A permissão da Springer e Elsevier para reimprimir figuras é muito reconhecido.

1. Isolamento de E. coli co-transformantes <ol><li> Prepare células eletro-competente do E. apropriado coli estirpe a ser transformada. Transferência para 1 ml de meio LB (triptona, extracto de levedura, NaCl a 10, 5 e 10 g / L, respectivamente) de uma única colónia da estirpe de escolha, e incubar a 37 ° C sob condições de agitação de 180 rpm. Dilui-se a pré-cultura de 1: 500 em 25 ml de meio LB fresco e incuba-se a cultura a 37 ° C. </li><li> Na fase de log (0,6 OD) centrifugar a suspensão de células em 5000 xg durante 20 min), e ressuspender o sedimento em 10%, v / v arrefecido em gelo estéril de glicerol-água em metade do volume de cultura original. Repita este passo 4 vezes, reduzir para metade a cada vez que o volume de ressuspensão. Finalmente, ressuspender o sedimento em glicerol / água e dividir a suspensão de células em alíquotas de 50 ul. Armazenar as alíquotas a -80 ° C até 6 meses. </li><li> Dissolve-se o plasmídeo pretendido em água estéril suplementado com 0,5 mM de EDTA. Thaw em ice uma alíquota de células eletro-competentes e misturar com uma quantidade apropriada (2,5-5 ng) de vector. Dispensar a mistura em uma solução de 0,1 cm de percurso óptico adequado para a electroporação, e aplicam-se 1,8 kV. </li><li> Transferir imediatamente as células electroporadas em 1 ml de meio SOC (meio LB suplementado com 0,2% w / v de glucose, 10 mM de MgCl2, 2,5 mM de KCl), incubar durante 1 h sob agitação, e, finalmente, transferi-se alíquotas de 100 ul para placas de Petri contendo LB agar com o antibiótico apropriado. Incubar O / N a 37 ° C. </li><li> Purifica-se os transformantes por estrias colónias únicas em placas de Petri. </li><li> Prepare células eletro-competente dos transformantes primários e repita os passos 1.1 a 1.5 para transformar com outros plasmídeos. </li><li> Prepare stocks de glicerol dos co-transformantes. Transferir uma única colónia em LB suplementado com os antibióticos apropriados, incubar a 37 ° C sob agitação, e a fase de log (0,6 OD) centrifugar a suspensão de células a 5000 x g durante 20 min. Resuspend o sedimento em meio LB-antibióticos contendo 15% v / v de glicerol. Dispensar em alíquotas e armazenar a - 80 ° C. </li></ol> 2. A co-expressão de α e ε Subunidades de E. coli ADN polimerase III <ol><li> Por meio de uma agulha esterilizada uma pequena quantidade do estoque de glicerol tensão adequada (por exemplo, TOP10 / pGOOD-ε243 e TOP10 / pGOOD-ε243-θ / pBADα1160, veja a Figura 1) em uma placa de Petri contendo meio LB e antibióticos. Deixe secar células suspensão na placa de Petri, e raia as células gota. Incubar a 37 ° CO / N. </li><li> Transferência, utilizando um palito estéril, uma única colónia para 1 ml de meio LB-antibióticos. Incubar 8 horas a 37 ° C. Dilui-se a pré-cultura de 1: 500 em meio fresco e incuba-se a 30 ° C durante 15 h. </li><li> Adicionar IPTG, arabinose, ou arabinose e IPTG 1 mM cada. Incubar a 30 ° C durante 2,5 h. Recolher as células, e armazenar os peletes a -20 ° C. </li><li> Descongelar as pelotas em gelo e ressuspender em tampão de lise (50 mM Tris-HCl, NaCl 50 mM, EDTA 1 mM, ditiotreitol 2,5 mM, fluoreto de fenilmetilsulfonilo 1 mM (PMSF), pH 8). Gentilmente homogeneizar a suspensão células com um oleiro vidro frio. </li><li> Sonicar a 15 W durante 15 segundos, seguido por 15 segundos de intervalo de arrefecimento (4 ciclos). </li><li> Centrifuga-se os extractos de proteína total a 10.000 xg durante 20 min, a 4 ° C para recuperar a fracção solúvel. </li><li> Analisar por alíquotas de cada extracto de proteína solúvel em SDS-PAGE (12,5% de acrilamida). Para este objectivo, transferir 20 ul de cada extracto de proteína solúvel para tubos Eppendorf contendo 60 ul de H2O e 20 ul de tampão de carga (250 mM de Tris-HCl pH 6,8, 10 mM β-mercaptoetanol, 10% (w / v) SDS, 50% (v / v) de glicerol, 0,25% (w / v) de azul de bromofenol) e ferver durante 5 min. Carga de 18 ul de cada amostra e realizar a electroforese a 140 V durante cerca de 1,5 h. </li></ol> 3. Co-expressão de α Subunidade de Deinococradiodurans cus ADN Polimerase III e ε Subunidade de E. coli