Os autores agradecem ao Dr. Thomas Rademacher por fornecer o ppam planta vetor de expressão e Ibrahim Al Amedi para cultivar as plantas de tabaco utilizados neste estudo. Gostaríamos de agradecer ao Dr. Richard M. Twyman por sua ajuda com a edição do manuscrito. Este trabalho foi em parte financiado pelo Conselho Europeu de Investigação Avançada Grant &quot;Future-Pharma&quot;, número da proposta 269110 eo Fraunhofer Zukunftsstiftung (Fraunhofer Fundação Futuro).

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A taxa de publicação foi parcialmente patrocinado pelas empresas Statease, Inc. (EUA) e Statcon (Alemanha), que não estavam envolvidos no envolvidos na preparação do manuscrito ou responsáveis ​​por qualquer dos seus conteúdos.

Cada experimento requer um planejamento cuidadoso, porque os recursos são muitas vezes escasso e caro. Isto é particularmente verdadeiro para as estratégias DoE porque erros durante a fase de planejamento (por exemplo, a seleção de um modelo de base que não cobre todas as interações fator significativo) pode diminuir substancialmente a capacidade de previsão dos modelos resultantes e, assim, desvalorizar todo o experimento. No entanto, esses erros podem ser facilmente evitado, seguindo procedimentos b?...

