Eu gostaria de agradecer Olga de Souza Silva para assistência técnica. Este trabalho foi financiado pela Swiss National Science Foundation (SNSF) Grant 31003A_127029.

O método TransFLP (Figura 1) é exemplificado por três abordagens diferentes: I) a eliminação de dois genes adjacentes que codificam determinantes de virulência de V. cholerae (por exemplo, ΔctxAB), II) a remoção de uma ilha de patogenicidade (por exemplo, ΔVPI-1), e III) a integração de uma polimerase de RNA dependente de sequência do promotor de T7 a montante de um gene de interesse, o que permite a sua subsequentemente expressão artificial no fundo estirpe respectivo (por exemplo, T7-TFox) (Figura 2). 1. Preparação de Quitina Flakes <ol><li> 50-80 mg de peso de flocos de quitina de um padrão de tubo de plástico de 1,5 ml. Isto pode ser preparado em grandes quantidades. Quitina flocos estão comercialmente disponíveis a partir de Sigma (cat. número C9213). </li><li> Autoclavar os flocos mantendo as tampas dos tubos aberto. </li><li> Imediatamente fechar as pálpebras após a autoclave tenha arrefecido. </li><li> Loja autoclavado quitina flocosà temperatura ambiente. </li></ol> 2. Opcional: Transformação Artificial de V. cholerae com Flp Recombinase-encoding Plasmid <ol><li> Prepare electrocompetentes V. cholerae células utilizando métodos padrão 11. Armazenar aliquotas de células competentes a -80 ° C. </li><li> Adicionar 1-2 uL de uma regularidade de mini-preparação de plasmídeo pBR-flp 5 ao electrocompetentes V. células cholerae e transferir a mistura para uma cuvete de electroporação (0,2 largura da abertura cm). </li><li> Aplicar um pulso de 1,6 kV. </li><li> Adicionar 0,9 ml de meio SOC, gentilmente transferir células de um tubo de ml padrão 14. Incubar sem movimento durante 2,5 a 3 horas a 30 ° C. </li><li> Ul placa 100 e 300 ul em placas LB contendo 100 ug / ml de ampicilina e incubado a 30 ° C durante a noite. </li><li> Purificar único clone e armazenar como estoque de glicerol para experimentos TransFLP futuras. </li></ol> 3. Desenho oligonucleótido e Reacção em Cadeia da Polimerase <ol><li> Pelo menos seis oligonucleótidos são necessários para amplificar a região de ADN (s) de interesse, bem como o cassete de FRT-flanqueado antibiótico por PCR (Figura 3). Recomenda-se também incluir um par de oligonucleótidos escorva fora do fragmento de PCR inserido. Isto permite a verificação de integração e a excisão correcta da cassete de resistência FRT-flanqueado antibiótico (por exemplo, descrito como &quot;chk-up&quot; e primers &quot;chk-down&quot; nas Figuras 3 a 5). </li><li> Projetar os oligonucleótidos # 2, # 3, # 4 e # 5 com cuidado. Eles devem ser suficientemente capazes de hibridar (por exemplo, ~ 28 pb) com o DNA molde. O molde de ADN é ADN genómico (gDNA), para os iniciadores # 2 e # 5, e FRT-Kan-FRT contendo DNA de plasmídeo como pROD17 12 e pBR-FRT-KAN-FRT (neste estudo) para os iniciadores # 3 e # 4 ( Figura 3A). </li><li> Conceber os iniciadores # 2 a # 5 permitindo extensa de emparelhamento de bases na sua extremidade 5 &#39;entre # 2 / # 3 e # 4 / # 5, respectivamente (Figura 3A </strong&gt; inset). Duas rodadas de PCR seguir. </li><li> Realizar três reações independentes de PCR como uma rodada 1 º de PCR. A primeira vai amplificar a região a montante do gene / ADN da região-de-interesse com a ajuda de oligonucleótidos # 1 e # 2. A PCR deve resultar num fragmento de pelo menos 500 pb de comprimento que permitem a recombinação homóloga dentro das células. Fragmentos mais curtos (~ 250 pb) pode ser de 8 suficiente mas não são recomendados. </li><li> Em paralelo realizar a segunda PCR, que amplifica a cassete de FRT-flanqueado antibiótico. Para os exemplos fornecidos aqui nós usamos principalmente aph (kan R). Oligonucleotídeos uso # 3 e # 4 para essa reação (Figura 3A). </li><li> Concomitantemente, a amplificação da região de ADN a jusante através