ADN polimerase III <ol><li> Prepara-se uma pré-cultura de E. coli estirpe TOP10 / Dr pBAD-α / pGOOD-ε243 em 1 ml de meio LB-ampicilina-tetraciclina e incubar 8 horas a 37 ° C. Diluir 1: 1000 em meio fresco e incubam O / N a 37 ° C. </li><li> Dilui-se 1: 100 em 200 ml de meio fresco, incubar a 37 ° C durante 3 h, em seguida, induzir durante 3 horas com arabinose e IPTG, 1 mM de cada um. Colher as células, e armazenar o sedimento a -20 ° C. </li><li> Ressuspender o pellet em 50 mM Tris-HCl pH 8, NaCl 150 mM, EDTA 1 mM, PMSF 1 mM. Homogeneizar e perturbar as células conforme descrito em 2.4 e 2.5. </li><li> Imediatamente centrifugar as células lisado a 10.000 xg durante 20 min. Desprezar o pelete. Filtra-se o sobrenadante com um funil de Buchner equipado com 3 camadas de papel de filtro. Aplicar vácuo suave. Para evitar a formação excessiva de espuma, manter o vácuo no frasco Erlenmeyer de gelo durante a filtração. </li><li> Determinar a concentração de proteína de acordo com Bradford 14. </li></ol> 4. Cromatografia de Filtração em Gel <ol><li> Equilibrar um 16x70 encamisado-água (200) de coluna de filtração em gel com 50 mM Tris-HCl, NaCl 150 mM, EDTA 1 mM, pH 8. Carga na coluna o extracto de proteína solúvel. Para melhor resolução, use um loop de amostra de 1 ml. Realize a cromatografia em 0.6 ml / min. Manter a temperatura da coluna a 4 ° C durante toda. </li><li> Recolha 0,9 ml frações e analisá-los por SDS-PAGE. Transferir 20 uL de cada fracção relevante para tubos Eppendorf contendo 60 ul de H2O e 20 ul de tampão de carga (250 mM de Tris-HCl pH 6,8, 10 mM β-mercaptoetanol, 10% (w / v) de SDS, 50% ( v / v) de glicerol, 0,25% (w / v) de azul de bromofenol) e fervura durante 5 min. Carga de 18 ul de cada amostra e realizar a electroforese a 140 V durante cerca de 1,5 h. </li><li> Determinar a exonuclease 3&#39;-5 e a actividade de polimerase de ADN de cada fracção em 96 nósll microplacas. Ensaiar a actividade de exonuclease 5&#39;-timidina utilizando monofosfato de éster de p-nitrofenilo (p-NP PGT) como substrato 15. Estimar a actividade da polimerase de ADN, utilizando o PPX (pirofosfatase, purina nucleosídeo fosforilase, xantina oxidase) acoplado a enzima e ensaio de 16 2- (4-iodofenil) -3- (4-nitrofenil) -5-fenil-2H-tetrazólio (INT) como o aceitador de electrões 16. </li></ol> 5. Mutação análise de populações Co-expressam o Wild-tipo ε Subunidade de E. coli DNA polimerase III e mutagênicos εD12A Variant <ol><li> Transferir uma única colônia de E. coli TOP10 contendo o pBAD-ε e os pGOOD1-εD12A 11 vectores para 1 ml de meio LB-antibióticos. Incubar O / N a 37 ° C. </li><li> Dilui-se a pré-cultura de 1: 250 em três balões contendo meio fresco (10 ml), adicionar os indutores apropriados (arabinose, IPTG, ou arabinose e IPTG, 1 mM de cada um) e incubar a 37 ° Cdurante 8 h. Prepara-se culturas paralelas não induzidas. </li><li> Recolhe alíquotas de 1 ml, depois dilui-se 1: 500 em novos frascos contendo meio fresco (10 ml) suplementado ou não com os indutores apropriados. Incubar O / N a 37 ° C. </li><li> Repita os passos 5.2 e 5.3 e recolhem alíquotas de 1 ml. </li><li> Determinar o número de gerações ocorreu em cada cultura. Transferir em placas de LB, 100 ul de diluições em série apropriadas de inoculo e cultura. Incubar O / N a 37 ° C. Contar as colónias nas placas de LB, e calcular o logaritmo do número de células presentes no inoculo (log I) e na cultura no final do crescimento (log C). Para determinar o número de gerações, usar a fórmula: (log C - log I) /0.301. </li><li> Centrifugar a 5.000 xg durante 20 min e ressuspender as células em 1 ml de 50 mM Tris-HCl pH 7,6, NaCl 50 mM. Para permeabilizar as células, adicionar 2-3 gotas de clorofórmio e de vórtice durante 20 seg. </li><li> Determinar a β-actividade de glicosidase cada aliquota numa microplaca de 96 poços, utilizando-se p-nitrofenil-β-D-glucopiranósido (PNPGluc) como substrato. Adicionar a cada poço 100 ul de células permeabilizadas e 100 ul de substrato (16 mg / ml em solução de H2O). Tome cuidado para evitar bolhas de ar nos poços. Ler a absorvância a 420 nm, utilizando um leitor de microplacas e o filtro apropriado. </li></ol>