A produção de proteínas biofarmacêutica em plantas é vantajoso porque as plantas são baratos a crescer, a plataforma pode ser aumentado apenas por crescimento mais plantas, e os agentes patogénicos humanos são incapazes de se replicar de 1,2. Estratégias de expressão transitória por exemplo com base na infiltração das folhas com Agrobacterium tumefaciens proporciona benefícios adicionais, porque o tempo entre o ponto de libertação de ADN e a entrega de um produto purificado é reduzida de anos a menos do que 2 meses 3. A expressão transiente é também utilizado para a análise funcional, por exemplo, para testar os genes para a sua capacidade para complementar os mutantes de perda de função ou para investigar interacções proteína 4-6. No entanto, os níveis de expressão transiente tendem a mostrar maior variação de lote para lote do que os níveis de expressão em plantas transgénicas 7-9. Isto reduz a probabilidade de que os processos de fabricação de produtos biofarmacêuticos com base na expressão transitória wivai ser aprovada no contexto de boas práticas de fabricação (BPF), pois a reprodutibilidade é um atributo essencial de qualidade e está sujeita a avaliação de risco 10. Essa variação também pode mascarar qualquer interação que os pesquisadores pretendem investigar. Por isso, nos propusemos a identificar os principais fatores que afetam os níveis de expressão transiente em plantas e para a construção de um modelo preditivo quantitativa de alta qualidade. O (OFAT) abordagem de um fator-em-um-tempo é muitas vezes usado para caracterizar o impacto (efeito) de certos parâmetros (fatores) sobre o resultado (resposta) de um experimento 11. Mas isso é sub-ótima, pois os testes individuais (executado) no decurso do inquérito (experimento) serão alinhados como pérolas em um colar com a área potencial gerado por fatores que são testados (espaço de projeto). A cobertura do espaço de projeto e, portanto, o grau de informações derivadas da experiência ébaixo, como mostrado na Figura 1A 12. Além disso, as interdependências entre as diferentes factores (interacções do factor) pode permanecer oculto, resultando em modelos pobres e / ou a previsão de falsa óptimos, como mostrado na Figura 1B 13. As desvantagens acima descritas podem ser evitados através da utilização de uma concepção de experiências (DOE) de aproximação na qual as pistas de uma experiência são dispersas mais uniformemente por todo o espaço de criação, o que significa que mais do que um factor é variada entre duas pistas 14. Há projetos especializados para misturas, fatores (fatoriais) ea quantificação dos impactos do fator sobre as respostas (métodos de superfície de resposta, RSM s) 15 de triagem. Além disso, RSMs pode ser realizado como projetos centro-composto, mas também pode ser conseguido, usando software especializado que pode aplicar diferentes critérios para a seleção de pistas. Por exemplo, o assim chamado D-optimalitcritério y irá selecionar corridas de modo a minimizar o erro nos coeficientes do modelo resultante, enquanto que o critério IV-optimality seleciona corridas que atingem o menor previsão de variação ao longo do espaço de design 15,16. O MSE descrito aqui permite a quantificação precisa da expressão da proteína transiente em plantas, mas que pode ser facilmente transferido para qualquer sistema envolvendo vários (~ 5-8) factores numéricos (por exemplo, temperatura, tempo, de concentração) e alguns (~ 2 - 4) fatores categóricas (por exemplo, promotor, cor), em que uma descrição mecanicista não está disponível ou demasiado complexas para modelar. A abordagem DoE origem nas ciências agrícolas, mas se espalhou para outras áreas porque é transferível para qualquer situação em que é útil para reduzir o número de execuções necessárias para a obtenção de dados confiáveis ​​e gerar modelos descritivos de processos complexos. Isto, por sua vez, levou-se à inclusão de corça no &quot;Orientação paraIndústria, Q8 (R2) Pharmaceutical Development &quot;, publicado pela Conferência Internacional de Harmonização dos Requisitos Técnicos para Registro de Produtos Farmacêuticos para Uso Humano (ICH) 17. DoE é agora amplamente utilizado na investigação científica e na indústria 18. Contudo, cuidados devem ser tomados durante o planejamento e execução do experimento, pois a seleção de um polinômio de grau impróprio para o modelo de regressão linear múltiplo (modelo básico) pode apresentar uma necessidade de corridas adicionais para modelar todos os efeitos do fator corretamente. Além disso, dados corrompidos ou perdidos gerar modelos incorretos e falho previsões, e pode até mesmo impedir qualquer tentativa de construção do modelo, conforme descrito nas seções de protocolo e discussão 18. Na seção de protocolo, que inicialmente irá definir as etapas de planejamento mais importantes para uma experiência baseada em RSM e, em seguida, explicar o design baseado no DoE DesignExpert v8.1 software. Mas projetos similares podem ser construídas com outros includi softwareng JMP, Modde e STATISTICA. Os procedimentos experimentais são seguidas por instruções para a análise de dados e avaliação. <img alt="Figura 1" fo:content-width="5in" fo:src="/files/ftp_upload/51216/51216fig1highres.jpg" src="/files/ftp_upload/51216/51216fig1.jpg" /> Figura 1. Comparação de OFAT e DoE. Uma. Variação sequencial de um fator de cada vez (OFAT) em um experimento (círculos pretos, vermelhos e azuis) atinge uma baixa cobertura do espaço de projeto (regiões nascidos). Em contraste, a variação de mais do que um factor de cada vez utilizando o desenho de experiências de estratégia (DOE) (círculos verdes) aumenta a cobertura e, assim, a precisão dos modelos resultantes. B. A cobertura do espaço de projeto tendencioso significa que as experiências OFAT (círculos pretos) também pode deixar de identificar as regiões operacionais ótimas (vermelho) e prever soluções sub-ótimas (grande círculo preto), enquanto DoE estrategicamentees (estrelas negras) são mais propensos a identificar as condições preferíveis (grande estrela preta).

