs and greater fold changes over basal expression than the T7 system across rich and minimal media. In addition, we show that they outperformed the T7 system when we used an engineered metabolic pathway to produce lycopene. IMPORTANCE Genetic systems for protein overexpression are required tools in microbiological and biochemical research. Ideally, these systems include standardized genetic parts with predictable behavior, enabling the construction of stable expression systems in the host organism. Modularity of a genetic system is advantageous, so that the expression system can be easily moved into a host that best suits the needs of a given experiment. The T7 expression system lacks both predictability and stability and requires special host strains to function. Despite these limitations, it remains one of the most popular systems for protein overproduction. This study directly compared the T7 system to four inducible systems from our broad-host-range plasmid toolbox and demonstrated these alternative expression systems have distinct advantages over the T7. The systems are entirely plasmid-based and not constrained to a specific bacterial host, expanding the options for high-level protein expression across strains.
Asunto(s)
ARN Polimerasas Dirigidas por ADN , Escherichia coli , Escherichia coli/genética , Escherichia coli/metabolismo , ARN Polimerasas Dirigidas por ADN/genética , ARN Polimerasas Dirigidas por ADN/metabolismo , Plásmidos/genética , Regiones Promotoras Genéticas , Regulación de la Expresión GénicaRESUMEN
BACKGROUND & AIMS: Retreatment with glecaprevir/pibrentasvir (G/P) resulted in a rate of sustained virologic response 12 weeks after treatment completion (SVR12) of >90% in HCV genotype 1 (GT1) patients who previously failed a regimen of sofosbuvir plus an NS5A inhibitor (NS5Ai). This study investigated the prevalence and impact of baseline NS3 and NS5A resistance-associated substitutions (RASs) on the efficacy of G/P in prior GT1 sofosbuvir+NS5Ai failures and the persistence of treatment-emergent RASs. METHODS: Longitudinal samples from 177 patients enrolled in a phase IIIb, randomized pragmatic clinical trial were analyzed. Patients without cirrhosis were randomized to 12 or 16 weeks of G/P, and patients with compensated cirrhosis were randomized to G/P and ribavirin for 12 weeks or G/P for 16 weeks. Linkage of RAS was identified using Primer-ID next-generation sequencing at a 15% cut-off. RESULTS: Of 177 patients, 169 (95.5%) were PI-naïve. All 33 GT1b-infected patients achieved SVR12. In GT1a-infected patients, baseline NS5A RASs were prevalent (74.5%, 105/141) but NS3 RASs were uncommon. Baseline NS3 RASs had no impact on G/P efficacy and patients with baseline NS5A RASs showed a numerically but not statistically significantly lower SVR12 rate compared to those without NS5A RASs (89% vs. 97%). SVR12 was achieved in 34 of 35 (97%) patients without NS5A baseline substitution, and 53 of 57 (93%), 35 of 40 (88%), 5 of 8 (63%) with single, double-linked, and triple-linked NS5A substitutions, respectively. Among 13 patients with virologic failure, 4 acquired treatment-emergent NS3 RASs and 10 acquired NS5A RASs. CONCLUSION: Baseline NS5A RASs were highly prevalent. The presence of an increasing number of linked NS5A RASs in GT1a showed a trend in decreasing SVR12 rates, although no specific NS5A RASs or their linkage pattern were associated with lower SVR12 rates. LAY SUMMARY: Direct-acting antivirals have revolutionized the treatment of chronic hepatitis C infection, but treatment failure occurs in some patients. Retreatment of patients who previously failed a regimen consisting of sofosbuvir and an NS5A inhibitor with a regimen of glecaprevir and pibrentasvir (G/P) is >90% effective. Herein, we analyzed samples from these patients and showed that retreatment efficacy with G/P is lower in patients with double- or triple-linked NS5A resistance mutations than in patients with single or no NS5A resistance mutations. CLINICAL TRIAL NUMBER: NCT03092375.