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The authors declare that they have no competing financial interests.

As proteínas podem ser intrinsecamente desordenados, caracteriza regiões cuja estrutura terciária não está restrita a um número limitado de conformações 25. Estas proteínas são geralmente desordenados propensos a agregação de 25, e o seu isolamento e caracterização pode representar uma tarefa difícil. A subunidade de E. ε DNA polimerase III coli apresenta dois domínios distintos 26,27, a saber: i) o domínio N-ter, tendo o &#39;exonuclease 3&#39;-5, e c...

A introdução de tecnologias superexpressão potenciado quer os estudos bioquímicos de baixo número de cópias enzimas Número e a produção industrial de proteínas farmacologicamente activas (por exemplo, insulina). Desde o advento das tecnologias, avanços significativos foram conseguidos para aumentar o rendimento e a qualidade de proteínas recombinantes. Além disso, 1,2 procariotas e eucariotas 3 sistemas superexpressão foram desenvolvidos ao longo dos anos, oferecendo alternativas úteis para o &quot;cavalo de trabalho&quot; da biotecnologia proteína, ou seja, Escherichia coli. Em particular, a disponibilidade de plataformas alternativas para E. coli levou à produção de péptidos recombinantes ou proteínas de rolamento modificações pós-traducionais. No entanto, deve ser mencionado que a E. coli ainda representa o organismo de eleição para a produção de proteína recombinante. Isso se deve a vários fatores, entre os quais o mais relevante pode serconsiderado: i) a disponibilidade de um grande número de sistemas de sobre-expressão (vectores de expressão e estirpes) para E. coli 1,2; ii) vezes a curto geração e de alto rendimento de biomassa, de E. coli em uma variedade de mídia rica e sintética; iii) a manipulação fácil, quer a bioquímica e genética ao nível deste microrganismo; iv) o isolamento das estirpes capazes de produção de proteínas tóxicas 4; v) a construção de cepas que caracterizam indução homogênea a nível da população 5,6. Além disso, demonstrou-se recentemente que os sistemas de expressão adequados para a produção em E. coli de proteínas modificadas pós-tradução podem ser concebidos e construídos 2. Actualmente, a sobre-expressão da proteína é utilizada, principalmente, para obter proteínas monoméricas ou homo-oligoméricas, cuja hypersynthesis pode ser realizado com um único gene clonado num plasmídeo adequado. No entanto, a atenção foi recentementepago para a construção de E. sistemas de proteína co-expressão de E. coli, desafiando a produção, in vivo, de complexos hetero-oligomérica 2. Curiosamente, os primeiros experimentos de proteína co-expressão se dirigiu à assembléia inter-espécies de grandes e pequenas subunidades de carboxylase cianobactérias ribulose-1,5-bisfosfato / oxigenase 7,8, ea associação de formas truncadas e de corpo inteiro de HIV-1 transcriptase reversa 9. Estes estudos pioneiros demonstrado que a co-expressão da proteína representa uma poderosa alternativa ao tradicional na reconstituição in vitro. Além disso, a proteína co-expressão em E. coli foi utilizado para produzir diferentes proteínas que ostentam modificações pós-traducionais 2, para se obter proteínas que