de PCR (amostra III). O fragmento de PCR também deve ser de pelo menos 500 pb de comprimento. Realizar a PCR com gDNA como molde e os oligonucleotídeos # 5 e # 6 (Figura 3A). </li><li> Após a purificação de todos os três PCRfragmentos, realizar a 2 ª rodada de PCR. Use uma mistura igual de todos os três fragmentos obtidos no primeiro turno como modelo. A amplificação é catalisada por oligonucleótidos # 1 e # 6. O fragmento de PCR resultante (Figura 3B), que serve como a transformação de ADN na experiência de transformação natural (Figura 1). </li></ol> 4. Quitina induzida Transformação Natural <ol><li> Crescer V. células cholerae aerobicamente em meio rico a 30 ° C até atingirem uma densidade óptica a 600 nm de aproximadamente 0,5. </li><li> Colheita das bactérias por centrifugação. Lavar pelota uma vez em meio artificial definido (DASW 6) antes de ressuspender as células em 2 volumes de DASW. Adicionar 1 ml da cultura para 50-80 mg de estéril quitina flocos (explicado no ponto 1). Se as bactérias porto plasmídeo pBR-flp (opcional ponto 2), adicionar ampicilina (50 ug / ml) à cultura. Incubar a 30 ° C durante 16-24 h sem movimento. </li><li> Adicionar &amp; ge; 200 ng do fragmento 2 ª rodada de PCR-derivada (explicado no ponto 3). Misturar cuidadosamente, sem extensivamente destacando as bactérias da superfície da quitina. Incubar a 30 ° C durante 24 horas sem movimento. </li><li> Vortex a cultura extensivamente para ≥ 30 seg. Espalhe 100-300 ul em placas LB seletivos médio (por exemplo, kanamicina ou gentamicina placas contendo LB para exemplos fornecidos aqui). Utilizar placas selectivas duas vezes se as bactérias abrigam o plasmídeo pBR-flp adicionando ampicilina concomitantemente com outros antibióticos. Incubar as placas a 30 ° C durante 16-24 horas ou até que as colónias são visíveis. </li><li> Isolar transformantes individuais de placas selectivas. Tais transformantes ter substituído o locus cromossómico original, pelo fragmento de PCR devido a um evento de crossover duplo. Estas estirpes podem ser directamente utilizados para experiências posteriores, no caso da remoção da cassete de antibiótico não é necessário. </li></ol> 5. Remoção de Cassete Seletivo (s) por FlpA recombinação <ol><li> Artificialmente transformar seus transformantes com plasmídeo pBR flp, conforme descrito no ponto 2 se não for feito antes do ensaio de transformação natural. </li><li> Crescer as bactérias em placas de agar LB contendo ampicilina a 37 ° C durante 16-24 hr. Opções: mudar a temperatura entre 2-3 horas para a 40 ° C, como expressão de flp de plasmídeo pBR-flp é de-reprimida em 5,10 altas temperaturas, bactérias transferência para placas frescas após aprox. 8 horas de incubação. </li><li> Ensaio de sensibilidade aos antibióticos (demonstrado aqui para canamicina; Figura 1) por restreaking os clones em paralelo em placas de agar contendo antibióticos e livre de antibióticos. Isolar o único colônias sensíveis. Congelar ampicilina resistente clone como estoque de glicerol se você pretende modificar ainda mais a tensão com a supressão outro / inserção usando TransFLP. </li></ol> 6. Cura plasmídeo <ol><li> Crescer a cultura durante a noite sob condições aeróbias ea 30 ° C. Use meio rico sem a adição de qualquer antibiótico. </li><li> Opcional: Crescer cultura fresco por 3-6 horas, diluindo a 1:100 cultura durante a noite em meio livre de antibióticos fresco. </li><li> Placa raia ou diluição (s) de cultura em placa de ágar LB simples (s) e incubar a 30 ° C durante 8-16 horas (até que as colónias visíveis). </li><li> Teste para ampicilina sensibilidade dos clones por restreaking os clones em paralelo em placas de ágar contendo antibiótico e antibiótico-livre (Figura 1). Congelar ampicilina cepa sensíveis como o estoque de glicerol. </li></ol>

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Não há conflitos de interesse declarados.

