Agradecemos ao Dr. Dr. Michael Cashel (National Institutes of Health, Bethesda, MA, EUA) por enviar cepas de bactérias, plasmídeos, e protocolos necessários. Agradecemos ao Dr. John Glover (Department of Biochemistry, University of Toronto) para uso do leitor de placas SpecraMax. Reino Unido é o destinatário de uma National Ciências e Pesquisa de Engenharia Council of Canada (NSERC) Bolsa de Pós-Graduação, a Canadian Institutes of Health Programa de Formação de Investigação Estratégica na Biologia Estrutural de Proteínas de Membrana ligados à doença, e da Universidade de Toronto Fellowship Open. Este trabalho foi financiado por uma doação do Canadian Institutes of Health Research (MOP-67210) para WAH.

1) Reagentes e Equipamentos <ol><li> Primeiro, prepare as três seguintes soluções: 1 mL de uma solução que é composta de 8 mM L-lisina, 100 mM de sódio 2 - (N-Morfolino) ethanesulphonic ácido (MES) pH 6,5, 0,2 mM de nucleotídeos, onde o nucleotídeo é ou: difosfato de guanosina (GDP), o trifosfato de guanosina (GTP), guanosina 5&#39;-difosfato, difosfato-3&#39;(ppGpp), ou guanosina 5&#39;-trifosfato, difosfato-3&#39;(pppGpp), 0,1 mM piridoxal-5&#39;-fosfato (PLP), e 1 mM β-mercaptoetanol / (β-ME). 1 mL da solução B, que consiste em sódio 100 mM MES pH 6,5, 0,1 mM PLP, 1 mM β-ME, e 50 nM LdcI. LdcI foi purificada como descrito no Snider et al. 6 e Kanjee et al. 14. 1 mL da solução C que é idêntico a Solução B, mas não contém LdcI. </li><li> Prepare 100 mL da solução de parada consistindo de um carbonato de sódio M (10,6 g/100 mL) e alíquota de 50 mL em cada poço de uma placa de poliestireno de 96 poços, utilizando uma pipeta multi-canal. Adicionar 30 mL de água para a placa e em seguida, cobrir. Note que a soma do volume de água adicionada e do volume de amostra extraída durante a reação enzimática deve ser igual a 50 mL. Neste caso, 20 mL de amostra reação enzimática será removido. </li><li> Prepare 5 mL de solução TNBS em 10 mM diluindo 294 mL de 5% (w / v) ações TNBS com 4706 mL de água, embrulhe em papel alumínio e mantenha no gelo. </li><li> Equilibrar a ThermoStat Eppendorf Além disso, a 37 ° C. O Plus ThermoStat tem 24 poços em seis colunas. Veja a Figura 2 para esquemática. Rótulo 1,5 mL tubos Eppendorf indicando a identidade do nucleotídeo, que será testado (um por coluna): GTP (coluna A), o PIB (coluna B), ppGpp (coluna C), pppGpp (coluna D), não nucleotídeo (coluna E ), e amostras de proteínas (coluna F). </li><li> Caixas de rack de várias dicas de 200 mL de tal forma que cada linha alternativa é omitido dando um total de quatro linhas de dicas. Isso é necessário para o uso da pipeta multi-canal com o termostato heatblock Além disso, durante o ensaio enzimático. </li><li> Equilibrar Digital Heatblock (VWR) a 42 ° C. </li><li> Coloque um de 96 poços da placa de polipropileno 2,0 mL sobre o gelo para esfriar. </li></ol> 2) Ensaio LdcI <ol><li> Alíquota de 50 mL de uma solução com o nucleotídeo apropriada para tubos de 1,5 mL Eppendorf em cada uma das cinco colunas do termostato Eppendorf. </li><li> Adicionar 330 mL da solução B para três tubos na coluna F e adicione 330 ml de solução C (o controle não-proteína) ao tubo finais na coluna F. </li><li> Equilibrar as soluções a 37 ° C por cinco minutos. Dica: depois de 5 minutos, corte as tampas dos tubos no centro do bloco de aquecimento para impedi-los de interferir com a pipeta multicanal para ser utilizado próximo. </li><li> Usando um 50-50 mL VWR multi-canal de pipeta, 50 mL de transferência dos tubos na coluna F para cada um dos tubos em colunas AE. Iniciar o cronômetro assim que o primeiro tubo é misturado. </li><li> Aos 2, 4 e 6 minutos retirar 20 mL de amostra usando a pipeta multi-canal e adicionar à solução de parada. </li><li> Nota: as amostras em solução de parada pode ser percorrida em filme plástico e congeladas a -20 ° C para posterior processamento. As placas devem ser descongeladas em temperatura ambiente antes da reação com TNBS. </li></ol> 3) Reação TNBS e Desenvolvimento Cor <ol><li> Adicionar 50 mL de solução de 10 mM TNBS para a solução parar de usar a pipeta multi-canal e, em seguida, incubar a 42 ° C por 6 minutos. A