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 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 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.

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 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 Development and Characterization of a Subtilin-Regulated Expression System in Bacillus subtilis: Strict Control of Gene Expression by Addition of Subtilin

2005 ◽  
Vol 71 (12) ◽  
pp. 8818-8824 ◽  
Author(s):  
Roger S. Bongers ◽  
Jan-Willem Veening ◽  
Maarten Van Wieringen ◽  
Oscar P. Kuipers ◽  
Michiel Kleerebezem
Keyword(s):  
Gene Expression ◽  
Bacillus Subtilis ◽  
Fluorescent Protein ◽  
Expression System ◽  
Functional Characterization ◽  
Industrial Applications ◽  
Wild Type ◽  
Control Of Gene Expression ◽  
Regulated Gene Expression

ABSTRACT A system for subtilin-regulated gene expression (SURE) in Bacillus subtilis that is based on the regulatory module involved in cell-density-dependent control of the production of subtilin is described. An integration vector for introduction of the essential sensor-regulator couple spaRK into the amyE locus of the B. subtilis chromosome and a B. subtilis 168-derived production host in which the spaRK genes were functionally introduced were constructed. Furthermore, several expression plasmids harboring the subtilin-inducible wild-type spaS promoter or a mutated derivative of this promoter were constructed, which facilitated both transcriptional and translational promoter-gene fusions. Functional characterization of both spaS promoters and the cognate expression host could be performed by controlled overproduction of the β-glucuronidase (GusA) and green fluorescent protein (GFP) reporters. Both spaS promoters exhibited very low levels of basal expression, while extremely high levels of expression were observed upon induction with subtilin. Moreover, the level of expression depended directly on the amount of inducer (subtilin) used. The wild-type spaS promoter appeared to be more strictly controlled by the addition of subtilin, while the highest levels of expression were obtained when the mutated spaS promoter was used. Induction by subtilin led to 110- and 80-fold increases in GusA activity for the spaS promoter and its mutant derivative, respectively. Since the SURE system has attractive functional characteristics, including promoter silence under noninducing conditions and a controlled and high level of expression upon induction, and since it is not subject to catabolite control, we anticipate that it can provide a suitable expression system for various scientific and industrial applications.

Chemically inducible gene expression in seeds before testa rupture

2015 ◽  
Vol 25 (3) ◽  
pp. 345-352 ◽  
Author(s):  
Mariko Nonogaki ◽  
Taira Sekine ◽  
Hiroyuki Nonogaki
Keyword(s):  
Gene Expression ◽  
Function Analysis ◽  
Expression System ◽  
Basic Research ◽  
Inducible Expression ◽  
Upstream Sequence ◽  
Dependent Manner ◽  
Inducible Gene Expression ◽  
Inducible Expression System ◽  
Inducible Gene

AbstractImpermeability of the testa hinders efficient penetration of some small chemicals, such as transcriptional inhibitors, through the endosperm and the embryo during seed experiments. InArabidopsisseeds, 5-bromo-4-chloro-3-indolyl β-d-glucuronic acid, a substrate for β-glucuronidase, did not permeate through the endosperm and embryo efficiently at the stages before testa rupture. TheArabidopsistesta also limited efficient entry of methoxyfenozide, a chemical ligand that was used for inducible gene expression experiments, into seeds. While the detection of a reporter gene at the early imbibitional stages could be replaced by reverse transcription-polymerase chain reaction (RT-PCR), the interference of entry of the chemical ligand into seeds by the testa was still problematic to gene induction experiments. To develop an efficient inducible expression system for gene function analysis in seeds, an inducible expression system with nitrate, which is a testa-permeable ligand, was examined. The vector containing the 2.1-kb upstream sequence ofNITRITE REDUCTASE 1was able to cause expression of a test gene (long non-coding RNA) in imbibed seeds at the stage before testa rupture in a nitrate-dependent manner. This system can be used not only for characterization of genes associated with seed dormancy and germination in basic research, but also for the development of germination recovery or enhancement technologies for agricultural applications.

scholarly journals Regulation of Gene Expression in Diverse Cyanobacterial Species by Using Theophylline-Responsive Riboswitches

2014 ◽  
Vol 80 (21) ◽  
pp. 6704-6713 ◽  
Author(s):  
Amy T. Ma ◽  
Calvin M. Schmidt ◽  
James W. Golden
Keyword(s):  
Gene Expression ◽  
Synthetic Biology ◽  
Atmospheric Carbon Dioxide ◽  
Fluorescent Protein ◽  
Genetic Manipulation ◽  
Yellow Fluorescent Protein ◽  
Regulation Of Gene Expression ◽  
Ease Of Use ◽  
Algal Biotechnology ◽  
Content Type

