scholarly journals A xylose-inducible expression system and a CRISPRi-plasmid for targeted knock-down of gene expression inClostridioides difficile

2018 ◽  
Author(s):  
Ute Müh ◽  
Anthony G. Pannullo ◽  
David S. Weiss ◽  
Craig D. Ellermeier
Keyword(s):  
Gene Expression ◽  
Cell Division ◽  
Fluorescent Protein ◽  
Cell Envelope ◽  
Expression System ◽  
Developed Countries ◽  
Inducible Expression ◽  
Guide Rna ◽  
Antibiotic Development ◽  
Clostridioides Difficile

AbstractHere we introduce plasmids for xylose-regulated expression and repression of genes inClostridioides difficile. The xylose-inducible expression vector allows for ~100-fold induction of anmCherryOptreporter gene. Induction is titratable and uniform from cell-to-cell. The gene repression plasmid is a CRISPR-interference (CRISPRi) system based on a nuclease-defective, codon-optimized allele of theStreptococcus pyogenesCas9 protein (dCas9) that is targeted to a gene of interest by a constitutively-expressed single guide RNA (sgRNA). Expression ofdCas9is induced by xylose, allowing investigators to control the timing and extent of gene-silencing, as demonstrated here by dose-dependent repression of a chromosomal gene for a red fluorescent protein (maximum repression ~100-fold). To validate the utility of CRISPRi for deciphering gene function inC. difficile, we knocked-down expression of three genes involved in biogenesis of the cell envelope: the cell division geneftsZ, the S-layer protein geneslpAand the peptidoglycan synthase genepbp-0712. CRISPRi confirmed known or expected phenotypes associated with loss of FtsZ and SlpA, and revealed that the previously uncharacterized peptidoglycan synthase PBP-0712 is needed for proper elongation, cell division and protection against lysis.ImportanceClostridioides difficilehas become the leading cause of hospital-acquired diarrhea in developed countries. A better understanding of the basic biology of this devastating pathogen might lead to novel approaches for preventing or treatingC. difficileinfections. Here we introduce new plasmid vectors that allow for titratable induction (Pxyl) or knockdown (CRISPRi) of gene expression. The CRISPRi plasmid allows for easy depletion of target proteins inC. difficile. Besides bypassing the lengthy process of mutant construction, CRISPRi can be used to study the function of essential genes, which are particularly important targets for antibiotic development.

scholarly journals A Xylose-Inducible Expression System and a CRISPR Interference Plasmid for Targeted Knockdown of Gene Expression in Clostridioides difficile

2019 ◽  
Vol 201 (14) ◽  
Author(s):  
Ute Müh ◽  
Anthony G. Pannullo ◽  
David S. Weiss ◽  
Craig D. Ellermeier
Keyword(s):  
Gene Expression ◽  
Cell Division ◽  
Fluorescent Protein ◽  
Expression System ◽  
Developed Countries ◽  
Inducible Expression ◽  
Content Type ◽  
Antibiotic Development ◽  
Crispr Interference ◽  
Clostridioides Difficile

ABSTRACT Here we introduce plasmids for xylose-regulated expression and repression of genes in Clostridioides difficile. The xylose-inducible expression vector allows for ∼100-fold induction of an mCherryOpt reporter gene. Induction is titratable and uniform from cell to cell. The gene repression plasmid is a CRISPR interference (CRISPRi) system based on a nuclease-defective, codon-optimized allele of the Streptococcus pyogenes Cas9 protein (dCas9) that is targeted to a gene of interest by a constitutively expressed single guide RNA (sgRNA). Expression of dCas9 is induced by xylose, allowing investigators to control the timing and extent of gene silencing, as demonstrated here by dose-dependent repression of a chromosomal gene for a red fluorescent protein (maximum repression, ∼100-fold). To validate the utility of CRISPRi for deciphering gene function in C. difficile, we knocked down the expression of three genes involved in the biogenesis of the cell envelope: the cell division gene ftsZ, the S-layer protein gene slpA, and the peptidoglycan synthase gene pbp-0712. CRISPRi confirmed known or expected phenotypes associated with the loss of FtsZ and SlpA and revealed that the previously uncharacterized peptidoglycan synthase PBP-0712 is needed for proper elongation, cell division, and protection against lysis. IMPORTANCE Clostridioides difficile has become the leading cause of hospital-acquired diarrhea in developed countries. A better understanding of the basic biology of this devastating pathogen might lead to novel approaches for preventing or treating C. difficile infections. Here we introduce new plasmid vectors that allow for titratable induction (Pxyl) or knockdown (CRISPRi) of gene expression. The CRISPRi plasmid allows for easy depletion of target proteins in C. difficile. Besides bypassing the lengthy process of mutant construction, CRISPRi can be used to study the function of essential genes, which are particularly important targets for antibiotic development.