contêm aminoácidos não naturais 2, e para aumentar o rendimento de proteínas de membrana overexpressed 2. Além disso, o potencial de co-expressão da proteína como uma ferramenta para conferir a E. coli competirnce na secreção de proteínas está sob investigação ativa 2. Duas estratégias principais de co-expressão da proteína em E. coli pode ser prosseguido: i) o uso de um único plasmídeo para receber os diferentes genes a ser sobre-expresso; ii) a utilização de vários plasmídeos em células individuais para co-expressar as proteínas alvo. No primeiro caso, os critérios para a escolha do plasmídeo não diferem dos de experiências individuais tradicionais sobre-expressão da proteína, embora determinados plasmídeos contendo em tandem os elementos promotor / operador foram construídos para co-expressão de 10. Esta primeira abordagem é, portanto, bastante simples. No entanto, deve mencionar-se que o uso de um único plasmídeo de co-expressar proteínas diferentes enfrenta dois grandes problemas: i) a massa molecular do vector aumenta com o número de genes que alberguem, limitando o número de proteínas co-expressas; ii) quando vários genes foram clonados sob o controlo de um único promotor, de polaridade pode decreasum e a expressão dos genes distais do promotor. O uso de plasmídeos duplos ou múltiplos no único E. células de E. coli tem de realizar o grau de compatibilidade dos vectores de escolha, por conseguinte, a imposição de restrições para as combinações de plasmídeos elegíveis. No entanto, esta segunda estratégia de co-expressão apresenta a vantagem de conter a massa molecular de vectores, e limita polaridade. Construiu-se, recentemente, um sistema de co-expressão de proteínas concebido para facilitar o vaivém de genes entre os plasmídeos de co-expressão de 11. Em particular, nós construídos vectores da série pGOOD, as características relevantes dos quais são os seguintes: i) uma origem de replicação de p15A, para proporcionar a compatibilidade dos plasmídeos pGOOD com os vectores comerciais contendo a origem ColE1 (por exemplo, a série de pBAD 12); ii) uma cassete de resistência à tetraciclina; iii) a presença de lac -derived elementos reguladores, ou seja, o promotor-operador (O 1) casal eo lacI </em&gt; gene q. Usando um par pBAD-pGOOD disso, fomos capazes de overexpress o núcleo catalítico de E. coli ADN polimerase III, composto por três subunidades diferentes, ou seja, α (5&#39;-3 &#39;da polimerase), ε (3&#39;-5&#39; exonuclease) e θ (estabilizador ε) 13. Em particular, foi demonstrado que a co-expressão do complexo αεθ era estritamente dependente da adição à E. coli meio de cultura de ambos IPTG e arabinose, provocando a sobre-expressão de pBAD pGOOD e, respectivamente (Figura 1A). No presente relatório, que ilustram como proteína co-expressão pode eficientemente resolver dificuldades relacionadas com a baixa solubilidade de uma proteína subunidade complexo. Além disso, mostramos como em testes de complementação de proteína vivo pode ser realizada, e, finalmente, um relatório sobre a utilização de proteínas co-expressão a frequência de mutação sintonizar E. coleu. Para tal, utilizou-se casais pGOOD-pBAD adequados para ilustrar exemplos relevantes de cada estudo de caso.