solução vai virar uma cor amarelo escuro / laranja como o TNBS reage com a lisina ea cadaverina. </li><li> Após 6 minutos, arrefecer a placa de gelo para diminuir a reação. </li><li> Remover 100 mL de amostra com uma pipeta VWR 2-20 multi-canal L e lugar na 2 mL prato fundo bem que foi resfriado em gelo. Transferência balde de gelo para o fumehood. </li><li> Adicionar 500 mL de tolueno em cada cavidade utilizando um HandyStep (Marca) pipetador de repetição e 12,5 mL pipeta ponta (PLASTIBRAND). </li><li> Limpe qualquer excesso de tolueno usando um kimwipe e cobrir o prato com tiras de fita de embalagem. Certifique-se de pressionar firmemente para obter uma boa vedação. Cobrir a placa de 96 poços com uma tampa plana e, em seguida, agitar vigorosamente por 1 minuto e 30 segundos. </li><li> Deixe as soluções para resolver durante 5 minutos. O TNP-cadaverina está agora na fase superior tolueno, enquanto que o TNP-lisina permanece solúvel em água (ver Figura 1). </li><li> Remover 200 mL de tolueno usando a 20-200 pipeta multi-canal mL em uma placa de 96 poços para leitura. Nota: certifique-se de verificar que cada amostra está sendo removido é clara e não tem qualquer fundo da fase aquosa. Nota: Dicas de uso de barreira para evitar danos ao multi-canal de pipeta pelo tolueno. </li><li> Ler a absorvância a 340 nm em um leitor de placas SpectraMax 340. </li><li> Para limpar a placa de quartzo, lavar o tolueno com água e colocar a placa de quartzo em um grandebandeja de vidro na capela. Despeje sobre 100 mL de uma mistura 07:03 de 70% (v / v) de ácido nítrico: 95% (v / v) etanol e cobrir a placa de quartzo com uma bandeja de vidro segundo. Atenção: a reação é altamente exotérmica proteção contra o desgaste e às vezes explosiva apropriadas e assegurar que a folha capela está definido para o menor nível 6 &quot;Após arrefecimento, lavar o ácido nítrico com água e depois 95% de etanol.. </li></ol> 4) Resultados Representante 1) cadaverina Curva padrão (Figura 3) O ensaio foi realizado como descrito, mas sem qualquer L-lisina ou LdcI e concentrações em vez de vários cadaverina foram usados ​​para determinar empiricamente a faixa linear do ensaio. O ensaio foi linear para um OD 340 de 0,25, correspondendo a ~ 22 nmoles de cadaverina (Figura 3). Atividade 2) de LdcI (Figura 4) A atividade de LdcI sozinho estava determinado a ser 153,5 (± 18,1) nmoles cadaverina min-1 mg LdcI-1 em pH 6,5. A atividade de LdcI não é afetada na presença de 100 mM GTP ou GDP, mas é fortemente inibida (&gt; 10 vezes) na presença de pppGpp e ppGpp (Figura 4). <img src="/files/ftp_upload/2094/2094fig1.jpg" alt="Figura 1" /> Figura 1. Esquema da reação LdcI. A reação de descarboxilação de L-lisina para gerar CO 2 e cadaverina é mostrado, assim como a reação subseqüente com TNBS em pH elevado para gerar N, N&#39;-bistrinitrophenylcadaverine (TNP-cadaverina) e N, N &#39;-bistrinitrophenyllysine (TNP-lisina). Baseado em Phan et al.11 <img src="/files/ftp_upload/2094/2094fig2.jpg" alt="Figura 2" /> Figura 2. Configuração do termostato Eppendorf. O layout das amostras no ThermoStat Eppendorf de 24 poços é mostrada junto com a descrição das etapas do ensaio (indicado em negrito). As setas indicam a transferência de soluções de reação de acordo com o protocolo. Remoção da solução de reação à solução de parada na placa de 96 poços também é indicado. <img src="/files/ftp_upload/2094/2094fig3.jpg" alt="Figura 3" /> Figura 3. Cadaverina curva padrão. Um gráfico de absorvância a 340 nm contra nmoles de cadaverina é mostrado. Barras de erro representam o desvio padrão de pelo menos três medições independentes. A linha de melhor ajuste também é mostrada e tem um R2 de 0,996. <img src="/files/ftp_upload/2094/2094fig4.jpg" alt="Figura 4" /> Figura 4. Os resultados do ensaio LdcI. Um gráfico da taxa de atividade LdcI (em nmoles cadaverina produzido min-1 g-1 LdcI) é mostrada na presença de 100 mM GTP, GDP, pppGpp, ppGpp e na ausência de nucleotídeos. Os nucleotídeos resposta rigorosa (p) ppGpp são capazes de inibir significativamente a atividade LdcI. As barras de erro representam o desvio padrão de pelo menos seis medições independentes.