O método TransFLP descrito acima e noutros locais 5 tem sido extensivamente utilizada no nosso laboratório. O tipo de manipulações genéticas que são viáveis ​​incluem, entre outros: a eliminação de genes individuais e aglomerados de genes, a deleção de ilhas genómicas (por exemplo, VPI-1), a inserção de sequências a montante de um gene de interesse (por exemplo, sequências do promotor) e da inserção sequências na extremidade de um gene específico (por exemplo,</em...

<table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de catálogo </td><td> Comentários </td></tr><tr><td> Quitina flocos </td><td> Sigma </td><td> C9213 </td><td> Autoclavagem exigido </td></tr><tr><td> OPCIONAL: Micropulser (no caso cassete antibiótico deve ser extirpado por Flp recombinase codificado em pBR-flp) </td><td> Biorad </td><td> 165-2100 </td><td> Ou electroporators comparáveis </td></tr></tbody></table>

transflp &#x2014; a method to genetically modify vibrio cholerae based on natural transformation and flp-recombination

Vários métodos estão disponíveis para manipular cromossomas bacterianos 1-3. A maior parte destes protocolos contar com a inserção de plasmídeos replicativos condicionalmente (por exemplo, abrigando replicões pir-dependentes ou sensíveis à temperatura 1,2). Estes plasmídeos são integrados nos cromossomas bacterianos com base na homologia mediada por recombinação. Esses mutantes de inserção são muitas vezes usado diretamente em ambientes experimentais. Em alternativa, a selecção para a excisão do plasmídeo seguido por sua perda pode ser realizada, que para bactérias gram-negativas depende frequentemente da enzima sucrase contra-seleccionável de levana, codificada pelo gene sacB 4. A excisão pode restaurar o genótipo de pré-inserção, ou em resultado de uma troca entre o cromossoma e a cópia mediada por plasmídeos do gene modificado. Uma desvantagem desta técnica é que é demorado. O plasmídeo tem que ser clonado primeiro, que exige a transferência horizontal em V. cholerae (mais n<html> otably por acasalamento com um E. coli estirpe dadora) ou transformação artificial deste último, e da excisão do plasmídeo é aleatório e pode restaurar o genótipo inicial ou criar a modificação desejada se nenhuma selecção positiva é exercida. A seguir, apresentamos um processo para a manipulação rápida da V. cholerae cromossoma (s) 5 (Figura 1). Este método TransFLP baseia-se na descoberta recentemente indução mediada por quitina de competência natural nesse organismo representativo 6 e outros do género, tais como Vibrio V. fischeri 7. Competência natural permite a captação de ADN livre, incluindo fragmentos de PCR gerados pelo DNA. Uma vez absorvido, o ADN recombina com o cromossoma, dada a presença de um mínimo de 250-500 pb da região flanqueadora homólogo 8. Incluindo um marcador de selecção de entre estas regiões flanqueadoras permite a fácil detecção de transformantes que ocorrem com frequência. t &quot;&gt; Este método pode ser usado para diferentes manipulações genéticas de V. cholerae e potencialmente também outras bactérias naturalmente competentes. Fornecemos três exemplos novos do que pode ser conseguida por este método, além do nosso estudo publicado anteriormente em deleções de um único gene e do Além da afinidade tag-seqüências 5. passos de otimização Vários sobre o protocolo inicial de quitina induzida por transformação natural 6 são incorporados neste protocolo TransFLP. Estes incluem, entre outros, a substituição de fragmentos de conchas de caranguejos por disponível comercialmente quitina flocos 8, a doação de PCR locais derivado de ADN como a transformação de material 9, e a adição de sítios de recombinação FLP-alvo FRT () 5. DRF permitir a excisão sítio-dirigida do marcador de selecção mediada pela recombinase Flp 10.