scholarly journals Zn2+-Inducible Expression Platform for Synechococcus sp. Strain PCC 7002 Based on the smtA Promoter/Operator and smtB Repressor

2016 ◽  
Vol 83 (3) ◽  
Author(s):  
Adam A. Pérez ◽  
John P. Gajewski ◽  
Bryan H. Ferlez ◽  
Marcus Ludwig ◽  
Carol S. Baker ◽  
...  
Keyword(s):  
Gene Expression ◽  
Fluorescent Protein ◽  
Expression System ◽  
Yellow Fluorescent Protein ◽  
Background Level ◽  
Basic Research ◽  
Industrial Applications ◽  
Inducible Expression ◽  
Content Type ◽  
Expression Platform

ABSTRACT Synechococcus sp. strain PCC 7002 has been gaining significance as both a model system for photosynthesis research and for industrial applications. Until recently, the genetic toolbox for this model cyanobacterium was rather limited and relied primarily on tools that only allowed constitutive gene expression. This work describes a two-plasmid, Zn2+-inducible expression platform that is coupled with a zurA mutation, providing enhanced Zn2+ uptake. The control elements are based on the metal homeostasis system of a class II metallothionein gene (smtA 7942) and its cognate SmtB7942 repressor from Synechococcus elongatus strain PCC 7942. Under optimal induction conditions, yellow fluorescent protein (YFP) levels were about half of those obtained with the strong, constitutive phycocyanin (cpcBA 6803) promoter of Synechocystis sp. strain PCC 6803. This metal-inducible expression system in Synechococcus sp. strain PCC 7002 allowed the titratable gene expression of YFP that was up to 19-fold greater than the background level. This system was utilized successfully to control the expression of the Drosophila melanogaster β-carotene 15,15′-dioxygenase, NinaB, which is toxic when constitutively expressed from a strong promoter in Synechococcus sp. strain PCC 7002. Together, these properties establish this metal-inducible system as an additional useful tool that is capable of controlling gene expression for applications ranging from basic research to synthetic biology in Synechococcus sp. strain PCC 7002. IMPORTANCE This is the first metal-responsive expression system in cyanobacteria, to our knowledge, that does not exhibit low sensitivity for induction, which is one of the major hurdles for utilizing this class of genetic tools. In addition, high levels of expression can be generated that approximate those of established constitutive systems, with the added advantage of titratable control. Together, these properties establish this Zn2+-inducible system, which is based on the smtA 7942 operator/promoter and smtB 7942 repressor, as a versatile gene expression platform that expands the genetic toolbox of Synechococcus sp. strain PCC 7002.

scholarly journals Construction and Characterization of a Gradually Inducible Expression Vector for Halobacterium salinarum, Based on thekdpPromoter

2012 ◽  
Vol 78 (7) ◽  
pp. 2100-2105 ◽  
Author(s):  
Dorthe Kixmüller ◽  
Jörg-Christian Greie
Keyword(s):  
Gene Expression ◽  
Expression Vector ◽  
Expression System ◽  
Expression Patterns ◽  
Inducible Expression ◽  
Halobacterium Salinarum ◽  
Expression Vectors ◽  
Content Type ◽  
Inducible Expression System

ABSTRACTGradually inducible expression vectors which are governed by variations of growth conditions are powerful tools for gene expression of conditionally lethal mutants. Furthermore, controlled expression allows monitoring of overproduction of proteins at various stages in their expressing hosts. ForHalobacterium salinarum, which is often used as a paradigm for halophilic archaea, such an inducible expression system is not available to date. Here we show that thekdppromoter (Pkdp), which facilitates gene expression upon K+limitation, can be used to establish such a system for molecular applications. Pkdpfeatures a rather high expression rate, with an approximately 50-fold increase that can be easily varied by K+concentrations in the growth medium. Besides the construction of an expression vector, our work describes the characterization of expression patterns and, thus, offers a gradually inducible expression system to the scientific community.

