Development of a regulatable expression system for the functional study of Vibrio vulnificus essential genes

2017 ◽  
Vol 110 (4) ◽  
pp. 607-614 ◽  
Author(s):  
Yan-Jiao Zhang ◽  
Guozhong Chen ◽  
Huiyuan Lin ◽  
Pan Wang ◽  
Baozhi Kuang ◽  
...  
Keyword(s):  
Vibrio Vulnificus ◽  
Expression System ◽  
Essential Genes ◽  
Functional Study

scholarly journals Organelle Visualization With Multicolored Fluorescent Markers in Bamboo

2021 ◽  
Vol 12 ◽  
Author(s):  
Mengdi Zhang ◽  
Shuai Hu ◽  
Fang Yi ◽  
Yanli Gao ◽  
Dongmei Zhu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Subcellular Localization ◽  
Molecular Mechanisms ◽  
Expression System ◽  
Transient Gene Expression ◽  
Moso Bamboo ◽  
Functional Study ◽  
Efficient System ◽  
Gene Expression System ◽  
Fluorescent Markers

Bamboo is an important model plant to study the molecular mechanisms of rapid shoot growth and flowering once in a lifetime. However, bamboo research about protein functional characterization is largely lagged behind, mainly due to the lack of gene transformation platforms. In this study, a protoplast transient gene expression system in moso bamboo has been first established. Using this reliable and efficient system, we have generated a set of multicolored fluorescent markers based on the targeting sequences from endogenous proteins, which have been validated by their comparative localization with Arabidopsis organelle markers, in a combination with pharmaceutical treatments. Moreover, we further demonstrated the power of this multicolor marker set for rapid, combinatorial analysis of the subcellular localization of uncharacterized proteins, which may play potential functions in moso bamboo flowering and fast growth of shoots. Finally, this protoplast transient gene expression system has been elucidated for functional analysis in protein–protein interaction by fluorescence resonance energy transfer (FRET) and co-immunoprecipitation analysis. Taken together, in combination with the set of moso bamboo organelle markers, the protoplast transient gene expression system could be used for subcellular localization and functional study of unknown proteins in bamboo and will definitely promote rapid progress in diverse areas of research in bamboo plants.

scholarly journals Microcephaly family protein MCPH1 stabilizes RAD51 filaments

2020 ◽  
Vol 48 (16) ◽  
pp. 9135-9146
Author(s):  
Hao-Yen Chang ◽  
Chia-Yi Lee ◽  
Chih-Hao Lu ◽  
Wei Lee ◽  
Han-Lin Yang ◽  
...  
Keyword(s):  
Single Molecule ◽  
Expression System ◽  
Fold Increase ◽  
Functional Study ◽  
Dna Double Strand Breaks ◽  
Dsb Repair ◽  
Mammalian Expression ◽  
Contact Points ◽  
Family Protein ◽  
The Stability

Abstract Microcephalin 1 (MCPH1) was identified from genetic mutations in patients with primary autosomal recessive microcephaly. In response to DNA double-strand breaks (DSBs), MCPH1 forms damage-induced foci and recruits BRCA2–RAD51 complex, a key component of the DSB repair machinery for homologous recombination (HR), to damage sites. Accordingly, the efficiency of HR is significantly attenuated upon depletion of MCPH1. The biochemical characteristics of MCPH1 and its functional interaction with the HR machinery had remained unclear due to lack of highly purified MCPH1 recombinant protein for functional study. Here, we established a mammalian expression system to express and purify MCPH1 protein. We show that MCPH1 is a bona fide DNA-binding protein and provide direct biochemical analysis of this MCPH family protein. Furthermore, we reveal that MCPH1 directly interacts with RAD51 at multiple contact points, providing evidence for how MCPH1 physically engages with the HR machinery. Importantly, we demonstrate that MCPH1 enhances the stability of RAD51 on single-strand DNA, a prerequisite step for RAD51-mediated recombination. Single-molecule tethered particle motion analysis showed a ∼2-fold increase in the lifetime of RAD51–ssDNA filaments in the presence of MCPH1. Thus, our study demonstrates direct crosstalk between microcephaly protein MCPH1 and the recombination component RAD51 for DSB repair.

scholarly journals Multiplex CRISPRi System Enables the Study of Stage-Specific Biofilm Genetic Requirements in Enterococcus faecalis

mBio ◽  
2020 ◽  
Vol 11 (5) ◽  
Author(s):  
Irina Afonina ◽  
June Ong ◽  
Jerome Chua ◽  
Timothy Lu ◽  
Kimberly A. Kline
Keyword(s):  
Enterococcus Faecalis ◽  
Biofilm Formation ◽  
Pathogenic Bacteria ◽  
Expression System ◽  
Multidrug Resistant ◽  
Essential Genes ◽  
Host Bacterium ◽  
Content Type ◽  
Wide Range ◽  
Life Threatening