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Bencimidazoles/farmacología , Resistencia a Medicamentos/inmunología , Pirrolidinas/farmacología , Quinoxalinas/farmacología , ARN Polimerasa Dependiente del ARN/antagonistas & inhibidores , Sofosbuvir/metabolismo , Sulfonamidas/farmacología , Proteínas no Estructurales Virales/antagonistas & inhibidores , Adulto , Antivirales/administración & dosificación , Antivirales/metabolismo , Bencimidazoles/uso terapéutico , Combinación de Medicamentos , Femenino , Hepatitis C/tratamiento farmacológico , Hepatitis C/epidemiología , Hepatitis C/fisiopatología , Humanos , Estudios Longitudinales , Masculino , Persona de Mediana Edad , Pirrolidinas/uso terapéutico , Quinoxalinas/administración & dosificación , Quinoxalinas/uso terapéutico , ARN Polimerasa Dependiente del ARN/farmacología , Sofosbuvir/administración & dosificación , Sulfonamidas/uso terapéutico , Estados Unidos/epidemiología , Proteínas no Estructurales Virales/farmacologíaRESUMEN
Screening assays are used to test if one or more microbes suppress a pathogen of interest. In the presence of more than one microbe, the screening method must be able to accurately distinguish viable pathogen cells from non-viable and non-target microbes in a sample. Current screening methods are time-consuming and require special reagents to detect viability in mixed microbial communities. Screening assays performed using soil or other complex matrices present additional challenges for screening. Here, we develop an experimental workflow based on the most probable number (MPN) assay for testing the ability of synthetic microbial communities to suppress a soil-borne pathogen. Our approach, fluorMPN, uses a fluorescently labeled pathogen and microplate format to enable high-throughput comparative screening. In parallel, we developed a command-line tool, MicroMPN, which significantly reduces the complexity of calculating MPN values from microplates. We compared the performance of the fluorMPN assay with spotting on agar and found that both methods produced strongly correlated counts of equal precision. The suppressive effect of synthetic communities on the pathogen was equally recoverable by both methods. The application of this workflow for discriminating which communities lead to pathogen reduction helps narrow down candidates for additional characterization. Together, the resources offered here are meant to facilitate and simplify the application of MPN-based assays for comparative screening projects. IMPORTANCE: We created a unified set of software and laboratory protocols for screening microbe libraries to assess the suppression of a pathogen in a mixed microbial community. Existing methods of fluorescent labeling were combined with the most probable number (MPN) assay in a microplate format to enumerate the reduction of a pathogenic soil microbe from complex soil matrices. This work provides a fluorescent expression vector available from Addgene, step-by-step laboratory protocols hosted by protocols.io, and MicroMPN, a command-line software for processing plate reader outputs. MicroMPN simplifies MPN estimation from 96- and 384-well microplates. The microplate screening assay is amenable to robotic automation with standard liquid handling robots, further reducing the hands-on processing time. This tool was designed to evaluate synthetic microbial communities for use as microbial inoculates or probiotics. The fluorMPN method is also useful for screening chemical and antimicrobial libraries for pathogen suppression in complex bacterial communities like soil.
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Bacterias , Ensayos Analíticos de Alto Rendimiento , Programas Informáticos , Microbiología del Suelo , Ensayos Analíticos de Alto Rendimiento/métodos , Bacterias/aislamiento & purificación , Bacterias/genética , Bacterias/clasificación , MicrobiotaRESUMEN
Actinobacteria, the bacterial phylum most renowned for natural product discovery, has been established as a valuable source for drug discovery and biotechnology but is underrepresented within accessible genome and strain collections. Herein, we introduce the Natural Products Discovery Center (NPDC), featuring 122,449 strains assembled over eight decades, the genomes of the first 8490 NPDC strains (7142 Actinobacteria), and the online NPDC Portal making both strains and genomes publicly available. A comparative survey of RefSeq and NPDC Actinobacteria highlights the taxonomic and biosynthetic diversity within the NPDC collection, including three new genera, hundreds of new species, and ~7000 new gene cluster families. Selected examples demonstrate how the NPDC Portal's strain metadata, genomes, and biosynthetic gene clusters can be leveraged using genome mining approaches. Our findings underscore the ongoing significance of Actinobacteria in natural product discovery, and the NPDC serves as an unparalleled resource for both Actinobacteria strains and genomes.
RESUMEN
HIV drug resistance is a major threat to achieving long-term viral suppression in HIV-positive individuals. Drug resistant HIV variants, including minority variants, can compromise response to antiretroviral therapy. Many studies have investigated the clinical relevance of drug resistant minority variants, but the level at which minority variants become clinically relevant remains unclear. A combination of Primer-ID and deep sequencing is a promising approach that may quantify minority variants more accurately compared to standard deep sequencing. However, most studies that used the Primer-ID method have analyzed clinical samples directly. Thus, its sensitivity and quantitative accuracy have not been adequately validated using known controls. Here, we constructed defined proportions of artificial RNA and virus quasispecies and measured their relative proportions using the Primer-ID based, quantitative single-variant sequencing (qSVS) assay. Our results showed that minority variants present at 1% of quasispecies were detected reproducibly with minimal variations between technical replicates. In addition, the measured frequencies were comparable to the expected frequencies. These data validate the accuracy and reproducibility of the qSVS assay in quantifying authentic HIV minority variants, and support the use of this approach to examine the impacts of minority HIV variants on virologic response and clinical outcome.
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VIH-1/genética , Límite de Detección , Polimorfismo de Nucleótido Simple , Plásmidos/genéticaRESUMEN
Our objective was to identify drug interactions between ledipasvir (LDV) and sofosbuvir (SOF) against a genotype 1b replicon to determine optimal exposures for each agent that will maximize antiviral activity against susceptible and drug-resistant subpopulations. LDV and SOF were evaluated using a fully factorial experimental design in the BelloCell system. Replicon levels and drug-resistant variants were quantified at various times post-therapy for 14 days and a high-dimensional mathematical model was fit to the data. Mutations associated with SOF resistance were not detected; but LDV-resistant mutants were selected and mutant subpopulations increased as exposure intensity increased. Combination therapy was additive for the total replicon population and the LDV-resistant population, but a threshold concentration of 100 ng/ml of SOF must be attained to suppress LDV-resistant subpopulations. These novel findings hold important implications for not only improving therapeutic outcomes, but also maximizing the clinical utility of LDV and SOF combination regimens.