336 QUANTITATIVE ANALYSIS OF TETRACYCLINE-INDUCIBLE EXPRESSION OF THE GREEN FLUORESCENT PROTEIN GENE IN TRANSGENIC CHICKENS

2013 ◽  
Vol 25 (1) ◽  
pp. 315
Author(s):  
B. Koo ◽  
M. Kwon ◽  
J. Roh ◽  
J. Kim ◽  
T. Kim
Keyword(s):  
Fluorescent Protein ◽  
Expression System ◽  
Constitutive Expression ◽  
Transgenic Animals ◽  
Inducible Expression ◽  
Gfp Expression ◽  
Transgenic Chicken ◽  
Inducible Expression System ◽  
Green Fluorescent ◽  
Transgenic Chickens

The use of transgenic farm animals as bioreactors to address the growing demand for biopharmaceuticals, both in terms of increased quantity and greater number, represents a key development in the advancement of medical science. However, the potential for detrimental side effects as a result of uncontrolled constitutive expression of foreign genes in transgenic animals is a well-recognised limitation of such systems. Previously, using a tetracycline-inducible expression system, we demonstrated the induction of expression of a transgene encoding green fluorescent protein (GFP) in transgenic chickens by feeding with doxycycline, a tetracycline derivative; expression of GFP reverted to pre-induction levels when the inducer was removed from the diet (Kwon et al. 2011 Biochem. Biophys. Res. Commun. 410, 890–894). As a proof of principle study, however, quantitative assessment of expression was not possible, as only 1 G0 and 1 G1 transgenic chicken was obtained. In the current study, with 7 G2 transgenic chickens obtained from 1 G1 hen, we confirmed stable genomic integration of a single copy number of the transgene by Southern blot analysis. As we have observed in G1 transgenic chicken previously, all of the G2 transgenic chickens emitted a green fluorescence upon doxycycline feeding (50 mg kg–1 of formula feed). Fluorescence became detectable 4 days after starting doxycycline feeding, and maximum GFP expression was detected after 2 weeks. Removal of doxycycline from the diet after 14 days of induction feeding resulted in the return of external fluorescence to pre-induction levels after 39 days. Quantitative analysis of gene induction was done using protein and mRNA extracted from primary cultured cells derived from 6-day transgenic chicken embryos. The eggs were obtained by mating a nontransgenic wild-type hen with 1 of G2 transgenic roosters. Protein levels of GFP were analysed by immunoblot and quantified using a densitometer. In the absence of doxycycline, the amount of GFP per 1 µg of total protein was 0.2 ng. However, when the cells were treated with doxycycline for 6 days, the amount of GFP increased to 3.1 ng per 1 µg of total protein, which was 16-fold higher than that of the cells pre-treated with doxycycline. Switching to doxycycline-free medium after doxycycline induction resulted in significant abrogation of GFP expression in 6 days; the amount of GFP reduced from 3.1 to 0.5 ng, a 6.2-fold reduction. Transcription of the GFP gene was also assessed by Northern blot. The amount of GFP mRNA measured by band density increased as much as 20-fold (3.9/0.2) with 6 days of doxycycline induction and declined to 1/8 (3.9/0.5) when doxycycline was removed from the cell culture media for 6 days. The use of an inducible expression system that can be regulated by dietary supplementation could help mitigate the physiological disruption that can occur in transgenic animals as a result of uncontrolled constitutive expression of a transgene.

scholarly journals Levulinic Acid-Inducible and Tunable Gene Expression System for Methylorubrum extorquens