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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.

A subunidade de E. ε coli DNA polimerase III é composto por 243 aminoácidos e possui baixa solubilidade em 17,18, a menos que os resíduos 187-243 são eliminados 17. Contudo, mostrámos previamente 11 que o co-expressão de α de comprimento completo, ε, e subunidades θ produz DNA polimerase III solúvel do núcleo catalítico (Figura 1). Em particular, usando o sistema de pBAD-pGOOD co-expressão, demonstramos que: i) a sobre-expressão de subuni...

Watch this Scientific Journal Video about The Multifaceted Benefits of Protein Co-expression in Escherichia coli at JoVE.com

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 ...

the-multifaceted-benefits-of-protein-co-expression-in-escherichia-coli

Research

JoVE Journal

Biology

The Multifaceted Benefits of Protein Co-expression in Escherichia coli

11.8K visualizações.  University of Bologna. 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. 

Vídeo: The Multifaceted Benefits of Protein Co-expression in Escherichia coli

An Assay for Measuring the Activity of Escherichia coli Inducible Lysine 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 acid stresses including passage through the mammalian stomach where the pH can fall to as low as pH 1 - 22. To enable such a broad range of acidic pH survival, E. coli possesses four different inducible amino acid decarboxylases that decarboxylate their substrate amino acids in a proton-dependent manner thus raising the internal pH. The decarboxylases include the glutamic acid decarboxylases GadA and GadB3, the arginine decarboxylase AdiA4, the lysine decarboxylase LdcI5, 6 and the ornithine decarboxylase SpeF7. All of these enzymes utilize pyridoxal-5'-phospate as a co-factor8 and function together with inner-membrane substrate-product antiporters that remove decarboxylation products to the external medium in exchange for fresh substrate2. In the case of LdcI, the lysine-cadaverine antiporter is called CadB. Recently, we determined the X-ray crystal structure of LdcI to 2.0 &#197;, and we discovered a novel small-molecule bound to LdcI the stringent response regulator guanosine 5'-diphosphate,3'-diphosphate (ppGpp) 14. The stringent response occurs when exponentially growing cells experience nutrient deprivation or one of a number of other stresses9. As a result, cells produce ppGpp which leads to a signaling cascade culminating in the shift from exponential growth to stationary phase growth10. We have demonstrated that ppGpp is a specific inhibitor of LdcI 14. Here we describe the lysine decarboxylase assay, modified from the assay developed by Phan et al.11, that we have used to determine the activity of LdcI and the effect of pppGpp/ppGpp on that activity. The LdcI decarboxylation reaction removes the &alpha;-carboxy group of L-lysine and produces carbon dioxide and the polyamine cadaverine (1,5-diaminopentane)5. L-lysine and cadaverine can be reacted with 2,4,6-trinitrobenzensulfonic acid (TNBS) at high pH to generate N,N'-bistrinitrophenylcadaverine (TNP-cadaverine) and N,N&#8242;-bistrinitrophenyllysine (TNP-lysine), respectively11. The TNP-cadaverine can be separated from the TNP-lysine as the former is soluble in organic solvents such as toluene while the latter is not (See Figure 1). The linear range of the assay was determined empirically using purified cadaverine.

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

The activity of the inducible lysine decarboxylase is monitored by reacting the substrate L-lysine and the product cadaverine with 2,4,6-trinitrobenzensulfonic acid to form adducts that have differential solubility in toluene.

