Identification and application of a panel of constitutive promoters for gene overexpression in Staphylococcus aureus
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ABSTRACT:
PROVIDER: PRJNA783934 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR17048858_1.fastq.gz | Fastqsanger.gz | |||
| SRR17048858_2.fastq.gz | Fastqsanger.gz | |||
| SRR17048859_1.fastq.gz | Fastqsanger.gz | |||
| SRR17048859_2.fastq.gz | Fastqsanger.gz | |||
| SRR17048860_1.fastq.gz | Fastqsanger.gz |
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Project description:Staphylococcus aureus is a leading pathogen that is currently the most common cause of infection in hospitalized patients. An in-depth genetic analysis of S. aureus virulence genes contributing to pathogenesis is needed to develop novel antimicrobial therapies. However, tools for genetic manipulation in S. aureus are limited, particularly those for gene expression. Here, 38 highly expressed genes were identified in S. aureus USA300_FPR3757 via RNA-seq. Promoter regions from 30 of these genes were successfully cloned, of which 20 promoters exhibited a wide range of activity. By utilizing these active promoters, 20 S. aureus-E. coli shuttle vectors were constructed and evaluated by expressing an egfp reporter gene. Expression of egfp gene under the control of different promoters was confirmed and quantified by Western-blot and qPCR, which suggested that the activity of these promoters varied from 18% to 650% of the activity of PsarA, a widely used promoter for gene expression currently. In addition, our constructed vectors were verified to be of high compatibility on gene expression in different S. aureus strains. Furthermore, these vectors were evaluated and used in overexpressing two endogenous proteins in S. aureus, namely, catalase and the transcriptional repressor of purine biosynthesis (purR). Meanwhile, the physiological functions and phenotypes of overexpressed purR and catalase in S. aureus were validated. Altogether, this evidence indicates that our constructed vectors provide a wide range of promoter activity on gene expression in S. aureus. This developed system will provide a powerful tool for the direct analysis of target gene function in staphylococcal cells.
2021-11-30 | GSE189644 | GEO
Project description:Staphylococcus aureus is a leading pathogen that is currently the most common cause of infection in hospitalized patients. An in-depth genetic analysis of S. aureus virulence genes contributing to pathogenesis is needed to develop novel antimicrobial therapies. However, tools for genetic manipulation in S. aureus are limited, particularly those for gene expression. Here, 38 highly expressed genes were identified in S. aureus USA300_FPR3757 via RNA-seq. Promoter regions from 30 of these genes were successfully cloned, of which 20 promoters exhibited a wide range of activity. By utilizing these active promoters, 20 S. aureus-Escherichia coli shuttle vectors were constructed and evaluated by expressing an egfp reporter gene. Expression of the egfp gene under the control of different promoters was confirmed and quantified by Western blotting and qPCR, which suggested that the activity of these promoters varied from 18 to 650% of the activity of P sarA , a widely used promoter for gene expression. In addition, our constructed vectors were verified to be highly compatible with gene expression in different S. aureus strains. Furthermore, these vectors were evaluated and used to overexpress two endogenous proteins in S. aureus, namely, catalase and the transcriptional repressor of purine biosynthesis (PurR). Meanwhile, the physiological functions and phenotypes of overexpressed PurR and catalase in S. aureus were validated. Altogether, this evidence indicates that our constructed vectors provide a wide range of promoter activity on gene expression in S. aureus. This set of vectors carrying different constitutive promoters developed here will provide a powerful tool for the direct analysis of target gene function in staphylococcal cells.
| S-EPMC8918988 | biostudies-literature
Project description:A toolset of constitutive promoters for engineering Rhodosporidium toruloides
| PRJNA527258 | ENA
Project description:Background: A key prerequisite for pathway engineering is the development of genetic tools and resources. Rhodosporidium toruloides is emerging as a promising host for the production of bioproducts from lignocellulosic biomass. However, there is a lack of characterized promoters to drive expression of heterologous genes for strain engineering in R. toruloides. Results: The resulting data describes a set of native R. toruloides promoters, characterized over time in four media commonly used for this yeast. The promoter sequences were sorted using transcriptional analysis and several of them were found to drive expression bidirectionally. We measured promoter expression by flow cytometry using a dual fluorescent reporter system. From these analyses, we found a total of 20 constitutive promoters (12 monodirectional and 8 bidirectional), that are strong, stable, and can reliably be used for genetic manipulation of this emergent host. Conclusions: We are presenting a list of robust constitutive promoters that are native to the emergent bioconversion host R. toruloides which helps to fulfill the lack of existing tools for this yeast and that can be applied in future metabolic engineering studies.