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	<li>Gale, E. F., Epps, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+the+pH+of+the+medium+during+growth+on+the+enzymic+activities+of+bacteria+(Escherichia+coli+and+Micrococcus+lysodeikticus)+and+the+biological+significance+of+the+changes+produced.">The effect of the pH of the medium during growth on the enzymic activities of bacteria (Escherichia coli and Micrococcus lysodeikticus) and the biological significance of the changes produced.</a> Biochem J. 36, 600-618 (1942).</li><li>Foster, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Escherichia+coli+acid+resistance:+tales+of+an+amateur+acidophile.">Escherichia coli acid resistance: tales of an amateur acidophile.</a> Nat Rev Microbiol. 2, 898-907 (2004).</li><li>Castanie-Cornet, M. P., Penfound, T. A., Smith, D., Elliott, J. F., Foster, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+acid+resistance+in+Escherichia+coli.">Control of acid resistance in Escherichia coli.</a> J Bacteriol. 181, 3525-3535 (1999).</li><li>Iyer, R., Williams, C., Miller, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arginine-agmatine+antiporter+in+extreme+acid+resistance+in+Escherichia+coli.">Arginine-agmatine antiporter in extreme acid resistance in Escherichia coli.</a> J Bacteriol. 185, 6556-6561 (2003).</li><li>Sabo, D. L., Boeker, E. A., Byers, B., Waron, H., Fischer, E. 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No ensaio descarboxilase lisina, TNBS é reagido com as aminas primárias de L-lisina e cadaverina para formar TNP-lisina e cadaverina TNP-adutos (Figura 1). Devido à presença do grupo de ácido carboxílico em TNP-lisina, este permanece aduto solúvel em água, enquanto o TNP-cadaverina, faltando o grupo ácido carboxílico, é capaz de particionamento em tolueno 11. Este tipo de ensaio pode ser utilizado de forma mais ampla em outros tipos de aminoácidos, onde a perda de um grupo de ácido carboxílico o...

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		<th>Material Name</th>
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		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>2,4,6-trinitrobenzensulfonic acid </td>
<td>&nbsp;</td>
<td>Sigma Aldrich</td>
<td>P2297</td>
<td>&nbsp;</td>
<tr>
<td>0.1 mM pyridoxal 5'-phosphate (PLP)</td>
<td>&nbsp;</td>
<td>Sigma Aldrich</td>
<td>P9255</td>
<td>&nbsp;</td>
<tr>
<td>Cadaverine</td>
<td>&nbsp;</td>
<td>Sigma Aldrich</td>
<td>D22606</td>
<td>&nbsp;</td>
<tr>
<td>96 well polystyrene plates</td>
<td>&nbsp;</td>
<td>Sarstedt</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>96-well quartz plate</td>
<td>&nbsp;</td>
<td>Hellma</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>VWR Digital Heatblock</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>ThermoStat Plus</td>
<td>&nbsp;</td>
<td>Eppendorf</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>2.0 mL 96-well polypropylene plates</td>
<td>&nbsp;</td>
<td>Axygen</td>
<td>P-DW-20-C</td>
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<td>Handy Step Repeat Pipettor</td>
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<td>Brand</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>12.5 mL Repeat Pipettor Tips</td>
<td>&nbsp;</td>
<td>Brand (Plastibrand)</td>
<td>702378</td>
<td>&nbsp;</td>
<tr>
<td>SpectraMax 340PC Plate Reader</td>
<td>&nbsp;</td>
<td>SpectraMax</td>
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an assay for measuring the activity of escherichia coli inducible lysine decarboxyase