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 ...

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 ...

Mapping Bacterial Functional Networks and Pathways in Escherichia Coli using Synthetic Genetic Arrays

Phenotypes are determined by a complex series of physical (e.g. protein-protein) and functional (e.g. gene-gene or genetic) interactions (GI)1. While physical interactions can indicate which bacterial proteins are associated as complexes, they do not necessarily reveal pathway-level functional relationships1. GI screens, in which the growth of double mutants bearing two deleted or inactivated genes is measured and compared to the corresponding single mutants, can illuminate epistatic dependencies between loci and hence provide a means to query and discover novel functional relationships2. Large-scale GI maps have been reported for eukaryotic organisms like yeast3-7, but GI information remains sparse for prokaryotes8, which hinders the functional annotation of bacterial genomes. To this end, we and others have developed high-throughput quantitative bacterial GI screening methods9, 10.
Here, we present the key steps required to perform quantitative E. coli Synthetic Genetic Array (eSGA) screening procedure on a genome-scale9, using natural bacterial conjugation and homologous recombination to systemically generate and measure the fitness of large numbers of double mutants in a colony array format. Briefly, a robot is used to transfer, through conjugation, chloramphenicol (Cm) - marked mutant alleles from engineered Hfr (High frequency of recombination) 'donor strains' into an ordered array of kanamycin (Kan) - marked F- recipient strains. Typically, we use loss-of-function single mutants bearing non-essential gene deletions (e.g. the 'Keio' collection11) and essential gene hypomorphic mutations (i.e. alleles conferring reduced protein expression, stability, or activity9, 12, 13) to query the functional associations of non-essential and essential genes, respectively. After conjugation and ensuing genetic exchange mediated by homologous recombination, the resulting double mutants are selected on solid medium containing both antibiotics. After outgrowth, the plates are digitally imaged and colony sizes are quantitatively scored using an in-house automated image processing system14. GIs are revealed when the growth rate of a double mutant is either significantly better or worse than expected9. Aggravating (or negative) GIs often result between loss-of-function mutations in pairs of genes from compensatory pathways that impinge on the same essential process2. Here, the loss of a single gene is buffered, such that either single mutant is viable. However, the loss of both pathways is deleterious and results in synthetic lethality or sickness (i.e. slow growth). Conversely, alleviating (or positive) interactions can occur between genes in the same pathway or protein complex2 as the deletion of either gene alone is often sufficient to perturb the normal function of the pathway or complex such that additional perturbations do not reduce activity, and hence growth, further. Overall, systematically identifying and analyzing GI networks can provide unbiased, global maps of the functional relationships between large numbers of genes, from which pathway-level information missed by other approaches can be inferred9.

Mapping Bacterial Functional Networks and Pathways in Escherichia Coli using Synthetic Genetic Arrays

Systematic, large-scale synthetic genetic (gene-gene or epistasis) interaction screens can be used to explore genetic redundancy and pathway cross-talk. Here, we describe a high-throughput quantitative synthetic genetic array screening technology, termed eSGA that we developed for elucidating epistatic relationships and exploring genetic interaction networks in Escherichia coli.

Mapeamento bacterianas redes funcionais e vias<em&gt; Escherichia Coli</em&gt; Usando sintéticos matrizes genéticas

Phenotypes  are determined by a complex series of physical (e.g. protein-protein) and  functional (e.g. gene-gene or genetic) interactions (GI)1. While ...