<table border="0" cellpadding="0" cellspacing="0" width="946">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Design-Expert(R) 8</td>
			<td style="width:195px;">Stat-Ease, Inc.</td>
			<td style="width:119px;">n.a.</td>
			<td style="width:272px;">DoE software</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Tryptone</td>
			<td style="width:195px;">Carl Roth GmbH</td>
			<td style="width:119px;">8952.2</td>
			<td>Media component</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Yeast extract</td>
			<td style="width:195px;">Carl Roth GmbH</td>
			<td style="width:119px;">2363.2</td>
			<td>Media component</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Sodium chloride</td>
			<td style="width:195px;">Carl Roth GmbH</td>
			<td style="width:119px;">P029.2</td>
			<td>Media component</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Ampicillin</td>
			<td style="width:195px;">Carl Roth GmbH</td>
			<td style="width:119px;">K029.2</td>
			<td>Antibiotic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Agar-Agar</td>
			<td style="width:195px;">Carl Roth GmbH</td>
			<td style="width:119px;">5210.2</td>
			<td>Media component</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Escherichia coli K12 DH5a</td>
			<td style="width:195px;">Life technologies</td>
			<td style="width:119px;">18263-012</td>
			<td>Microorganism</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">pPAM</td>
			<td style="width:195px;">GenBank</td>
			<td style="width:119px;">AY027531</td>
			<td>Cloning/expression vector;&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">NucleoSpin Plasmid&#160;</td>
			<td style="width:195px;">MACHEREY-NAGEL GmbH</td>
			<td style="width:119px;">740588.250</td>
			<td>Plasmid DNA isolation kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">NucleoSpin Gel and PCR Clean-up</td>
			<td style="width:195px;">MACHEREY-NAGEL GmbH</td>
			<td style="width:119px;">740609.250</td>
			<td>Plasmid DNA purification kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">NanoDrop 2000</td>
			<td style="width:195px;">Thermo Scientific</td>
			<td style="width:119px;">n.a.</td>
			<td>Spectrophotometer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">NcoI</td>
			<td style="width:195px;">New England Biolabs Inc.</td>
			<td style="width:119px;">R3193L</td>
			<td>Restrictionendonuclease</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">EcoRI</td>
			<td style="width:195px;">New England Biolabs Inc.</td>
			<td style="width:119px;">R3101L</td>
			<td>Restrictionendonuclease</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">AscI</td>
			<td style="width:195px;">New England Biolabs Inc.</td>
			<td style="width:119px;">R0558L</td>
			<td>Restrictionendonuclease</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">NEB 4</td>
			<td style="width:195px;">New England Biolabs Inc.</td>
			<td style="width:119px;">B7004S</td>
			<td>Restrictionendonuclease buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">TRIS</td>
			<td style="width:195px;">Carl Roth GmbH</td>
			<td style="width:119px;">4855.3</td>
			<td>Media component</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Disodium tetraborate</td>
			<td style="width:195px;">Carl Roth GmbH</td>
			<td style="width:119px;">4403.3</td>
			<td>Media component</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">EDTA</td>
			<td style="width:195px;">Carl Roth GmbH</td>
			<td style="width:119px;">8040.2</td>
			<td>Media component</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Agarose</td>
			<td style="width:195px;">Carl Roth GmbH</td>
			<td style="width:119px;">6352.4</td>
			<td>Media component</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Bromophenol blue</td>
			<td style="width:195px;">Carl Roth GmbH</td>
			<td style="width:119px;">A512.1</td>
			<td>Color indicator</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Xylene cyanol</td>
			<td style="width:195px;">Carl Roth GmbH</td>
			<td style="width:119px;">A513.1</td>
			<td>Color indicator</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Glycerol</td>
			<td style="width:195px;">Carl Roth GmbH</td>
			<td style="width:119px;">7530.2</td>
			<td>Media component</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Mini-Sub Cell GT Cell</td>
			<td style="width:195px;">BioRad</td>
			<td style="width:119px;">170-4406</td>
			<td>Gel electrophoresis chamber</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Agrobacterium tumefaciens strain GV3101:pMP90RK</td>
			<td style="width:195px;">DSMZ</td>
			<td style="width:119px;">12365</td>
			<td>Microorganism</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Electroporator 2510</td>
			<td style="width:195px;">Eppendorf</td>
			<td style="width:119px;">4307000.658</td>
			<td>Electroporator</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Beef extract</td>
			<td style="width:195px;">Carl Roth GmbH</td>
			<td style="width:119px;">X975.2</td>
			<td>Media component</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Peptone</td>
			<td style="width:195px;">Carl Roth GmbH</td>
			<td style="width:119px;">2365.2</td>