ABSTRACT Enterococcus faecalis is an opportunistic pathogen, which can cause multidrug-resistant life-threatening infections. Gaining a complete understanding of enterococcal pathogenesis is a crucial step in identifying a strategy to effectively treat enterococcal infections. However, bacterial pathogenesis is a complex process often involving a combination of genes and multilevel regulation. Compared to established knockout methodologies, CRISPR interference (CRISPRi) approaches enable the rapid and efficient silencing of genes to interrogate gene products and pathways involved in pathogenesis. As opposed to traditional gene inactivation approaches, CRISPRi can also be quickly repurposed for multiplexing or used to study essential genes. Here, we have developed a novel dual-vector nisin-inducible CRISPRi system in E. faecalis that can efficiently silence via both nontemplate and template strand targeting. Since the nisin-controlled gene expression system is functional in various Gram-positive bacteria, the developed CRISPRi tool can be extended to other genera. This system can be applied to study essential genes, genes involved in antimicrobial resistance, and genes involved in biofilm formation and persistence. The system is robust and can be scaled up for high-throughput screens or combinatorial targeting. This tool substantially enhances our ability to study enterococcal biology and pathogenesis, host-bacterium interactions, and interspecies communication. IMPORTANCE Enterococcus faecalis causes multidrug-resistant life-threatening infections and is often coisolated with other pathogenic bacteria from polymicrobial biofilm-associated infections. Genetic tools to dissect complex interactions in mixed microbial communities are largely limited to transposon mutagenesis and traditional time- and labor-intensive allelic-exchange methods. Built upon streptococcal dCas9, we developed an easily modifiable, inducible CRISPRi system for E. faecalis that can efficiently silence single and multiple genes. This system can silence genes involved in biofilm formation and antibiotic resistance and can be used to interrogate gene essentiality. Uniquely, this tool is optimized to study genes important for biofilm initiation, maturation, and maintenance and can be used to perturb preformed biofilms. This system will be valuable to rapidly and efficiently investigate a wide range of aspects of complex enterococcal biology.

scholarly journals Identification of in vivo Essential Genes of Vibrio vulnificus for Establishment of Wound Infection by Signature-Tagged Mutagenesis

2019 ◽  
Vol 10 ◽  
Author(s):  
Kohei Yamazaki ◽  
Takashige Kashimoto ◽  
Mio Morita ◽  
Takehiro Kado ◽  
Kaho Matsuda ◽  
...  
Keyword(s):  
Wound Infection ◽  
Vibrio Vulnificus ◽  
Essential Genes

scholarly journals Clinical and Functional Characterization of a Novel URAT1 Dysfunctional Variant in a Pediatric Patient with Renal Hypouricemia

2019 ◽  
Vol 9 (17) ◽  
pp. 3479
Author(s):  
Blanka Stiburkova ◽  
Jana Bohata ◽  
Iveta Minarikova ◽  
Andrea Mancikova ◽  
Jiri Vavra ◽  
...  
Keyword(s):  
Pediatric Patient ◽  
Primary Care Physicians ◽  
Expression System ◽  
Functional Characterization ◽  
Functional Study ◽  
Sequencing Analysis ◽  
Coding Region ◽  
Cytoplasmic Staining ◽  
Renal Hypouricemia

Renal hypouricemia (RHUC) is caused by an inherited defect in the main (reabsorptive) renal urate transporters, URAT1 and GLUT9. RHUC is characterized by decreased concentrations of serum uric acid and an increase in its excretion fraction. Patients suffer from hypouricemia, hyperuricosuria, urolithiasis, and even acute kidney injury. We report the clinical, biochemical, and genetic findings of a pediatric patient with hypouricemia. Sequencing analysis of the coding region of SLC22A12 and SLC2A9 and a functional study of a novel RHUC1 variant in the Xenopus expression system were performed. The proband showed persistent hypouricemia (67–70 µmol/L; ref. range 120–360 µmol/L) and hyperuricosuria (24–34%; ref. range 7.3 ± 1.3%). The sequencing analysis identified common non-synonymous allelic variants c.73G > A, c.844G > A, c.1049C > T in the SLC2A9 gene and rare variants c.973C > T, c.1300C > T in the SLC22A12 gene. Functional characterization of the novel RHUC associated c.973C > T (p. R325W) variant showed significantly decreased urate uptake, an irregular URAT1 signal on the plasma membrane, and reduced cytoplasmic staining. RHUC is an underdiagnosed disorder and unexplained hypouricemia warrants detailed metabolic and genetic investigations. A greater awareness of URAT1 and GLUT9 deficiency by primary care physicians, nephrologists, and urologists is crucial for identifying the disorder.

scholarly journals Use of a Tetracycline-Inducible System for Conditional Expression in Mycobacterium tuberculosis and Mycobacterium smegmatis