Os resultados representativos das três exemplos são mostrados nas Figuras 4 a 6. A primeira abordagem, que visa suprimir os genes vizinhos ctxA e CtxB. Juntos, eles codificar o principal fator de virulência de V. cholerae, a toxina da cólera. Concebemos oligonucleótidos para o método TransFLP como descrito acima e de acordo com a Figura 3. A estirpe parental, a estirpe portadora do intermediário FRT-flanqueado cassete de resistência a antibióticos no ...

Watch this Scientific Journal Video about TransFLP â€” A Method to Genetically Modify Vibrio cholerae Based on Natural Transformation and FLP-recombination at JoVE.com

TransFLP &#x2014; A Method to Genetically Modify Vibrio cholerae Based on Natural Transformation and FLP-recombination

Um método rápido para modificar o genoma de<em&gt; V. cholerae</em&gt; É descrito. Essas modificações incluem a eliminação de genes individuais, grupos de genes e ilhas genômicas, bem como a integração de seqüências curtas (por exemplo, elementos promotor ou afinidade tag-seqüências). O método baseia-se na transformação de recombinação natural e FLP.

TransFLP - um método para modificar geneticamente<em&gt; Vibrio cholerae</em&gt; Com base Transformação Natural e recombinação FLP-

Several methods are available to manipulate bacterial chromosomes1-3. Most of these protocols rely on the insertion of conditionally replicative plasmids ...

transflp-method-to-genetically-modify-vibrio-cholerae-based-on

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

14.6K visualizações.  Ecole Polytechnique Fédérale de Lausanne (EPFL). Um método rápido para modificar o genoma de V. cholerae É descrito. Essas modificações incluem a eliminação de genes individuais, grupos de genes e ilhas genômicas, bem como a integração de seqüências curtas (por exemplo, elementos promotor ou afinidade tag-seqüências). O método baseia-se na transformação de recombinação natural e FLP.

Vídeo: TransFLP — A Method to Genetically Modify Vibrio cholerae Based on Natural Transformation and FLP-recombination

Conjugative Mating Assays for Sequence-specific Analysis of Transfer Proteins Involved in Bacterial Conjugation

The transfer of genetic material by bacterial conjugation is a process that takes place via complexes formed by specific transfer proteins. In Escherichia coli, these transfer proteins make up a DNA transfer machinery known as the mating pair formation, or DNA transfer complex, which facilitates conjugative plasmid transfer. The objective of this paper is to provide a method that can be used to determine the role of a specific transfer protein that is involved in conjugation using a series of deletions and/or point mutations in combination with mating assays. The target gene is knocked out on the conjugative plasmid and is then provided in trans through the use of a small recovery plasmid harboring the target gene. Mutations affecting the target gene on the recovery plasmid can reveal information about functional aspects of the target protein that result in the alteration of mating efficiency of donor cells harboring the mutated gene. Alterations in mating efficiency provide insight into the role and importance of the particular transfer protein, or a region therein, in facilitating conjugative DNA transfer. Coupling this mating assay with detailed three-dimensional structural studies will provide a comprehensive understanding of the function of the conjugative transfer protein as well as provide a means for identifying and characterizing regions of protein-protein interaction.

Here, we present a protocol to knockout a gene of interest involved in plasmid conjugation and subsequently analyze the impact of its absence using mating assays. The function of the gene is further explored to a specific region of its sequence using deletion or point mutations.

Conjugadas acasalamento Os ensaios para a análise específica da sequência de proteínas de transferência envolvidos na conjugação bacteriana

The transfer of genetic material by bacterial conjugation is a process that takes place via complexes formed by specific transfer proteins. In Escherichia ...