			<td>Media component</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Sucrose</td>
			<td style="width:195px;">Carl Roth GmbH</td>
			<td style="width:119px;">4621.2</td>
			<td>Media component</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Magnesium sulfate</td>
			<td style="width:195px;">Carl Roth GmbH</td>
			<td style="width:119px;">0261.3</td>
			<td>Media component</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Carbenicillin</td>
			<td style="width:195px;">Carl Roth GmbH</td>
			<td style="width:119px;">6344.2</td>
			<td>Antibiotic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Kanamycin</td>
			<td style="width:195px;">Carl Roth GmbH</td>
			<td style="width:119px;">T832.3</td>
			<td>Antibiotic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Rifampicin</td>
			<td style="width:195px;">Carl Roth GmbH</td>
			<td style="width:119px;">4163.2</td>
			<td>Antibiotic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">FWD primer</td>
			<td style="width:195px;">Eurofins MWG Operon</td>
			<td style="width:119px;">n.a.</td>
			<td>CCT CAG GAA GAG CAA TAC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">REV primer</td>
			<td style="width:195px;">Eurofins MWG Operon</td>
			<td style="width:119px;">n.a.</td>
			<td>CCA AAG CGA GTA CAC AAC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">2720 Thermal cycler</td>
			<td style="width:195px;">Applied Biosystems</td>
			<td style="width:119px;">4359659</td>
			<td>Thermocycler</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">RNAfold webserver</td>
			<td style="width:195px;">University of Vienna</td>
			<td style="width:119px;">n.a.</td>
			<td>Software</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Ferty 2 Mega</td>
			<td style="width:195px;">Kammlott</td>
			<td style="width:119px;">5.220072</td>
			<td>Fertilizer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Grodan Rockwool Cubes 10x10cm</td>
			<td style="width:195px;">Grodan</td>
			<td style="width:119px;">n.a.</td>
			<td>Rockwool block</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Greenhouse</td>
			<td style="width:195px;">n.a.</td>
			<td style="width:119px;">n.a.</td>
			<td>For plant cultivation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Phytotron</td>
			<td style="width:195px;">Ilka Zell</td>
			<td style="width:119px;">n.a.</td>
			<td>For plant cultivation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Omnifix-F Solo</td>
			<td style="width:195px;">B. Braun</td>
			<td style="width:119px;">6064204</td>
			<td>Syringe</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Murashige and Skoog salts</td>
			<td style="width:195px;">Duchefa</td>
			<td style="width:119px;">M 0222.0010</td>
			<td>Media component</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Glucose</td>
			<td style="width:195px;">Carl Roth GmbH</td>
			<td style="width:119px;">6780.2</td>
			<td>Media component</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Acetosyringone</td>
			<td style="width:195px;">Sigma-Aldrich</td>
			<td style="width:119px;">D134406-5G</td>
			<td>Phytohormon analogon</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">&#160;BioPhotometer plus</td>
			<td style="width:195px;">Eppendorf</td>
			<td style="width:119px;">&#160;6132 000.008</td>
			<td>Photometer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Osram cool white 36 W</td>
			<td style="width:195px;">Osram</td>
			<td style="width:119px;">4930440</td>
			<td>Light source</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Disodium phosphate</td>
			<td style="width:195px;">Carl Roth GmbH</td>
			<td style="width:119px;">&#160;4984.3&#160;</td>
			<td>Media component</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Centrifuge 5415D</td>
			<td style="width:195px;">Eppendorf</td>
			<td style="width:119px;">5424 000.410</td>
			<td>Centrifuge</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Forma -86C ULT freezer</td>
			<td style="width:195px;">ThermoFisher</td>
			<td style="width:119px;">88400</td>
			<td>Freezer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Synergy HT</td>
			<td style="width:195px;">BioTek</td>
			<td style="width:119px;">SIAFRT</td>
			<td>Fluorescence plate reader</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Biacore T200</td>
			<td style="width:195px;">GE Healthcare</td>
			<td style="width:119px;">n.a.</td>
			<td>SPR device</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Protein A</td>
			<td style="width:195px;">Life technologies</td>
			<td style="width:119px;">10-1006</td>
			<td>Antibody binding protein</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">HEPES</td>
			<td style="width:195px;">Carl Roth GmbH</td>
			<td style="width:119px;">9105.3</td>
			<td>Media component</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Tween-20</td>
			<td style="width:195px;">Carl Roth GmbH</td>
			<td style="width:119px;">9127.3</td>
			<td>Media component</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">2G12 antibody</td>
			<td style="width:195px;">Polymun</td>
			<td style="width:119px;">AB002</td>
			<td>Reference antibody</td>
		</tr>
	</tbody>
</table>