2021 ◽  
Vol 9 ◽  
Author(s):  
Chandran Sathesh-Prabu ◽  
Young Shin Ryu ◽  
Sung Kuk Lee
Keyword(s):  
Gene Expression ◽  
Levulinic Acid ◽  
Fluorescent Protein ◽  
Expression System ◽  
Constitutive Expression ◽  
Value Added ◽  
Total Protein Content ◽  
Reporter Protein ◽  
Gene Expression System ◽  
Wide Range

Methylorubrum extorquens AM1 is an efficient platform strain possessing biotechnological potential in formate- and methanol-based single carbon (C1) bioeconomy. Constitutive expression or costly chemical-inducible expression systems are not always desirable. Here, several glucose-, xylose-, and levulinic acid (LA)-inducible promoter systems were assessed for the induction of green fluorescent protein (GFP) as a reporter protein. Among them, the LA-inducible gene expression system (HpdR/PhpdH) showed a strong expression of GFP (51-fold) compared to the control. The system was induced even at a low concentration of LA (0.1 mM). The fluorescence intensity increased with increasing concentrations of LA up to 20 mM. The system was tunable and tightly controlled with meager basal expression. The maximum GFP yield obtained using the system was 42 mg/g biomass, representing 10% of the total protein content. The efficiency of the proposed system was nearly equivalent (90%–100%) to that of the widely used strong promoters such as PmxaF and PL/O4. The HpdR/PhpdH system worked equally efficiently in five different strains of M. extorquens. LA is a low-cost, renewable, and sustainable platform chemical that can be used to generate a wide range of products. Hence, the reported system in potent strains of M. extorquens is highly beneficial in the C1-biorefinery industry to produce value-added products and bulk chemicals.

scholarly journals Possible differences in efficiency of guide RNA with different spacer sequence in CRISPR knock-down or knock-out of particular gene

2019 ◽  
Author(s):  
Wenfa Ng
Keyword(s):  
Gene Expression ◽  
Genome Editing ◽  
Target Gene ◽  
Target Genes ◽  
Fluorescent Protein ◽  
Bacterial Cells ◽  
Fluorescent Intensity ◽  
Guide Rna ◽  
Targeting Efficiency ◽  
Template Dna

Cluster regularly interspersed short palindromic repeats (CRISPR) mediated genome editing has emerged as the dominant technique for modulating the expression of target genes. Specifically, when coupled with different effectors, CRISPR could be utilized to either activate or repress gene expression. Specificity of the CRISPR gene editing method arises from the unique spacer sequence in guide RNA that mediates the specific localization of Cas9 endonuclease to particular stretches of DNA. However, complementary base pairing between the guide RNA and template DNA depends critically on existence of protospacer adjacent motif (PAM) sequence immediately downstream of the spacer sequence. Such three nucleotide PAM sequence could be present at multiple loci in a given gene, which meant that different spacer sequence could be incorporated in guide RNA design to target the same gene. Given that different spacer sequences have different binding affinities to template DNA, differences could exist in the efficiency in which CRISPR-Cas9 could be guided to generate a double strand break in a particular gene locus. Using green fluorescent protein (GFP) reporter gene expressed in recombinant Escherichia coli as experimental system, this study sought to understand if differences in targeting efficiency exist between guide RNA with different spacer sequence that could target the same gene. Fluorescent intensity of cells at the population level would serve as readout of the targeting efficiency. For example, spacer sequence in guide RNA that could better activate the endonuclease activity of Cas9 would result in lower fluorescent intensity of GFP. To check for the effect of expression mode on targeting efficiency of guide RNA, GFP gene would be expressed on a plasmid in E. coli as well as integrated into the genome of the bacterium. Doing so would provide critical information on whether the CRISPR-Cas9 system has differentiated efficacy in generating double strand breaks in genomic versus plasmid DNA. Such information would inform future experimental design involving CRISPR-Cas9 genome editing technology as well as hold implications on how CRISPR evolved as an adaptive immune system in defending bacterial cells against foreign DNA. Given the goal of the study to understand the relative extent in which a target gene would be disrupted by CRISPR-Cas9 guided by different spacer sequence on guide RNA, no repair module for the target gene would be provided. Collectively, multiple occurrence of PAM sequence in a target gene meant that different spacer sequences could be used in CRISPR-Cas9 to downregulate gene expression. Relative efficacies of different spacer sequence in guide RNA in achieving targeted gene inactivation remain poorly understood and constitutes the basis of this study, which hopefully would provide guidance on the selection of specific spacer sequence that would yield the most efficacious disruption of gene expression at the genome and plasmid level.

