scholarly journals Optogenetic manipulation of medullary neurons in the locust optic lobe

2018 ◽  
Author(s):  
Hongxia Wang ◽  
Richard B. Dewell ◽  
Markus U. Ehrengruber ◽  
Eran Segev ◽  
Jacob Reimer ◽  
...  
Keyword(s):  
Gene Expression ◽  
Neural Circuits ◽  
Optic Lobe ◽  
Fluorescent Protein ◽  
Semliki Forest Virus ◽  
Yellow Fluorescent Protein ◽  
Movement Detector ◽  
Laser Illumination ◽  
Exogenous Gene ◽  
Medullary Neurons

AbstractLocust is a widely used animal model for studying sensory processing and its relation to behavior. Due to the lack of genomic information, genetic tools to manipulate neural circuits in locusts are not yet available. We examined whether Semliki Forest virus is suitable to mediate exogenous gene expression in neurons of the locust optic lobe. We subcloned a channelrhodopsin variant and the yellow fluorescent protein Venus into a Semliki Forest virus vector and injected the virus into the optic lobe of locusts (Schistocerca americana). Fluorescence was observed in all injected optic lobes. Most neurons that expressed the recombinant proteins were located in the first two neuropils of the optic lobe, the lamina and medulla. Extracellular recordings demonstrated that laser illumination increased the firing rate of medullary neurons expressing channelrhodopsin. The optogenetic activation of the medullary neurons also triggered firing of a postsynaptic, looming-sensitive neuron, the Lobula Giant Movement Detector (LGMD). These results indicate that Semliki Forest virus is efficient at mediating transient exogenous gene expression and provides a tool to manipulate neural circuits in the locust nervous system and likely other insects.New and NoteworthyUsing Semliki Forest virus, we efficiently delivered channelrhodopsin into neurons of the locust optic lobe. We demonstrate that laser illumination increases the firing of the medullary neurons expressing channelrhodopsin and of an identified postsynaptic target neuron, the LGMD neuron. This technique allows to manipulate the neuronal activity in locust neural circuits using optogenetics.

scholarly journals Optogenetic manipulation of medullary neurons in the locust optic lobe

2018 ◽  
Vol 120 (4) ◽  
pp. 2049-2058 ◽  
Author(s):  
Hongxia Wang ◽  
Richard B. Dewell ◽  
Markus U. Ehrengruber ◽  
Eran Segev ◽  
Jacob Reimer ◽  
...  
Keyword(s):  
Gene Expression ◽  
Neural Circuits ◽  
Optic Lobe ◽  
Semliki Forest Virus ◽  
Movement Detector ◽  
Excitatory Postsynaptic Potentials ◽  
Postsynaptic Potentials ◽  
Laser Illumination ◽  
Exogenous Gene ◽  
Medullary Neurons

The locust is a widely used animal model for studying sensory processing and its relation to behavior. Due to the lack of genomic information, genetic tools to manipulate neural circuits in locusts are not yet available. We examined whether Semliki Forest virus is suitable to mediate exogenous gene expression in neurons of the locust optic lobe. We subcloned a channelrhodopsin variant and the yellow fluorescent protein Venus into a Semliki Forest virus vector and injected the virus into the optic lobe of locusts ( Schistocerca americana). Fluorescence was observed in all injected optic lobes. Most neurons that expressed the recombinant proteins were located in the first two neuropils of the optic lobe, the lamina and medulla. Extracellular recordings demonstrated that laser illumination increased the firing rate of medullary neurons expressing channelrhodopsin. The optogenetic activation of the medullary neurons also triggered excitatory postsynaptic potentials and firing of a postsynaptic, looming-sensitive neuron, the lobula giant movement detector. These results indicate that Semliki Forest virus is efficient at mediating transient exogenous gene expression and provides a tool to manipulate neural circuits in the locust nervous system and likely other insects.NEW & NOTEWORTHY Using Semliki Forest virus, we efficiently delivered channelrhodopsin into neurons of the locust optic lobe. We demonstrate that laser illumination increases the firing of the medullary neurons expressing channelrhodopsin and elicits excitatory postsynaptic potentials and spiking in an identified postsynaptic target neuron, the lobula giant movement detector neuron. This technique allows the manipulation of neuronal activity in locust neural circuits using optogenetics.

scholarly journals Construction and Characterization of a Lactose-Inducible Promoter System for Controlled Gene Expression inClostridium perfringens