ABSTRACTCyanobacteria are photosynthetic bacteria that are currently being developed as biological production platforms. They derive energy from light and carbon from atmospheric carbon dioxide, and some species can fix atmospheric nitrogen. One advantage of developing cyanobacteria for renewable production of biofuels and other biological products is that they are amenable to genetic manipulation, facilitating bioengineering and synthetic biology. To expand the currently available genetic toolkit, we have demonstrated the utility of synthetic theophylline-responsive riboswitches for effective regulation of gene expression in four diverse species of cyanobacteria, including two recent isolates. We evaluated a set of six riboswitches driving the expression of a yellow fluorescent protein reporter inSynechococcus elongatusPCC 7942,Leptolyngbyasp. strain BL0902,Anabaenasp. strain PCC 7120, andSynechocystissp. strain WHSyn. We demonstrated that riboswitches can offer regulation of gene expression superior to that of the commonly used isopropyl-β-d-thiogalactopyranoside induction of alacIq-Ptrcpromoter system. We also showed that expression of the toxic protein SacB can be effectively regulated, demonstrating utility for riboswitch regulation of proteins that are detrimental to biomass accumulation. Taken together, the results of this work demonstrate the utility and ease of use of riboswitches in the context of genetic engineering and synthetic biology in diverse cyanobacteria, which will facilitate the development of algal biotechnology.

scholarly journals Development and Application of an Arabinose-Inducible Expression System by Facilitating Inducer Uptake in Corynebacterium glutamicum

2012 ◽  
Vol 78 (16) ◽  
pp. 5831-5838 ◽  
Author(s):  
Yun Zhang ◽  
Xiuling Shang ◽  
Shujuan Lai ◽  
Guoqiang Zhang ◽  
Yong Liang ◽  
...  
Keyword(s):  
Corynebacterium Glutamicum ◽  
Fluorescent Protein ◽  
Expression System ◽  
Carbon Sources ◽  
Inducible Expression ◽  
Gene Encoding ◽  
Content Type ◽  
E Coli ◽  
Inducible Expression System ◽  
Green Fluorescent

ABSTRACTCorynebacterium glutamicumis currently used for the industrial production of a variety of biological materials. Many available inducible expression systems in this species uselac-derived promoters fromEscherichia colithat exhibit much lower levels of inducible expression and leaky basal expression. We developed an arabinose-inducible expression system that contains thel-arabinose regulator AraC, thePBADpromoter from thearaBADoperon, and thel-arabinose transporter AraE, all of which are derived fromE. coli. The level of induciblePBAD-based expression could be modulated over a wide concentration range from 0.001 to 0.4%l-arabinose. This system tightly controlled the expression of the uracil phosphoribosyltransferase without leaky expression. When the gene encoding green fluorescent protein (GFP) was under the control ofPBADpromoter, flow cytometry analysis showed that GFP was expressed in a highly homogeneous profile throughout the cell population. In contrast to the case inE. coli,PBADinduction was not significantly affected in the presence of different carbon sources inC. glutamicum, which makes it useful in fermentation applications. We used this system to regulate the expression of theodhIgene fromC. glutamicum, which encodes an inhibitor of α-oxoglutarate dehydrogenase, resulting in high levels of glutamate production (up to 13.7 mM) under biotin nonlimiting conditions. This system provides an efficient tool available for molecular biology and metabolic engineering ofC. glutamicum.

scholarly journals Improving Baculovirus Infectivity by Efficiently Embedding Enhancing Factors into Occlusion Bodies

2017 ◽  
Vol 83 (14) ◽  
Author(s):  
Shili Yang ◽  
Lijuan Zhao ◽  
Ruipeng Ma ◽  
Wei Fang ◽  
Jia Hu ◽  
...  
Keyword(s):  
Protein Expression ◽  
Fluorescent Protein ◽  
Expression System ◽  
Agrotis Segetum ◽  
Foreign Protein ◽  
Content Type ◽  
Occlusion Bodies ◽  
Foreign Proteins ◽  
Novel Strategy