scholarly journals Tetracycline-Inducible Gene Expression and Gene Deletion in Candida albicans

2005 ◽  
Vol 4 (8) ◽  
pp. 1328-1342 ◽  
Author(s):  
Yang-Nim Park ◽  
Joachim Morschhäuser
Keyword(s):  
Gene Expression ◽  
Candida Albicans ◽  
Gene Function ◽  
High Efficiency ◽  
Expression System ◽  
Inducible Expression ◽  
Induced Expression ◽  
Inducible Gene Expression ◽  
Gene Expression System ◽  
Inducible Gene

ABSTRACT The genetic analysis of Candida albicans, the major fungal pathogen of humans, is hampered by its diploid genome, the absence of a normal sexual cycle, and a nonstandard codon usage. Although effective methods to study gene function have been developed in the past years, systems to control gene expression in C. albicans are limited. We have established a system that allows induction of gene expression in C. albicans by the addition of tetracycline (Tet). By fusing genetically modified versions of the reverse Tet repressor from Escherichia coli and the transcription activation domain of the Gal4 protein from Saccharomyces cerevisiae, a C. albicans-adapted reverse Tet-dependent transactivator (rtTA) was created that was expressed from the constitutive ADH1 or the opaque-specific OP4 promoter. To monitor Tet-inducible gene expression, the caGFP reporter gene was placed under the control of a Tet-dependent promoter, obtained by fusing a minimal promoter from C. albicans to seven copies of the Tet operator sequence. Fluorescence of the cells demonstrated that gene expression could be efficiently induced by the addition of doxycycline in yeast, hyphal, and opaque cells of C. albicans. The Tet-inducible gene expression system was then used to manipulate the behavior of the various growth forms of C. albicans. Tet-induced expression of a dominant-negative CDC42 allele resulted in growth arrest as large, multinucleate cells. Filamentous growth was efficiently inhibited under all tested hyphal-growth-promoting conditions by Tet-inducible expression of the NRG1 repressor. Tet-induced expression of the MTL a 1 gene in opaque cells of an MTLα strain forced the cells to switch to the white phase, whereas Tet-induced expression of the MTL a 2 transcription factor induced shmooing. When the ecaFLP gene, encoding the site-specific recombinase FLP, was placed under the control of the Tet-dependent promoter, Tet-inducible deletion of genes which were flanked by the FLP target sequences was achieved with high efficiency to generate conditional null mutants. In combination with the dominant selection marker caSAT1, the Tet-inducible gene expression system was also applied in C. albicans wild-type strains, including drug-resistant clinical isolates that overexpressed the MDR1, CDR1, and CDR2 multidrug efflux pumps. This system, therefore, allows a growth medium-independent, Tet-inducible expression and deletion of genes in C. albicans and provides a convenient, versatile new tool to study gene function and manipulate cellular behavior in this model pathogenic fungus.

scholarly journals Cell-Based Assay To Identify Inhibitors of the Hfq-sRNA Regulatory Pathway

2014 ◽  
Vol 58 (9) ◽  
pp. 5500-5509 ◽  
Author(s):  
Shaima A. El-Mowafi ◽  
John N. Alumasa ◽  
Sarah E. Ades ◽  
Kenneth C. Keiler
Keyword(s):  
Gene Expression ◽  
Cyclic Peptides ◽  
Fluorescent Protein ◽  
Cyclic Peptide ◽  
Yellow Fluorescent Protein ◽  
Mobility Shift ◽  
Content Type ◽  
Antibiotic Development ◽  
Split Intein ◽  
Gel Mobility Shift