2010 ◽  
Vol 77 (2) ◽  
pp. 471-478 ◽  
Author(s):  
Andrea H. Hartman ◽  
Hualan Liu ◽  
Stephen B. Melville
Keyword(s):  
Gene Expression ◽  
Fluorescent Protein ◽  
Shuttle Vector ◽  
Yellow Fluorescent Protein ◽  
Inducible Promoter ◽  
Gliding Motility ◽  
Glucuronidase Activity ◽  
Atpase Gene ◽  
Regulated Gene Expression ◽  
Promoter System

ABSTRACTClostridium perfringensis a Gram-positive anaerobic pathogen which causes many diseases in humans and animals. While some genetic tools exist for working withC. perfringens, a tightly regulated, inducible promoter system is currently lacking. Therefore, we constructed a plasmid-based promoter system that provided regulated expression when lactose was added. This plasmid (pKRAH1) is anEscherichia coli-C. perfringensshuttle vector containing the gene encoding a transcriptional regulator, BgaR, and a divergent promoter upstream of genebgaL(bgaR-PbgaL). To measure transcription at thebgaLpromoter in pKRAH1, theE. colireporter genegusA, encoding β-glucuronidase, was placed downstream of the PbgaLpromoter to make plasmid pAH2. When transformed into three strains ofC. perfringens, pAH2 exhibited lactose-inducible expression.C. perfringensstrain 13, a commonly studied strain, has endogenous β-glucuronidase activity. We mutated genebglR, encoding a putative β-glucuronidase, and observed an 89% decrease in endogenous activity with no lactose. This combination of a system for regulated gene expression and a mutant of strain 13 with low β-glucuronidase activity are useful tools for studying gene regulation and protein expression in an important pathogenic bacterium. We used this system to express theyfp-pilBgene, comprised of a yellow fluorescent protein (YFP)-encoding gene fused to an assembly ATPase gene involved in type IV pilus-dependent gliding motility inC. perfringens. Expression in the wild-type strain showed that YFP-PilB localized mostly to the poles of cells, but in apilCmutant it localized throughout the cell, demonstrating that the membrane protein PilC is required for polar localization of PilB.

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 PENYISIPAN GEN WARNA PADA IKAN Carassius auratus MENGGUNAKAN METODE ELEKTROFORASI DALAM UPAYA MENINGKATKAN KUALITAS IKAN HIAS

2016 ◽  
Vol 5 (3) ◽  
pp. 335
Author(s):  
Wartono Hadie ◽  
Eni Kusrini ◽  
Agus Priyadi ◽  
Alimuddin Alimuddin
Keyword(s):  
Gene Expression ◽  
Survival Rate ◽  
Carassius Auratus ◽  
Fluorescent Protein ◽  
Ornamental Fish ◽  
Japanese Flounder ◽  
Pcr Product ◽  
Result Show ◽  
Green Fluorescent ◽  
Exogenous Gene

Tujuan penelitian ini adalah untuk mendapatkan teknik rekayasa penyisipan gen warna pada ikan hias dan pengetahuan tentang pola pewarisannya yang akan dapat membantu perbaikan mutu ikan. Metode yang diterapkan adalah elektroforasi. Ikan hias yang digunakan dalam penelitian adalah ikan komet (Carassius auratus) sedangkan gen pemendar yang digunakan adalah GFP (green fluorescent protein) dengan konstruksi DNA yang digunakan berbentuk plasmid yang dikontrol oleh promoter b-aktin dari ikan Japanese flounder (ikan sebelah) dengan panjang fragmen pKer-GFP 6,0 kb. Sintasan dan keberadaan gen GFP diamati mulai dari telur menetas. Ekspresi gen dapat diamati setelah fase terbentuknya sirip dan dilakukan secara deskriptif (performa) dan PCR. Hasil yang diperoleh gen GFP terekspresi mulai pembentukan sirip dan hasil cek PCR semua ketiga konsentrasi DNA yang dicobakan mempunyai ekspresi yang sama. Untuk dapat mengetahui ekspresi gen GFP sampai pada F0 masih menunggu ikan dewasa dan matang gonad.This research was aimed to obtain a method for introduction of exogenous gene of green flourescent protein (GFP) in order to improve the ornamental fish color appearance. The method used were electroporation. The Carassius auratus were used in this research. The construct of flourescent gene of pKer-GFP as DNA construct of plasmid controlled by b-actin of Japanese flounder with pKer-GFP 6.0 kb in length. Survival rate and gene expression of GFP assessed right after the eggs hatched. Gene expression was observed using PCR product and direct observation. The result show that the expression of GFP from all of three treatments were observed but not significantly different. This expression however should be wait until the fish mature.