A Escherichia coli é uma bactéria entérica que é capaz de crescer em uma ampla faixa de valores de pH (pH 5-9) 1 e, incrivelmente, é capaz de sobreviver ácido extremo frisa, incluindo a passagem pelo estômago de mamíferos, onde o pH pode cair para valores tão baixos como pH 1 - 2 2. Para habilitar a um leque tão amplo de sobrevivência pH ácido, E. coli possui quatro diferentes induzível decarboxylases aminoácido que decarboxylate ácidos seu substrato aminoácidos de uma maneira próton-dependentes elevando o pH interno. O decarboxylases incluem o ácido glutâmico decarboxylases Gada e GadB 3, a arginina descarboxilase Adia 4, a lisina descarboxilase LdcI 5, 6 e ornitina decarboxilase SpeF 7. Todas essas enzimas utilizam piridoxal-5&#39;-fosfato como um co-fator 8 e funcionam em conjunto com a membrana interna-substrato-produto antiporters que remover produtos descarboxilação ao meio externo, em troca de substrato fresco 2. No caso de LdcI, o antiporter lisina cadaverina é chamado CADB. Recentemente, nós determinamos a estrutura de cristal de raios-X de LdcI a 2,0 Å, e nós descobrimos um romance de pequenas moléculas obrigado a LdcI o regulador de guanosina 5&#39;-difosfato rigorosos resposta, difosfato-3&#39;(ppGpp) 14. A resposta severas ocorre quando as células em crescimento exponencial experiência de privação de nutrientes ou uma de uma série de outros estresses 9. Como resultado, as células produzem ppGpp que leva a uma cascata de sinalização que culminou com a mudança de crescimento exponencial para o crescimento da fase estacionária 10. Nós demonstramos que ppGpp é um inibidor específico da LdcI 14. Aqui nós descrevemos o ensaio descarboxilase lisina, modificado a partir do ensaio desenvolvido por Phan et al. 11, que temos usado para determinar a atividade de LdcI eo efeito de pppGpp / ppGpp essa actividade. A reação de descarboxilação LdcI remove o grupo α-carboxila de L-lisina e produz dióxido de carbono ea cadaverina poliamina (1,5-diaminopentane) 5. L-lisina e cadaverina pode ser reagido com ácido 2,4,6-trinitrobenzensulfonic (TNBS) em pH elevado para gerar N, N&#39;-bistrinitrophenylcadaverine (TNP-cadaverina) e N, N&#39;-bistrinitrophenyllysine (TNP-lisina), respectivamente 11. O TNP-cadaverina podem ser separados do TNP-lisina como o primeiro é solúvel em solventes orgânicos como o tolueno, enquanto o último não é (ver Figura 1). A faixa linear do ensaio foi determinada empiricamente usando purificada cadaverina.

Watch this Scientific Journal Video about An Assay for Measuring the Activity of Escherichia coli Inducible Lysine Decarboxyase at JoVE.com

An Assay for Measuring the Activity of Escherichia coli Inducible Lysine Decarboxyase

A atividade da descarboxilase induzível lisina é monitorado pela reação do substrato L-lisina e cadaverina o produto com 2,4,6 trinitrobenzensulfonic-ácido para formar adutos que têm solubilidade diferencial em tolueno.