Genética

Site-specific Bacterial Chromosome Engineering: &#934;C31 Integrase Mediated Cassette Exchange (IMCE)

The bacterial chromosome may be used to stably maintain foreign DNA in the mega-base range1. Integration into the chromosome circumvents issues such as plasmid replication, plasmid stability, plasmid incompatibility, and plasmid copy number variance. This method uses the site-specific integrase from the Streptomyces phage (&Phi;) C312,3. The &Phi;C31 integrase catalyzes a direct recombination between two specific DNA sites: attB and attP (34 and 39 bp, respectively)4. This recombination is stable and does not revert5. A "landing pad" (LP) sequence consisting of a spectinomycin- resistance gene, aadA (SpR), and the E. coli &szlig;-glucuronidase gene (uidA) flanked by attP sites has been integrated into the chromosomes of Sinorhizobium meliloti, Ochrobactrum anthropi, and Agrobacterium tumefaciens in an intergenic region, the ampC locus, and the tetA locus, respectively. S. meliloti is used in this protocol. Mobilizable donor vectors containing attB sites flanking a stuffer red fluorescent protein (rfp) gene and an antibiotic resistance gene have also been constructed. In this example the gentamicin resistant plasmid pJH110 is used. The rfp gene6 may be replaced with a desired construct using SphI and PstI. Alternatively a synthetic construct flanked by attB sites may be sub-cloned into a mobilizable vector such as pK19mob7. The expression of the &Phi;C31 integrase gene (cloned from pHS628) is driven by the lac promoter, on a mobilizable broad host range plasmid pRK78139.
A tetraparental mating protocol is used to transfer the donor cassette into the LP strain thereby replacing the markers in the LP sequence with the donor cassette. These cells are trans-integrants. Trans-integrants are formed with a typical efficiency of 0.5%. Trans-integrants are typically found within the first 500-1,000 colonies screened by antibiotic sensitivity or blue-white screening using 5-bromo-4-chloro-3-indolyl-beta-D-glucuronic acid (X-gluc). This protocol contains the mating and selection procedures for creating and isolating trans-integrants.

A quick and efficient method to integrate foreign DNA of interest into pre-made acceptor strains, termed landing pad strains, is described. The method allows site-specific integration of a DNA cassette into the engineered landing pad locus of a given strain, through conjugation and expression of the &#934;C31 integrase.

Site-specific Engenharia cromossomo bacteriano: ΦC31 Integrase Mediated Cassette Exchange (IMCE)

The bacterial chromosome may be used to stably maintain foreign DNA in the mega-base range1. Integration into the chromosome circumvents issues such as ...

Bioengenharia

A Fluorescence-based Method to Study Bacterial Gene Regulation in Infected Tissues

Bacterial virulence genes are often regulated at the transcriptional level by multiple factors that respond to different environmental signals. Some factors act directly on virulence genes; others control pathogenesis by adjusting the expression of downstream regulators or the accumulation of signals that affect regulator activity. While regulation has been studied extensively during in vitro growth, relatively little is known about how gene expression is adjusted during infection. Such information is important when a particular gene product is a candidate for therapeutic intervention. Transcriptional approaches like quantitative, real-time RT-PCR and RNA-Seq are powerful ways to examine gene expression on a global level but suffer from many technical challenges including low abundance of bacterial RNA compared to host RNA, and sample degradation by RNases. Evaluating regulation using fluorescent reporters is relatively easy and can be multiplexed with fluorescent proteins with unique spectral properties. The method allows for single-cell, spatiotemporal analysis of gene expression in tissues that exhibit complex three-dimensional architecture and physiochemical gradients that affect bacterial regulatory networks. Such information is lost when data are averaged over the bulk population. Herein, we describe a method for quantifying gene expression in bacterial pathogens in situ. The method is based on simple tissue processing and direct observation of fluorescence from reporter proteins. We demonstrate the utility of this system by examining the expression of Staphylococcus aureus thermonuclease (nuc), whose gene product is required for immune evasion and full virulence ex vivo and in vivo. We show that nuc-gfp is strongly expressed in renal abscesses and reveal heterogeneous gene expression due in part to apparent spatial regulation of nuc promoter activity in abscesses fully engaged with the immune response. The method can be applied to any bacterium with a manipulatable genetic system and any infection model, providing valuable information for preclinical studies and drug development.