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 Insertion of an EYFP-pp71 (UL82) Coding Sequence into the Human Cytomegalovirus Genome Results in a Recombinant Virus with Enhanced Viral Growth

2008 ◽  
Vol 82 (21) ◽  
pp. 10543-10555 ◽  
Author(s):  
Nina Tavalai ◽  
Martina Kraiger ◽  
Nina Kaiser ◽  
Thomas Stamminger
Keyword(s):  
Gene Expression ◽  
Human Cytomegalovirus ◽  
Recombinant Virus ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Immune Defense ◽  
Tegument Protein ◽  
Wild Type ◽  
Coding Sequence ◽  
Protein Levels

ABSTRACT The human cytomegalovirus (HCMV) UL82-encoded tegument protein pp71 has recently been shown to activate viral immediate-early (IE) gene expression by neutralizing a cellular intrinsic immune defense instituted by the ND10 protein hDaxx. Pp71 localizes to ND10 upon infection and induces the degradation of hDaxx. Here, we report the successful generation of a recombinant HCMV expressing enhanced yellow fluorescent protein (EYFP) fused to the N terminus of pp71. Intriguingly, insertion of the EYFP-UL82 coding sequence into the HCMV AD169 genome gave rise to a recombinant virus, termed AD169/EYFP-pp71, that replicates to significantly higher titers than wild-type AD169. In particular, we noticed strongly increased protein levels of pp71 after AD169/EYFP-pp71 inoculation. Although the high abundance of pp71 resulted in augmented packaging of the tegument protein into viral particles, no increased hDaxx degradation was detectable upon AD169/EYFP-pp71 infection. In contrast, further investigation revealed a significantly enhanced viral DNA replication compared to wild-type AD169. Thus, we hypothesize that an as-yet-unidentified function of pp71 contributes to the enhanced infectivity of AD169/EYFP-pp71. This assumption is additionally supported by the observation that increased early and late gene expression after AD169/EYFP-pp71 infection occurs independent of elevated IE protein levels. Finally, immunofluorescence analyses confirmed that hDaxx determines the ND10-localization of pp71 upon infection, since pp71 exhibited a nucleolar distribution in the absence of hDaxx. Taken together, we generated a recombinant HCMV that constitutes a useful tool not only to dissect the in vivo dynamics of pp71 subnuclear localization more precisely but also to explore new features of this viral transactivator.

scholarly journals Golden Gate Assembly of Aerobic and Anaerobic Microbial Bioreporters

2021 ◽  
Author(s):  
Aaron J. Hinz ◽  
Benjamin Stenzler ◽  
Alexandre J. Poulain
Keyword(s):  
Gene Expression ◽  
Escherichia Coli ◽  
Synthetic Biology ◽  
Reporter Gene ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Fluorescent Microscopy ◽  
Genetic Circuit ◽  
Regulatory Sequences ◽  
Golden Gate

Microbial bioreporters provide direct insight into cellular processes by producing a quantifiable signal dictated by reporter gene expression. The core of a bioreporter is a genetic circuit in which a reporter gene (or operon) is fused to promoter and regulatory sequences that govern its expression. In this study, we develop a system for constructing novel Escherichia coli bioreporters based on Golden Gate assembly, a synthetic biology approach for the rapid and seamless fusion of DNA fragments. Gene circuits are generated by fusing promoter and reporter sequences encoding yellow fluorescent protein, mCherry, bacterial luciferase, and an anaerobically active flavin-based fluorescent protein. We address a barrier to the implementation of Golden Gate assembly by designing a series of compatible destination vectors that can accommodate the assemblies. We validate the approach by measuring the activity of constitutive bioreporters and mercury and arsenic biosensors in quantitative exposure assays. We also demonstrate anaerobic quantification of mercury and arsenic in biosensors that produce flavin-based fluorescent protein, highlighting the expanding range of redox conditions that can be examined by microbial bioreporters. IMPORTANCE Microbial bioreporters are versatile genetic tools with wide-ranging applications, particularly in the field of environmental toxicology. For example, biosensors that produce a signal output in the presence of a specific analyte offer less costly alternatives to analytical methods for the detection of environmental toxins such as mercury and arsenic. Biosensors of specific toxins can also be used to test hypotheses regarding mechanisms of uptake, toxicity, and biotransformation. In this study, we develop an assembly platform that uses a synthetic biology technique to streamline construction of novel Escherichia coli bioreporters that produce fluorescent or luminescent signals either constitutively or in response to mercury and arsenic exposure. Beyond the synthesis of novel biosensors, our assembly platform can be adapted for numerous applications, including labelling bacteria for fluorescent microscopy, developing gene expression systems, and modifying bacterial genomes.