Um ensaio para medir a atividade do Escherichia coli Induzível Lisina Decarboxyase

Escherichia coli is an enteric bacterium that is capable of growing over a wide range of pH values (pH 5 - 9)1 and, incredibly, is able to survive extreme ...

an-assay-for-measuring-activity-escherichia-coli-inducible-lysine

Research

JoVE Journal

Biology

An Assay for Measuring the Activity of Escherichia coli Inducible Lysine Decarboxyase

14.1K visualizações.  University of Toronto. A atividade da descarboxilase induzível lisina é monitorado pela reação do substrato L-lisina e cadaverina o produto com 2,4,6 trinitrobenzensulfonic-ácido para formar adutos que têm solubilidade diferencial em tolueno.

Vídeo: An Assay for Measuring the Activity of Escherichia coli Inducible Lysine Decarboxyase

Measuring the Bending Stiffness of Bacterial Cells Using an Optical Trap

We developed a protocol to measure the bending rigidity of filamentous rod-shaped bacteria. Forces are applied with an optical trap, a microscopic three-dimensional spring made of light that is formed when a high-intensity laser beam is focused to a very small spot by a microscope's objective lens. To bend a cell, we first bind live bacteria to a chemically-treated coverslip. As these cells grow, the middle of the cells remains bound to the coverslip but the growing ends are free of this restraint. By inducing filamentous growth with the drug cephalexin, we are able to identify cells in which one end of the cell was stuck to the surface while the other end remained unattached and susceptible to bending forces. A bending force is then applied with an optical trap by binding a polylysine-coated bead to the tip of a growing cell. Both the force and the displacement of the bead are recorded and the bending stiffness of the cell is the slope of this relationship.

We present a protocol for bending filamentous bacterial cells attached to a cover-slip surface with an optical trap to measure the cellular bending stiffness.

Medir a rigidez à flexão de células bacterianas Usando uma armadilha óptica

We developed a protocol to measure the bending rigidity of filamentous rod-shaped bacteria. Forces are applied with an optical trap, a microscopic ...

Biologia

High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli

Cellulase enzymes (endoglucanases, cellobiohydrolases, and &beta;-glucosidases) hydrolyze cellulose into component sugars, which in turn can be converted into fuel alcohols1. The potential for enzymatic hydrolysis of cellulosic biomass to provide renewable energy has intensified efforts to engineer cellulases for economical fuel production2. Of particular interest are fungal cellulases3-8, which are already being used industrially for foods and textiles processing.
Identifying active variants among a library of mutant cellulases is critical to the engineering process; active mutants can be further tested for improved properties and/or subjected to additional mutagenesis. Efficient engineering of fungal cellulases has been hampered by a lack of genetic tools for native organisms and by difficulties in expressing the enzymes in heterologous hosts. Recently, Morikawa and coworkers developed a method for expressing in E. coli the catalytic domains of endoglucanases from H. jecorina3,9, an important industrial fungus with the capacity to secrete cellulases in large quantities. Functional E. coli expression has also been reported for cellulases from other fungi, including Macrophomina phaseolina10 and Phanerochaete chrysosporium11-12. 
We present a method for high throughput screening of fungal endoglucanase activity in E. coli. (Fig 1) This method uses the common microbial dye Congo Red (CR) to visualize enzymatic degradation of carboxymethyl cellulose (CMC) by cells growing on solid medium. The activity assay requires inexpensive reagents, minimal manipulation, and gives unambiguous results as zones of degradation (&#x201C;halos&#x201D;) at the colony site. Although a quantitative measure of enzymatic activity cannot be determined by this method, we have found that halo size correlates with total enzymatic activity in the cell. Further characterization of individual positive clones will determine , relative protein fitness. 
Traditional bacterial whole cell CMC/CR activity assays13 involve pouring agar containing CMC onto colonies, which is subject to cross-contamination, or incubating cultures in CMC agar wells, which is less amenable to large-scale experimentation. Here we report an improved protocol that modifies existing wash methods14 for cellulase activity: cells grown on CMC agar plates are removed prior to CR staining. Our protocol significantly reduces cross-contamination and is highly scalable, allowing the rapid screening of thousands of clones. In addition to H. jecorina enzymes, we have expressed and screened endoglucanase variants from the Thermoascus aurantiacus and Penicillium decumbens (shown in Figure 2), suggesting that this protocol is applicable to enzymes from a range of organisms.