Described here is a method for analyzing bacterial gene expression in animal tissues at a cellular level. This method provides a resource for studying the phenotypic diversity occurring within a bacterial population in response to the tissue environment during an infection.

Um método baseado em fluorescência para estudar o Regulamento de Gene bacteriano em tecidos infectados

Bacterial virulence genes are often regulated at the transcriptional level by multiple factors that respond to different environmental signals. Some ...

Methodology for the Efficient Generation of Fluorescently Tagged Vaccinia Virus Proteins

Tagging of viral proteins with fluorescent proteins has proven an indispensable approach to furthering our understanding of virus-host interactions. Vaccinia virus (VACV), the live vaccine used in the eradication of smallpox, is particularly amenable to fluorescent live-cell microscopy owing to its large virion size and the ease with which it can be engineered at the genome level. We report here an optimized protocol for generating recombinant viruses. The minimal requirements for targeted homologous recombination during vaccinia replication were determined, which allows the simplification of construct generation. This enabled the alliance of transient dominant selection (TDS) with a fluorescent reporter and metabolic selection to provide a rapid and modular approach to fluorescently label viral proteins. By streamlining the generation of fluorescent recombinant viruses, we are able to facilitate downstream applications such as advanced imaging analysis of many aspects of the virus-host interplay that occurs during virus replication.

A rapid and modular protocol for the generation of recombinant vaccinia viruses expressing fluorescently&#160;tagged proteins simultaneously using the method of transient dominant selection is described here.

Metodologia para a Geração Eficiente de Proteínas do Vírus da Vaccinia marcadas fluorescentes

Tagging of viral proteins with fluorescent  proteins has proven an indispensable approach to furthering our understanding  of virus-host interactions. ...

Imunologia e Infecção

Engineering 'Golden' Fluorescence by Selective Pressure Incorporation of Non-canonical Amino Acids and Protein Analysis by Mass Spectrometry and Fluorescence

Fluorescent proteins are fundamental tools for the life sciences, in particular for fluorescence microscopy of living cells. While wild-type and engineered variants of the green fluorescent protein from Aequorea victoria (avGFP) as well as homologs from other species already cover large parts of the optical spectrum, a spectral gap remains in the near-infrared region, for which avGFP-based fluorophores are not available. Red-shifted fluorescent protein (FP) variants would substantially expand the toolkit for spectral unmixing of multiple molecular species, but the naturally occurring red-shifted FPs derived from corals or sea anemones have lower fluorescence quantum yield and inferior photo-stability compared to the avGFP variants. Further manipulation and possible expansion of the chromophore&#39;s conjugated system towards the far-red spectral region is also limited by the repertoire of 20 canonical amino acids prescribed by the genetic code. To overcome these limitations, synthetic biology can achieve further spectral red-shifting via insertion of non-canonical amino acids into the chromophore triad. We describe the application of SPI to engineer avGFP variants with novel spectral properties. Protein expression is performed in a tryptophan-auxotrophic E. coli strain and by supplementing growth media with suitable indole precursors. Inside the cells, these precursors are converted to the corresponding tryptophan analogs and incorporated into proteins by the ribosomal machinery in response to UGG codons. The replacement of Trp-66 in the enhanced &#34;cyan&#34; variant of avGFP (ECFP) by an electron-donating 4-aminotryptophan results in GdFP featuring a 108 nm Stokes shift and a strongly red-shifted emission maximum (574 nm), while being thermodynamically more stable than its predecessor ECFP. Residue-specific incorporation of the non-canonical amino acid is analyzed by mass spectrometry. The spectroscopic properties of GdFP are characterized by time-resolved fluorescence spectroscopy as one of the valuable applications of genetically encoded FPs in life sciences.

Synthetic biology enables the engineering of proteins with unprecedented properties using the co-translational insertion of non-canonical amino acids. Here, we presented how a spectrally red-shifted variant of a GFP-type fluorophore with novel fluorescence spectroscopic properties, termed &#34;gold&#34; fluorescent protein (GdFP), is produced in E. coli via selective pressure incorporation (SPI).