ABSTRACTNoncoding small RNAs (sRNAs) act in conjunction with the RNA chaperone Hfq to regulate gene expression in bacteria. Because Hfq is required for virulence in several bacterial pathogens, the Hfq-sRNA system is an attractive target for antibiotic development. A reporter strain in which the expression of yellow fluorescent protein (YFP) is controlled by Hfq-sRNA was engineered to identify inhibitors of this system. A reporter that is targeted by Hfq in conjunction with the RybB sRNA was used in a genetic screen to identify inhibitors from a library of cyclic peptides produced inEscherichia coliusing split-intein circular ligation of peptides and proteins (SICLOPPS), an intein-based technology. One cyclic peptide identified in this screen, RI20, inhibited Hfq-mediated repression of gene expression in conjunction with both RybB and an unrelated sRNA, MicF. Gel mobility shift assays showed that RI20 inhibited binding of Hfq to RybB and MicF with similarKivalues. These data suggest that RI20 inhibits Hfq activity by blocking interactions with sRNAs and provide a paradigm for inhibiting virulence genes in Gram-negative pathogens.

scholarly journals Use of mCherry Red Fluorescent Protein for Studies of Protein Localization and Gene Expression in Clostridium difficile

2014 ◽  
Vol 81 (5) ◽  
pp. 1652-1660 ◽  
Author(s):  
Eric M. Ransom ◽  
Craig D. Ellermeier ◽  
David S. Weiss
Keyword(s):  
Gene Expression ◽  
Clostridium Difficile ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Protein Localization ◽  
Anaerobic Bacteria ◽  
Gc Content ◽  
Inducible Promoter ◽  
Developed Countries ◽  
Content Type

ABSTRACTFluorescent proteins are powerful reporters in biology, but most require O2for chromophore maturation, making them inherently difficult to use in anaerobic bacteria.Clostridium difficile, a strict anaerobe with a genomic GC content of only 29%, is the leading cause of hospital-acquired diarrhea in developed countries, and new methods for studying this pathogen are sorely needed. We recently demonstrated that a cyan fluorescent protein called CFPoptthat has been codon optimized for production in low-GC bacteria can be used to study protein localization inC. difficileprovided the cells are fixed prior to exposure to air. We describe here a codon-optimized variant of mCherry (mCherryOpt) that exhibits faster acquisition of fluorescence and a better signal-to-noise ratio than CFPopt. We utilizedmCherryOptto construct plasmids for studying protein localization (pRAN473) and gene expression (pDSW1728) inC. difficile. Plasmid pRAN473 is anmCherryOptfusion vector with a tetracycline-inducible promoter. To document its biological utility, we demonstrated septal localization of two cell division proteins, MldA and ZapA. Plasmid pDSW1728 is designed for cloning a promoter of interest upstream ofmCherryOpt. As proof of principle, we studied the expression of thepdaVoperon, which is required for lysozyme resistance. In confirmation and extension of previous reports, we found that expression of thepdaVoperon requires the alternative sigma factor σvand that induction by lysozyme is dose dependent and uniform across the population of lysozyme-treated cells.

scholarly journals Identification of Promoters for Efficient Gene Expression in Magnetospirillum gryphiswaldense

2009 ◽  
Vol 75 (12) ◽  
pp. 4206-4210 ◽  
Author(s):  
Claus Lang ◽  
Anna Pollithy ◽  
Dirk Schüler
Keyword(s):  
Gene Expression ◽  
Escherichia Coli ◽  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Expression System ◽  
Antibody Binding ◽  
Magnetotactic Bacterium ◽  
Magnetospirillum Gryphiswaldense ◽  
Efficient Gene ◽  
Green Fluorescent

ABSTRACT To develop an expression system for the magnetotactic bacterium Magnetospirillum gryphiswaldense, we compared gene expression from the widely used Escherichia coli P lac promoter with that from known and predicted genuine M. gryphiswaldense promoters. With the use of green fluorescent protein as a reporter, the highest expression level was observed with the magnetosomal P mamDC promoter. We demonstrate that this promoter can be used for the expression of modified magnetosome proteins to generate “antibody-binding” magnetosomes.

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