High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli

We describe a low cost, high throughput method to screen for fungal endoglucanase activity in E. coli. The method relies on a simple visual readout of substrate degradation, does not require enzyme purification, and is highly scalable. This allows for the rapid screening of large libraries of enzyme variants.

High Throughput Screening da atividade endoglucanase de fungos em Escherichia coli</em

Cellulase enzymes (endoglucanases, cellobiohydrolases, and &beta;-glucosidases) hydrolyze cellulose into component sugars, which in turn can be converted ...

Application of MassSQUIRM for Quantitative Measurements of Lysine Demethylase Activity

Recently, epigenetic regulators have been discovered as key players in many different diseases 1-3. As a result, these enzymes are prime targets for small molecule studies and drug development 4. Many epigenetic regulators have only recently been discovered and are still in the process of being classified. Among these enzymes are lysine demethylases which remove methyl groups from lysines on histones and other proteins. Due to the novel nature of this class of enzymes, few assays have been developed to study their activity. This has been a road block to both the classification and high throughput study of histone demethylases. Currently, very few demethylase assays exist. Those that do exist tend to be qualitative in nature and cannot simultaneously discern between the different lysine methylation states (un-, mono-, di- and tri-). Mass spectrometry is commonly used to determine demethylase activity but current mass spectrometric assays do not address whether differentially methylated peptides ionize differently. Differential ionization of methylated peptides makes comparing methylation states difficult and certainly not quantitative (Figure 1A). Thus available assays are not optimized for the comprehensive analysis of demethylase activity.
Here we describe a method called MassSQUIRM (mass spectrometric quantitation using isotopic reductive methylation) that is based on reductive methylation of amine groups with deuterated formaldehyde to force all lysines to be di-methylated, thus making them essentially the same chemical species and therefore ionize the same (Figure 1B). The only chemical difference following the reductive methylation is hydrogen and deuterium, which does not affect MALDI ionization efficiencies. The MassSQUIRM assay is specific for demethylase reaction products with un-, mono- or di-methylated lysines. The assay is also applicable to lysine methyltransferases giving the same reaction products. Here, we use a combination of reductive methylation chemistry and MALDI mass spectrometry to measure the activity of LSD1, a lysine demethylase capable of removing di- and mono-methyl groups, on a synthetic peptide substrate 5. This assay is simple and easily amenable to any lab with access to a MALDI mass spectrometer in lab or through a proteomics facility. The assay has ~8-fold dynamic range and is readily scalable to plate format 5.

We present a method for using MALDI mass spectrometry and reductive methylation chemistry to quantify changes in lysine methylation.

Aplicação de Medidas para MassSQUIRM quantitativa da actividade demetilase Lisina

Recently, epigenetic regulators have been discovered as key players in many different diseases 1-3. As a result, these enzymes are prime targets for small ...

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 ...

Protocols for Implementing an Escherichia coli Based TX-TL Cell-Free Expression System for Synthetic Biology

Ideal cell-free expression systems can theoretically emulate an in vivo cellular environment in a controlled in vitro platform.1 This is useful for expressing proteins and genetic circuits in a controlled manner as well as for providing a prototyping environment for synthetic biology.2,3 To achieve the latter goal, cell-free expression systems that preserve endogenous Escherichia coli transcription-translation mechanisms are able to more accurately reflect in vivo cellular dynamics than those based on T7 RNA polymerase transcription. We describe the preparation and execution of an efficient endogenous E. coli based transcription-translation (TX-TL) cell-free expression system that can produce equivalent amounts of protein as T7-based systems at a 98% cost reduction to similar commercial systems.4,5 The preparation of buffers and crude cell extract are described, as well as the execution of a three tube TX-TL reaction. The entire protocol takes five days to prepare and yields enough material for up to 3000 single reactions in one preparation. Once prepared, each reaction takes under 8 hr from setup to data collection and analysis. Mechanisms of regulation and transcription exogenous to E. coli, such as lac/tet repressors and T7 RNA polymerase, can be supplemented.6 Endogenous properties, such as mRNA and DNA degradation rates, can also be adjusted.7 The TX-TL cell-free expression system has been demonstrated for large-scale circuit assembly, exploring biological phenomena, and expression of proteins under both T7- and endogenous promoters.6,8 Accompanying mathematical models are available.9,10 The resulting system has unique applications in synthetic biology as a prototyping environment, or "TX-TL biomolecular breadboard."