Engenharia 'Dourada' fluorescência por pressão seletiva de incorporação de aminoácidos não-canônicos e análise de proteínas por espectrometria de massa e fluorescência

Fluorescent proteins are fundamental tools for the life sciences, in particular for fluorescence microscopy of living cells. While wild-type and ...

Laboratory Techniques Used to Maintain and Differentiate Biotypes of Vibrio cholerae Clinical and Environmental Isolates

The aquatic Gram-negative bacterium Vibrio cholerae is the etiological agent of the infectious gastrointestinal disease cholera. Due to the global prevalence and severity of this disease, V. cholerae has been extensively studied in both environmental and laboratory settings, requiring proper maintenance and culturing techniques. Classical and El Tor are two main biotypes that compose the V. cholerae O1 serogroup, each displaying unique genotypic and phenotypic characteristics that provide reliable mechanisms for biotype characterization, and require distinct virulence inducing culturing conditions. Regardless of the biotype of the causative strain for any given infection or outbreak, the standard treatment for the disease involves rehydration therapy supplemented with a regimen of antibiotics. However, biotype classification may be necessary for laboratory studies and may have broader impacts in the biomedical field. In the early 2000&#39;s clinical isolates were identified which exhibit genotypic and phenotypic traits from both classical and El Tor biotypes. The newly identified hybrids, termed El Tor variants, have caused clinical and environmental isolate biotype identification to become more complex than previous traditional single assay identification protocols. In addition to describing V. cholerae general maintenance and culturing techniques, this manuscript describes a series of gene specific (ctxB and tcpA) PCR-based genetic screens and phenotypic assays (polymyxin B resistance, citrate metabolism, proteolytic activity, hemolytic activity, motility, and glucose metabolism via Voges-Proskauer assay) collectively used to characterize and/or distinguish between classical and El Tor biotypes. Together, these assays provide an efficient systematic approach to be used as an alternative, or in addition, to costly, labor-intensive experiments in the characterization of V. cholerae clinical (and environmental) isolates.

Laboratory Techniques Used to Maintain and Differentiate Biotypes of Vibrio cholerae Clinical and Environmental Isolates

This manuscript describes proper Vibrio cholerae maintenance techniques in addition to a series of biochemical assays, collectively utilized for quick and reliable differentiation between clinical and environmental V. cholerae biotypes in a laboratory setting.

Técnicas de laboratório usadas para manter e diferenciar biótipos de<em&gt; Vibrio cholerae</em&gt; Isolados clínicos e ambientais

The aquatic Gram-negative bacterium Vibrio cholerae is the etiological agent of the infectious gastrointestinal disease cholera. Due to the global ...

Coincubation Assay for Quantifying Competitive Interactions between Vibrio fischeri Isolates

This manuscript describes a culture-based, coincubation assay for detecting and characterizing competitive interactions between two bacterial populations. This method employs stable plasmids that allow each population to be differentially tagged with distinct antibiotic resistance capabilities and fluorescent proteins for selection and visual discrimination of each population, respectively. Here, we describe the preparation and coincubation of competing Vibrio fischeri strains, fluorescence microscopy imaging, and quantitative data analysis. This approach is simple, yields quick results, and can be used to determine whether one population kills or inhibits the growth of another population, and whether competition is mediated through a diffusible molecule or requires direct cell-cell contact. Because each bacterial population expresses a different fluorescent protein, the assay permits the spatial discrimination of competing populations within a mixed colony. Although the described methods are performed with the symbiotic bacterium V. fischeri using conditions optimized for this species, the protocol can be adapted for most culturable bacterial isolates.

Coincubation Assay for Quantifying Competitive Interactions between Vibrio fischeri Isolates

Bacteria encode diverse mechanisms for engaging in interbacterial competition. Here, we present a culture-based protocol for characterizing competitive interactions between bacterial isolates and how they impact the spatial structure of a mixed population.

Ensaio de coincubação para quantificar interações competitivas entre isolados de Vibrio fischeri

This manuscript describes a culture-based, coincubation assay for detecting and characterizing competitive interactions between two bacterial populations. ...