characterization of complex systems using the design of experiments approach: transient protein expression in tobacco as a case study

As plantas fornecem benefícios múltiplos para a produção de biofármacos incluindo os custos baixos, escalabilidade e segurança. A expressão transiente oferece a vantagem adicional de curto desenvolvimento e tempos de produção, mas os níveis de expressão podem variar significativamente entre os lotes, dando assim origem a preocupações regulatórias no contexto das boas práticas de fabrico. Foi utilizado um planejamento de experimentos (DoE) abordagem para determinar o impacto dos principais fatores, como elementos reguladores na construção de expressão, o crescimento das plantas e os parâmetros de desenvolvimento e as condições de incubação durante a expressão, sobre a variabilidade de expressão entre os lotes. Foram testadas as plantas que expressam um anticorpo modelo anti-HIV anticorpo monoclonal (2G12) e uma proteína de marcador fluorescente (DsRed). Discutimos a justificativa para a seleção de certas propriedades do modelo e identificar suas possíveis limitações. A abordagem geral pode ser facilmente transferido para outros problemas, porque os princípios do modelo de umre amplamente aplicável: seleção de parâmetros baseada no conhecimento, a redução da complexidade, dividindo o problema inicial em módulos menores, de configuração guiada por software de combinações experimentais óptimas e aumento do projeto passo a passo. Portanto, a metodologia é útil não só para caracterizar a expressão da proteína em plantas, mas também para a investigação de outros sistemas complexos que faltam uma descrição mecanicista. As equações de previsão que descrevem a interligação entre os parâmetros podem ser utilizados para estabelecer modelos mecanísticos para outros sistemas complexos.

Um modelo descritivo para acumulação durante DsRed expressão transitória utilizando diferentes promotores e 5&#39;UTRs DsRed fluorescência nos extractos de folhas foi utilizado para indicar o nível de expressão da proteína recombinante e, assim, foi utilizada como a resposta na estratégia DOE. A diferença mínima detectável foram consideradas relevantes foi de 20 ug / ml e o desvio padrão estimado do sistema foi de 8 ug / ml com base em experiências iniciais. Fa...

Watch this Scientific Journal Video about Characterization of Complex Systems Using the Design of Experiments Approach: Transient Protein Expression in Tobacco as a Case Study at JoVE.com

Characterization of Complex Systems Using the Design of Experiments Approach: Transient Protein Expression in Tobacco as a Case Study

Descreve-se um desenho de experiências abordagem que pode ser utilizada para determinar e modelar a influência dos elementos de regulação de transgenes, de crescimento e de desenvolvimento de parâmetros de planta, e as condições de incubação sobre a expressão transiente de anticorpos monoclonais e proteínas repórter em plantas.