2005 ◽  
Vol 71 (6) ◽  
pp. 3077-3084 ◽  
Author(s):  
Paul Carroll ◽  
D. G. Niranjala Muttucumaru ◽  
Tanya Parish
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Reporter Gene ◽  
Mycobacterium Smegmatis ◽  
Expression System ◽  
Inducible Promoter ◽  
Inducible Expression ◽  
Essential Genes ◽  
In Vivo Studies ◽  
Conditional Expression

ABSTRACT A number of essential genes have been identified in mycobacteria, but methods to study these genes have not been developed, leaving us unable to determine the function or biology of the genes. We investigated the use of a tetracycline-inducible expression system in Mycobacterium tuberculosis and Mycobacterium smegmatis. Using a reporter gene which encodes an unstable variant of GFP, we showed that tetracycline-inducible expression occurred in M. smegmatis and that expression levels were titratable to some extent by varying the concentration of tetracycline. The removal of tetracycline led to cessation of GFP expression, and we showed that this was a controllable on/off switch for fluorescence upon addition and removal of the antibiotic inducer. The system also functioned in M. tuberculosis, giving inducible expression of the reporter gene. We used homologous recombination to construct a strain of M. tuberculosis that expressed the only copy of the tryptophan biosynthetic enzyme, TrpD, from the tetracycline-inducible promoter. This strain was conditionally auxotrophic, showing auxotrophy only in the absence of tetracycline, confirming that trpD was tightly controlled by the foreign promoter. This is the first demonstration of the use of an inducible promoter to generate a conditional auxotroph of M. tuberculosis. The ability to tightly regulate genes now gives us the possibility to define the functions of essential genes by switching them off under defined conditions and paves the way for in vivo studies.

scholarly journals A genome-scale yeast library with inducible expression of individual genes

2020 ◽  
Author(s):  
Yuko Arita ◽  
Griffin Kim ◽  
Zhijian Li ◽  
Helena Friesen ◽  
Gina Turco ◽  
...  
Keyword(s):  
Single Gene ◽  
Expression System ◽  
Essential Genes ◽  
Dynamic Changes ◽  
A Genome ◽  
Powerful Approach ◽  
Molecular Barcode ◽  
Yeast Genes ◽  
Genome Scale ◽  
Lower Expression

AbstractThe ability to switch a gene from off to on and monitor dynamic changes provides a powerful approach for probing gene function and elucidating causal regulatory relationships, including instances of feedback control. Here, we developed and characterized YETI (Yeast Estradiol strains with Titratable Induction), a collection in which 5,687 yeast genes are engineered for transcriptional inducibility with single-gene precision at their native loci and without plasmids. Each strain contains Synthetic Genetic Array (SGA) screening markers and a unique molecular barcode, enabling high-throughput yeast genetics. We characterized YETI using quantitative growth phenotyping and pooled BAR-seq screens, and we used a YETI allele to characterize the regulon of ROF1, showing that it is a transcriptional repressor. We observed that strains with inducible essential genes that have low native expression can often grow without inducer. Analysis of data from other eukaryotic and prokaryotic systems shows that low native expression is a critical variable that can bias promoter-perturbing screens, including CRISPRi. We engineered a second expression system, Z3EB42, that gives lower expression than Z3EV, a feature enabling both conditional activation and repression of lowly expressed essential genes that grow without inducer in the YETI library.

scholarly journals Expression, Purification and Sublocalization of SARS-CoV Nucleocapsid Protein in Insect Cells

2004 ◽  
Vol 36 (11) ◽  
pp. 754-758 ◽  
Author(s):  
Ai-Xia Ren ◽  
You-Hua Xie ◽  
Yu-Ying Kong ◽  
Guan-Zhen Yang ◽  
Yao-Zhou Zhang ◽  
...  
Keyword(s):  
Nucleocapsid Protein ◽  
Trichoplusia Ni ◽  
Insect Cells ◽  
Expression System ◽  
Recombinant Baculovirus ◽  
Baculovirus Expression System ◽  
Functional Study ◽  
Expression And Purification ◽  
N Protein ◽  
Baculovirus Expression

Abstract The causative agent of severe acute respiratory syndrome (SARS) is a previously unidentified coronavirus, SARS-CoV. The nucleocapsid (N) protein of SARS-CoV is a major viral protein recognized by acute and early convalescent sera from SARS patients. To facilitate the studies on the function and structure of the N protein, this report describe the expression and purification of recombinant SARS-CoV N protein using the baculovirus expression system. Recombinant hexa-histidine-tagged N protein with a molecular mass of 47 kD was produced in insect cells. Recombinant N protein was purified to near homogeneity by Ni2+-NTA affinity chromatography. In addition, we examined the subcellular localization of the N protein by confocal microscopy in Trichoplusia ni BT1 Tn 5B1–4 cells infected with recombinant baculovirus. The N protein was found localized in the cytoplasm as well as in the nucleolus. The purified recombinant N protein can be used in further functional study of SARS-CoV.

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