Rapid Protocol for Preparation of Electrocompetent Escherichia coli and Vibrio cholerae

Electroporation has become a widely used method for rapidly and efficiently introducing foreign DNA into a wide range of cells. Electrotransformation has become the method of choice for introducing DNA into prokaryotes that are not naturally competent. Electroporation is a rapid, efficient, and streamlined transformation method that, in addition to purified DNA and competent bacteria, requires commercially available gene pulse controller and cuvettes. In contrast to the pulsing step, preparation of electrocompetent cells is time consuming and labor intensive involving repeated rounds of centrifugation and washes in decreasing volumes of sterile, cold water, or non-ionic buffers of large volumes of cultures grown to mid-logarithmic phase of growth. Time and effort can be saved by purchasing electrocompetent cells from commercial sources, but the selection is limited to commonly employed E. coli laboratory strains. We are hereby disseminating a rapid and efficient method for preparing electrocompetent E. coli, which has been in use by bacteriology laboratories for some time, can be adapted to V. cholerae and other prokaryotes. While we cannot ascertain whom to credit for developing the original technique, we are hereby making it available to the scientific community.

Rapid Protocol for Preparation of Electrocompetent Escherichia coli and Vibrio cholerae

Electroporation is a commonly employed method for introducing DNA into bacteria in a process known as transformation. Traditional protocols for the preparation of electrocompetent cells are time consuming and labor intensive. This article describes an alternate, rapid, and efficient method for the preparation of electrocompetent cells presently employed by some laboratories.

Protocolo Rápido para Preparação de electrocompetentes<em&gt; Escherichia coli</em&gt; E<em&gt; Vibrio cholerae</em

Electroporation has become a widely used method for rapidly and efficiently introducing foreign DNA into a wide range of cells. Electrotransformation has ...

Biologia

Integração gênica estável em Chlorella vulgaris usando Agrobacterium tumefaciens

Agrobacterium tumefaciens-Mediated Genetic Engineering of Green Microalgae, Chlorella vulgaris

Agrobacterium tumefaciens-mediated transformation (AMT) serves as a widely employed tool for manipulating plant genomes. However, A. tumefaciens exhibit the capacity for gene transfer to a diverse array of species. Numerous microalgae species lack well-established methods for reliably integrating genes of interest into their nuclear genome. To harness the potential benefits of microalgal biotechnology, simple and efficient genome manipulation tools are crucial. Herein, an optimized AMT protocol is presented for the industrial microalgae species Chlorella vulgaris, utilizing the reporter green fluorescent protein (mGFP5) and the antibiotic resistance marker for Hygromycin B. Mutants are selected through plating on Tris-Acetate-Phosphate (TAP) media containing Hygromycin B and cefotaxime. Expression of mGFP5 is quantified via fluorescence after over ten generations of subculturing, indicating the stable transformation of the T-DNA cassette. This protocol allows for the reliable generation of multiple transgenic C. vulgaris colonies in under two weeks, employing the commercially available pCAMBIA1302 plant expression vector.

Autor em destaque: Protocolo de transformação otimizado para Chlorella vulgaris usando Agrobacterium tumefaciens 

This protocol outlines the utilization of Agrobacterium tumefaciens-mediated transformation (AMT) for integrating gene(s) of interest into the nuclear genome of the green microalgae Chlorella vulgaris, leading to the production of stable transformants.

Engenharia Genética Mediada por Agrobacterium tumefaciens de Microalgas Verdes, Chlorella vulgaris

Agrobacterium tumefaciens-mediated transformation (AMT) serves as a widely employed tool for manipulating plant genomes. However, A. tumefaciens exhibit ...

TransFLP - um método para modificar geneticamente

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Um método baseado em fluorescência para estudar o Regulamento de Gene bacteriano em tecidos infectados

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Protocolo Rápido para Preparação de electrocompetentes

Engenharia Genética Mediada por Agrobacterium tumefaciens de Microalgas Verdes, Chlorella vulgaris

Engenharia Genética Mediada por Agrobacterium tumefaciens de Microalgas Verdes, Chlorella vulgaris