2020-03-02 | GSE128360 | GEO
Project description:Rhodosporidium toruloides is an oleaginous yeast under development with promising industrial applications. Since promoters of different strengths have been demonstrated as an efficient strategy to fine-tune gene expression in synthetic biology, a set of constitutive promoters with strengths varying over 2 orders of magnitude were identified in R. toruloides through transcriptome analysis under different growth conditions. Promoter candidates were first cloned and characterized using an enhanced green fluorescent protein (EGFP) as a reporter under eight conditions, and 31 promoters were identified with strength varied from 0.1 to 19.0 folds of the commonly used strong promoter of the glyceraldehyde-3-phosphate dehydrogenase gene (PGPD1). The resultant promoters were then used to optimize the linoleic acid biosynthetic pathway in R. toruloides in different media, including the use of lignocellulosic hydrolysate as the fermentation substrate, and improved the production of linoleic acid by up to 214.2% in minimal medium, with the highest production of 350.3 mg/L in Yeast Peptone Dextrose medium. This work has enriched the promoter library of R. toruloides, and helped develop R. toruloides as a platform organism for applications in biomanufacturing and synthetic biology.
| S-EPMC9908808 | biostudies-literature
Project description:Proteomes of Mycoplasma gallisepticum strains with overexpression and knockdown of WhiA transcription factor. The overexpression was introduced on a transposon vector carrying M. gallisepticum whiA gene with strong constitutive promoter and strong SD sequence. The knockdown was made by CRISPRi. dCas9 protein and sgRNA against whiA gene were introduced on a transposon vector.
2021-06-25 | PXD026928 | Pride
Project description:ProSys(1-178) constitutive expression
| PRJNA795968 | ENA
Project description:Cowpea (Vigna unguiculata) is an important protein source in Sub-Saharan Africa. Optimizing resilience and productivity through genetic engineering in cowpea has been slow due in part to a lack of defined species-specific regulatory elements and difficulty testing gene function within the native system. In many plant species, Agrobacterium-mediated transient gene expression is widely used to validate constructs before investing in transgenic lines, but its implementation in legumes has been challenging. In this study, we optimized an in planta agroinfiltration assay in trifoliate cowpea leaves using a betalain reporter. To demonstrate the “intact plant” aspect of this system, we used this assay to characterize drought-inducible promoters by challenging cowpea plants with drought stress. Subsequently, to identify and broaden the pool of native promoters known in cowpea, we developed a user-friendly web application, CowPEAsy, allowing users to interrogate gene expression from our canopy-level, developmental-series RNA-Seq dataset. Finally, using CowPEAsy, we identified six promoters that showed constitutive expression across all conditions and verified these promoters with our transient system. This work provides an in vivo platform for preliminary validation of regulatory elements in cowpea and other legumes and enhances current genetic resources by identifying a suite of physiologically relevant promoters of varying strengths.
2025-02-13 | GSE280644 | GEO
Project description:Attenuated Listeria monocytogenes could be a potential vaccine vector for the immunotherapy of tumors or pathogens. However, the lack of reliable promoters has limited its ability to express foreign antigens. In the present study, 21 promoters were identified from Listeria monocytogenes through RNA-seq analysis under two pH conditions of pH 7.4 and pH 5.5. Based on the constructed fluorescence report system, 7 constitutive promoters exhibited higher strength than Phelp (1.8-fold to 5.4-fold), a previously reported strong promoter. Furthermore, the selected 5 constitutive promoters exhibited higher UreB production activity than Phelp (1.1-fold to 8.3-fold). Notably, a well-characterized constitutive promoter P18 was found with the highest activity of fluorescence intensity and UreB production. In summary, the study provides a panel of strong constitutive promoters for Listeria monocytogenes and offers a theoretical basis for mining constitutive promoters in other organisms. KEY POINTS: • Twenty-one promoters were identified from L. monocytogenes through RNA-seq. • Fluorescent tracer of L. monocytogenes (P18) was performed in vitro and in vivo. • A well-characterized constitutive promoter P18 could improve the expression level of a foreign antigen UreB in L. monocytogenes.
| S-EPMC8175932 | biostudies-literature
Project description:Genome-wide, little is understood about how proteins organize at inducible promoters before and after in-duction, and to what extent inducible and constitutive architectures depend on cofactors. We report that se-quence-specific transcription factors and their tethered cofactors (e.g., SAGA, Mediator, TUP, NuA4, SWI/SNF, RPD3-L) are already bound to promoters prior to induction (“poised”), rather than recruited upon induction, whereas induction recruits the pre-initiation complex (PIC). Through depletion and/or deletion ex-periments we show that SAGA does not function at constitutive promoters, although a SAGA-independent Gcn5 does acetylate +1 nucleosomes there. At poised promoters, SAGA catalyzes +1 nucleosome acetylation but not PIC assembly. At induced promoters, SAGA catalyzes acetylation, deubiquitylation, and PIC assembly. Surprisingly, SAGA mediates induction by creating a PIC that allows TFIID to stably associate, rather than creating a TFIID-independent PIC, as is generally thought. These findings suggest that inducible systems, where present, evolved on top of constitutive systems.
2022-10-27 | GSE212654 | GEO