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the identification of the subunit composition of multi-protein complexes can provide insight into gene function and enhance understanding of biological systems1, 2. Physical interactions can be mapped with high confidence vialarge-scale isolation and characterization of endogenous protein complexes under near-physiological conditions based on affinity purification of chromosomally-tagged proteins in combination with mass spectrometry (APMS). This approach has been successfully applied in evolutionarily diverse organisms, including yeast, flies, worms, mammalian cells, and bacteria1-6. In particular, we have generated a carboxy-terminal Sequential Peptide Affinity (SPA) dual tagging system for affinity-purifying native protein complexes from cultured gram-negative Escherichia coli, using genetically-tractable host laboratory strains that are well-suited for genome-wide investigations of the fundamental biology and conserved processes of prokaryotes1, 2, 7. Our SPA-tagging system is analogous to the tandem affinity purification method developed originally for yeast8, 9, and consists of a calmodulin binding peptide (CBP) followed by the cleavage site for the highly specific tobacco etch virus (TEV) protease and three copies of the FLAG epitope (3X FLAG), allowing for two consecutive rounds of affinity enrichment. After cassette amplification, sequence-specific linear PCR products encoding the SPA-tag and a selectable marker are integrated and expressed in frame as carboxy-terminal fusions in a DY330 background that is induced to transiently express a highly efficient heterologous bacteriophage lambda recombination system10. Subsequent dual-step purification using calmodulin and anti-FLAG affinity beads enables the highly selective and efficient recovery of even low abundance protein complexes from large-scale cultures. Tandem mass spectrometry is then used to identify the stably co-purifying proteins with high sensitivity (low nanogram detection limits).
Here, we describe detailed step-by-step procedures we commonly use for systematic protein tagging, purification and mass spectrometry-based analysis of soluble protein complexes from E. coli, which can be scaled up and potentially tailored to other bacterial species, including certain opportunistic pathogens that are amenable to recombineering. The resulting physical interactions can often reveal interesting unexpected components and connections suggesting novel mechanistic links. Integration of the PPI data with alternate molecular association data such as genetic (gene-gene) interactions and genomic-context (GC) predictions can facilitate elucidation of the global molecular organization of multi-protein complexes within biological pathways. The networks generated for E. coli can be used to gain insight into the functional architecture of orthologous gene products in other microbes for which functional annotations are currently lacking.

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Affinity purification of tagged proteins in combination with mass spectrometry (APMS) is a powerful method for the systematic mapping of protein interaction networks and for investigating the mechanistic basis of biological processes. Here, we describe an optimized sequential peptide affinity (SPA) APMS procedure developed for the bacterium Escherichia coli that can be used to isolate and characterize stable multi-protein complexes to near homogeneity even starting from low copy numbers per cell.

Identificação de complexos de proteínas em<em&gt; Escherichia coli</em&gt; Utilizando a purificação por afinidade sequencial Peptide em combinação com espectrometria de massa tandem

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the ...

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 ...

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. ...

Non-chromatographic Purification of Recombinant Elastin-like Polypeptides and their Fusions with Peptides and Proteins from Escherichia coli

Elastin-like polypeptides are repetitive biopolymers that exhibit a lower critical solution temperature phase transition behavior, existing as soluble unimers below a characteristic transition temperature and aggregating into micron-scale coacervates above their transition temperature. The design of elastin-like polypeptides at the genetic level permits precise control of their sequence and length, which dictates their thermal properties. Elastin-like polypeptides are used in a variety of applications including biosensing, tissue engineering, and drug delivery, where the transition temperature and biopolymer architecture of the ELP can be tuned for the specific application of interest. Furthermore, the lower critical solution temperature phase transition behavior of elastin-like polypeptides allows their purification by their thermal response, such that their selective coacervation and resolubilization allows the removal of both soluble and insoluble contaminants following expression in Escherichia coli. This approach can be used for the purification of elastin-like polypeptides alone or as a purification tool for peptide or protein fusions where recombinant peptides or proteins genetically appended to elastin-like polypeptide tags can be purified without chromatography. This protocol describes the purification of elastin-like polypeptides and their peptide or protein fusions and discusses basic characterization techniques to assess the thermal behavior of pure elastin-like polypeptide products.

Non-chromatographic Purification of Recombinant Elastin-like Polypeptides and their Fusions with Peptides and Proteins from Escherichia coli

Elastin-like polypeptides are stimulus-responsive biopolymers with applications ranging from recombinant protein purification to drug delivery. This protocol describes the purification and characterization of elastin-like polypeptides and their peptide or protein fusions from Escherichia coli using their lower critical solution temperature phase transition behavior as a simple alternative to chromatography.