Protocols for Implementing an Escherichia coli Based TX-TL Cell-Free Expression System for Synthetic Biology

This five-day protocol outlines all steps, equipment, and supplemental software necessary for creating and running an efficient endogenous Escherichia coli based TX-TL cell-free expression system from scratch. With reagents, the protocol takes 8 hours or less to setup a reaction, collect, and process data.

Protocolos para implementação de uma<em&gt; Escherichia coli</em&gt; Com base TX-TL Expression System-Free Cell para Biologia Sintética

Ideal cell-free expression systems can theoretically emulate an in vivo cellular environment in a controlled in vitro platform.1 This is useful for ...

Detection of Live Escherichia coli O157:H7 Cells by PMA-qPCR

A unique open reading frame (ORF) Z3276 was identified as a specific genetic marker for E. coli O157:H7. A qPCR assay was developed for detection of E. coli O157:H7 by targeting ORF Z3276. With this assay, we can detect as low as a few copies of the genome of DNA of E. coli O157:H7. The sensitivity and specificity of the assay were confirmed by intensive validation tests with a large number of E. coli O157:H7 strains (n = 369) and non-O157 strains (n = 112). Furthermore, we have combined propidium monoazide (PMA) procedure with the newly developed qPCR protocol for selective detection of live cells from dead cells. Amplification of DNA from PMA-treated dead cells was almost completely inhibited in contrast to virtually unaffected amplification of DNA from PMA-treated live cells. Additionally, the protocol has been modified and adapted to a 96-well plate format for an easy and consistent handling of a large number of samples. This method is expected to have an impact on accurate microbiological and epidemiological monitoring of food safety and environmental source.

Detection of Live Escherichia coli O157:H7 Cells by PMA-qPCR

A qPCR assay was developed for detection of Escherichia coli O157:H7 targeting a unique genetic marker, Z3276. The qPCR was combined with propidium monoazide (PMA) treatment for live cell detection. This protocol has been modified and adapted to a 96-well plate format for easy and consistent handling of numerous samples

Detecção de Vivo<em&gt; Escherichia coli</em&gt; O157: H7 Células por PMA-qPCR

A unique open reading frame (ORF) Z3276 was identified as a specific genetic marker for E. coli O157:H7. A qPCR assay was developed for detection of E. ...

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

A streamlined approach to screening for the expression of recombinant membrane proteins in Escherichia coli based on fusion to green fluorescent protein is presented.

Verde Fluorescente à base de proteínas Expressão Rastreio de Proteínas de Membrana em<em&gt; Escherichia coli</em

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and ...

The Multifaceted Benefits of Protein Co-expression in Escherichia coli

We report here that the expression of protein complexes in vivo in Escherichia coli can be more convenient than traditional reconstitution experiments in vitro. In particular, we show that the poor solubility of Escherichia coli DNA polymerase III &#949; subunit (featuring 3&#8217;-5&#8217; exonuclease activity) is highly improved when the same protein is co-expressed with the &#945; and &#952; subunits (featuring DNA polymerase activity and stabilizing &#949;, respectively). We also show that protein co-expression in E. coli can be used to efficiently test the competence of subunits from different bacterial species to associate in a functional protein complex. We indeed show that the &#945; subunit of Deinococcus radiodurans DNA polymerase III can be co-expressed in vivo with the &#949; subunit of E. coli. In addition, we report on the use of protein co-expression to modulate mutation frequency in E. coli. By expressing the wild-type &#949; subunit under the control of the araBAD promoter (arabinose-inducible), and co-expressing the mutagenic D12A variant of the same protein, under the control of the lac promoter (inducible by isopropyl-thio-&#946;-D-galactopyranoside, IPTG), we were able to alter the E. coli mutation frequency using appropriate concentrations of the inducers arabinose and IPTG. Finally, we discuss recent advances and future challenges of protein co-expression in E. coli.

The Multifaceted Benefits of Protein Co-expression in Escherichia coli

Protein co-expression is a powerful alternative to the reconstitution in vitro of protein complexes, and is of help in performing biochemical and genetic tests in vivo. Here we report on the use of protein co-expression in Escherichia coli to obtain protein complexes, and to tune the mutation frequency of cells.