scholarly journals Programmed hierarchical patterning of bacterial populations

2017 ◽  
Author(s):  
Christian R. Boehm ◽  
Paul K. Grant ◽  
Jim Haseloff
Keyword(s):  
Gene Expression ◽  
Rna Polymerase ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Homoserine Lactone ◽  
T7 Rna Polymerase ◽  
Bacteriophage T7 ◽  
Surface Culture ◽  
Bacterial Populations ◽  
Bacterial Gene

AbstractModern genetic tools allow the dissection and emulation of fundamental mechanisms shaping morphogenesis in multicellular organisms. Several synthetic genetic circuits for control of multicellular patterning have been reported to date. However, hierarchical induction of gene expression domains has received little attention from synthetic biologists, despite its importance in biological self-organization. We report the first synthetic genetic system implementing population-based AND logic for programmed autonomous induction of bacterial gene expression domains. We develop a ratiometric assay for bacteriophage T7 RNA polymerase activity and use it to systematically characterize different intact and split enzyme variants. We then utilize the best-performing variant to build a three-color patterning system responsive to two different homoserine lactones. We validate the AND gate-like behavior of this system both in cell suspension and in surface culture. Finally, we use the synthetic circuit in a membrane-based spatial assay to demonstrate programmed hierarchical patterning of gene expression across bacterial populations.Abbreviations3OC6HSLN-(3-oxohexanoyl)-L-homoserine lactone3OC12HSLN-(3-oxododecanoyl)-L-homoserine lactoneCFPcyan fluorescent proteinIPTGisopropyl-β-D-thiogalactopyranosideParts RegistryMIT Registry of Standard Biological PartsPIpositional informationRDreaction-diffusionRFPred fluorescent proteinRFUrelative fluorescence unitss.d.standard deviationT7RNAPbacteriophage T7 RNA polymeraseYFPyellow fluorescent protein

Disulphide-less crotamine is effective for formation of DNA–peptide complex but is unable to improve bovine embryo transfection

Zygote ◽  
2019 ◽  
Vol 28 (1) ◽  
pp. 72-79
Author(s):  
Vicente J.F. Freitas ◽  
Iana S. Campelo ◽  
Mirelly M.A.S. Silva ◽  
Camila M. Cavalcanti ◽  
Dárcio I.A. Teixeira ◽  
...  
Keyword(s):  
Gene Expression ◽  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Peptide Sequence ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Gfp Expression ◽  
Green Fluorescent ◽  
Exogenous Gene

SummaryThis study aimed to investigate the ability of disulphide-less crotamine (dLCr) to complex DNA and to evaluate whether the DNA–dLCr complex is capable of improving transfection in bovine embryos. Three experiments were performed to: (i) evaluate the formation and stability of the DNA–dLCr complex; (ii) assess the dLCr embryotoxicity by exposure of bovine embryos to dLCr; and (iii) assess the efficiency of bovine embryo transfection after microinjection of the DNA–dLCr complex or green fluorescent protein (GFP) plasmid alone (control). DNA complexation by dLCr after 30 min of incubation at 1:100 and 1:50 proportions presented higher efficiency (P < 0.05) than the two controls: native crotamine (NCr) 1:10 and lipofectamine. There was no difference between DNA–dLCr 1:25 and the controls. The DNA–dLCr complexation was evaluated at different proportions and times. In all, at least half of maximum complexation was achieved within the initial 30 min. No embryotoxicity of dLCr was verified after exposure of in vitro fertilized embryos to different concentrations of the peptide. The effectiveness of dLCr to improve exogenous gene expression was evaluated by microinjection of the DNA–dLCr complex into in vitro fertilized zygotes, followed by verification of both embryo development and GFP expression. From embryos microinjected with DNA only, 4.6% and 2.8% expressed the GFP transgene at day 5 and day 7, respectively. The DNA–dLCr complex did not increase the number of GFP-positive embryos. In conclusion, dLCr forms a complex with DNA and its application in in vitro culture is possible. However, the dLCr peptide sequence should be redesigned to improve GFP expression.

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.

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