ABSTRACT The relatively low infectivity of baculoviruses to their host larvae limits their use as insecticidal agents on a larger scale. In the present study, a novel strategy was developed to efficiently embed foreign proteins into Autographa californica multiple nucleopolyhedrovirus (AcMNPV) occlusion bodies (OBs) to achieve stable expression of foreign proteins and to improve viral infectivity. A recombinant AcMNPV bacmid was constructed by expressing the 150-amino-acid (aa) N-terminal segment of polyhedrin under the control of the p10 promoter and the remaining C-terminal 95-aa segment under the control of the polyhedrin promoter. The recombinant virus formed OBs in Spodoptera frugiperda 9 cells, in which the occlusion-derived viruses were embedded in a manner similar to that for wild-type AcMNPV. Next, the 95-aa polyhedrin C terminus was fused to enhanced green fluorescent protein, and the recombinant AcMNPV formed fluorescent green OBs and was stably passaged in vitro and in vivo. The AcMNPV recombinants were further modified by fusing truncated Agrotis segetum granulovirus enhancin or truncated Cydia pomonella granulovirus ORF13 (GP37) to the C-terminal 95 aa of polyhedrin, and both recombinants were able to form normal OBs. Bioactivity assays indicated that the median lethal concentrations of these two AcMNPV recombinants were 3- to 5-fold lower than that of the control virus. These results suggest that embedding enhancing factors in baculovirus OBs by use of this novel technique may promote efficient and stable foreign protein expression and significantly improve baculovirus infectivity. IMPORTANCE Baculoviruses have been used as bioinsecticides for over 40 years, but their relatively low infectivity to their host larvae limits their use on a larger scale. It has been reported that it is possible to improve baculovirus infectivity by packaging enhancing factors within baculovirus occlusion bodies (OBs); however, so far, the packaging efficiency has been low. In this article, we describe a novel strategy for efficiently embedding foreign proteins into AcMNPV OBs by expressing N- and C-terminal (dimidiate) polyhedrin fragments (150 and 95 amino acids, respectively) as fusions to foreign proteins under the control of the p10 and polyhedrin promoters, respectively. When this strategy was used to embed an enhancing factor (enhancin or GP37) into the baculovirus OBs, 3- to 5-fold increases in baculoviral infectivity were observed. This novel strategy has the potential to create an efficient protein expression system and a highly efficient virus-based system for insecticide production in the future.

scholarly journals CsrA (BB0184) Is Not Involved in Activation of the RpoN-RpoS Regulatory Pathway in Borrelia burgdorferi

2014 ◽  
Vol 82 (4) ◽  
pp. 1511-1522 ◽  
Author(s):  
Zhiming Ouyang ◽  
Jianli Zhou ◽  
Michael V. Norgard
Keyword(s):  
Gene Expression ◽  
Borrelia Burgdorferi ◽  
Animal Studies ◽  
Expression System ◽  
Regulatory Pathway ◽  
Content Type ◽  
Mouse Tissues ◽  
Differential Gene ◽  
Carbon Storage Regulator

ABSTRACTBorrelia burgdorferiencodes a homologue of the bacterial carbon storage regulator A (CsrA). Recently, it was reported that CsrA contributes toB. burgdorferiinfectivity and is required for the activation of the central RpoN-RpoS regulatory pathway. However, many questions concerning the function of CsrA inB. burgdorferigene regulation remain unanswered. In particular, there are conflicting reports concerning the molecular details of how CsrA may modulaterpoSexpression and, thus, how CsrA may influence the RpoN-RpoS pathway inB. burgdorferi. To address these key discrepancies, we examined the role of CsrA in differential gene expression in the Lyme disease spirochete. Upon engineering an induciblecsrAexpression system inB. burgdorferi, controlled hyperexpression of CsrA in a merodiploid strain did not significantly alter the protein and transcript levels ofbosR,rpoS, and RpoS-dependent genes (such asospCanddbpA). In addition, we constructed isogeniccsrAmutants in two widely used infectiousB. burgdorferistrains. When expression ofbosR,rpoS,ospC, anddbpAwas compared between thecsrAmutants and their wild-type counterparts, no detectable differences were observed. Finally, animal studies indicated that thecsrAmutants remained infectious for and virulent in mice. Analyses ofB. burgdorferigene expression in mouse tissues showed comparable levels ofrpoStranscripts by thecsrAmutants and the parental strains. Taken together, these results constitute compelling evidence that CsrA is not involved in activation of the RpoN-RpoS pathway and is dispensable for mammalian infectious processes carried out byB. burgdorferi.

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