As multifacetadas benefícios da proteína Co-expressão em<em&gt; Escherichia coli</em

We report here that the expression of protein complexes in vivo in Escherichia coli can be more convenient than traditional reconstitution experiments in ...

Residue-specific Incorporation of Noncanonical Amino Acids into Model Proteins Using an Escherichia coli Cell-free Transcription-translation System

The canonical set of amino acids leads to an exceptionally wide range of protein functionality. Nevertheless, the set of residues still imposes limitations on potential protein applications. The incorporation of noncanonical amino acids can enlarge this scope. There are two complementary approaches for the incorporation of noncanonical amino acids. For site-specific incorporation, in addition to the endogenous canonical translational machineries, an orthogonal aminoacyl-tRNA-synthetase-tRNA pair must be provided that does not interact with the canonical ones. Consequently, a codon that is not assigned to a canonical amino acid, usually a stop codon, is also required. This genetic code expansion enables the incorporation of a noncanonical amino acid at a single, given site within the protein. The here presented work describes residue-specific incorporation where the genetic code is reassigned within the endogenous translational system. The translation machinery accepts the noncanonical amino acid as a surrogate to incorporate it at canonically prescribed locations, i.e., all occurrences of a canonical amino acid in the protein are replaced by the noncanonical one. The incorporation of noncanonical amino acids can change the protein structure, causing considerably modified physical and chemical properties. Noncanonical amino acid analogs often act as cell growth inhibitors for expression hosts since they modify endogenous proteins, limiting in vivo protein production. In vivo incorporation of toxic noncanonical amino acids into proteins remains particularly challenging. Here, a cell-free approach for a complete replacement of L-arginine by the noncanonical amino acid L-canavanine is presented. It circumvents the inherent difficulties of in vivo expression. Additionally, a protocol to prepare target proteins for mass spectral analysis is included. It is shown that L-lysine can be replaced by L-hydroxy-lysine, albeit with lower efficiency. In principle, any noncanonical amino acid analog can be incorporated using the presented method as long as the endogenous in vitro translation system recognizes it.

Residue-specific Incorporation of Noncanonical Amino Acids into Model Proteins Using an Escherichia coli Cell-free Transcription-translation System

An easy-to-use, cell-free expression protocol for the residue-specific incorporation of noncanonical amino acid analogs into proteins, including downstream analysis, is presented for medical, pharmaceutic, structural and functional studies.

Incorporação específico de resíduos de não-canônicos Aminoácidos em proteínas modelo Usando um<em&gt; Escherichia coli</em&gt; Sistema de transcrição-tradução livre-Cell

The canonical set of amino acids leads to an exceptionally wide range of protein functionality. Nevertheless, the set of residues still imposes ...

Measuring Lactase Enzymatic Activity in the Teaching Lab

Understanding how enzymes work, and relating this to real life examples, is critical to a wide range of undergraduate degrees in the biological and biomedical sciences. This easy to follow protocol was developed for first year undergraduate pharmacy students and provides an entry-level introduction to enzyme reactions and analytical procedures for enzyme analysis. The enzyme of choice is lactase, as this represents an example of a commercially available enzyme relevant to human disease/pharmaceutical practice. Lactase is extracted from dietary supplement tablets, and assessed using a colorimetric assay based upon hydrolysis of an artificial substrate for lactase (ortho-nitrophenol-beta-D-galactopyranoside, ONPG). Release of ortho-nitrophenol following the hydrolytic cleavage of ONPG by lactase is measured by a change in absorbance at 420 nm, and the effect of the temperature on the enzymatic reaction is evaluated by carrying out the reaction on ice, at room temperature and at 37 &#176;C. More advanced analysis can be implemented using this protocol by assessing the enzyme activity under different conditions and using different reagents.

The enzymatic activity of lactase is essential for the catabolic processing of the disaccharide lactose. Here, the activity of lactase found in dietary supplements is assayed using a colorimetric assay. This provides students with an experimental platform for understanding the activity of lactase and enzyme kinetics.

Medindo a atividade enzimática de Lactase no laboratório de ensino

Understanding how enzymes work, and relating this to real life examples, is critical to a wide range of undergraduate degrees in the biological and ...