A purificação cromatográfica do não-polipéptidos recombinantes Elastina semelhantes e suas fusões com Peptídeos e Proteínas de<em&gt; Escherichia coli</em

Elastin-like polypeptides are repetitive biopolymers that exhibit a lower critical solution temperature phase transition behavior, existing as soluble ...

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 ...

Gap Junctional Intercellular Communication: A Functional Biomarker to Assess Adverse Effects of Toxicants and Toxins, and Health Benefits of Natural Products

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction channels, which is a major intercellular process by which tissue homeostasis is maintained. Interruption of gap junctional intercellular communication (GJIC) by toxicants, toxins, drugs, etc. has been linked to numerous adverse health effects. Many genetic-based human diseases have been linked to mutations in gap junction genes. The SL-DT technique is a simple functional assay for the simultaneous assessment of GJIC in a large population of cells. The assay involves pre-loading cells with a fluorescent dye by briefly perturbing the cell membrane with a scalpel blade through a population of cells. The fluorescent dye is then allowed to traverse through gap junction channels to neighboring cells for a designated time. The assay is then terminated by the addition of formalin to the cells. The spread of the fluorescent dye through a population of cells is assessed with an epifluorescence microscope and the images are analyzed with any number of morphometric software packages that are available, including free software packages found on the public domain. This assay has also been adapted for in vivo studies using tissue slices from various organs from treated animals. Overall, the SL-DT assay can serve a broad range of in vitro pharmacological and toxicological needs, and can be potentially adapted for high throughput set-up systems with automated fluorescence microscopy imaging and analysis to elucidate more samples in a shorter time.

This protocol describes a scalpel loading-fluorescent dye transfer technique that measures intercellular communication through gap junction channels. Gap junctional intercellular communication is a major cellular process by which tissue homeostasis is maintained and disruption of this cell signaling has adverse health effects.

Gap juncional intercelular comunicação: um biomarcador funcional para avaliar os efeitos adversos de tóxicos e toxinas, e Benefícios para a saúde de produtos naturais

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction ...

Method for Labeling Transcripts in Individual Escherichia coli Cells for Single-molecule Fluorescence In Situ Hybridization Experiments

A method is described for labeling individual messenger RNA (mRNA) transcripts in fixed bacteria for use in single-molecule fluorescence in situ hybridization (smFISH) experiments in E. coli. smFISH allows the measurement of cell-to-cell variability in mRNA copy number of genes of interest, as well as the subcellular location of the transcripts. The main steps involved are fixation of the bacterial cell culture, permeabilization of cell membranes, and hybridization of the target transcripts with sets of commercially available short fluorescently-labeled oligonucleotide probes. smFISH can allow the imaging of the transcripts of multiple genes in the same cell, with limitations imposed by the spectral overlap between different fluorescent markers. Following completion of the protocol illustrated below, cells can be readily imaged using a microscope coupled with a camera suitable for low-intensity fluorescence. These images, together with cell contours obtained from segmentation of phase contrast frames, or from cell membrane staining, allow the calculation of the mRNA copy number distribution of a sample of cells using open-source or custom-written software. The labeling method described here can also be applied to image transcripts with stochastic optical reconstruction microscopy (STORM).

Method for Labeling Transcripts in Individual Escherichia coli Cells for Single-molecule Fluorescence In Situ Hybridization Experiments

This manuscript describes a method for labeling individual messenger RNA (mRNA) transcripts with fluorescently-labeled DNA probes, for use in single-molecule fluorescence in situ hybridization (smFISH) experiments in E. coli. smFISH is a visualization method that allows the simultaneous detection, localization, and quantification of single mRNA molecules in fixed individual cells.

Método para a rotulagem transcritos em células individuais Escherichia coli para único-molécula fluorescência In Situ as experiências de hibridação