Caracterização de Sistemas Complexos Usando o Projeto de Experimentos Abordagem: Transient Protein Expression em tabaco como um Estudo de Caso

Plants provide multiple benefits for the production of biopharmaceuticals including low costs, scalability, and safety. Transient expression offers the ...

characterization-complex-systems-using-design-experiments-approach

Research

JoVE Journal

Bioengineering

16.4K visualizações.  RWTH Aachen University.  Descreve-se um desenho de experiências abordagem que pode ser utilizada para determinar e modelar a influência dos elementos de regulação de transgenes, de crescimento e de desenvolvimento de parâmetros de planta, e as condições de incubação sobre a expressão transiente de anticorpos monoclonais e proteínas repórter em plantas. 

Vídeo: Characterization of Complex Systems Using the Design of Experiments Approach: Transient Protein Expression in Tobacco as a Case Study

High-throughput Protein Expression Generator Using a Microfluidic Platform

Rapidly increasing fields, such as systems biology, require the development and implementation of new technologies, enabling high-throughput and high-fidelity measurements of large systems. Microfluidics promises to fulfill many of these requirements, such as performing high-throughput screening experiments on-chip, encompassing biochemical, biophysical, and cell-based assays1. Since the early days of microfluidics devices, this field has drastically evolved, leading to the development of microfluidic large-scale integration2,3. This technology allows for the integration of thousands of micromechanical valves on a single device with a postage-sized footprint (Figure 1). We have developed a high-throughput microfluidic platform for generating in vitro expression of protein arrays (Figure 2) named PING (Protein Interaction Network Generator). These arrays can serve as a template for many experiments such as protein-protein 4, protein-RNA5 or protein-DNA6 interactions.
The device consist of thousands of reaction chambers, which are individually programmed using a microarrayer. Aligning of these printed microarrays to microfluidics devices programs each chamber with a single spot eliminating potential contamination or cross-reactivity Moreover, generating microarrays using standard microarray spotting techniques is also very modular, allowing for the arraying of proteins7, DNA8, small molecules, and even colloidal suspensions. The potential impact of microfluidics on biological sciences is significant. A number of microfluidics based assays have already provided novel insights into the structure and function of biological systems, and the field of microfluidics will continue to impact biology.

We present a microfluidic approach for the expression of protein arrays. The device consists of thousands of reaction chambers controlled by micro-mechanical valves. The microfluidic device is mated to a microarray-printed gene library. These genes are then transcribed and translated on-chip, resulting in a protein array ready for experimental use.

Alto rendimento Gerador Protein Expression Usando uma plataforma microfluídica

Rapidly increasing fields, such as systems biology, require the development and implementation of new technologies, enabling high-throughput and ...

Bioengenharia

Environmentally-controlled Microtensile Testing of Mechanically-adaptive Polymer Nanocomposites for ex vivo Characterization

Implantable microdevices are gaining significant attention for several biomedical applications1-4. Such devices have been made from a range of materials, each offering its own advantages and shortcomings5,6. Most prominently, due to the microscale device dimensions, a high modulus is required to facilitate implantation into living tissue. Conversely, the stiffness of the device should match the surrounding tissue to minimize induced local strain7-9. Therefore, we recently developed a new class of bio-inspired materials to meet these requirements by responding to environmental stimuli with a change in mechanical properties10-14. Specifically, our poly(vinyl acetate)-based nanocomposite (PVAc-NC) displays a reduction in stiffness when exposed to water and elevated temperatures (e.g. body temperature). Unfortunately, few methods exist to quantify the stiffness of materials in vivo15, and mechanical testing outside of the physiological environment often requires large samples inappropriate for implantation. Further, stimuli-responsive materials may quickly recover their initial stiffness after explantation. Therefore, we have developed a method by which the mechanical properties of implanted microsamples can be measured ex vivo, with simulated physiological conditions maintained using moisture and temperature control13,16,17. 
To this end, a custom microtensile tester was designed to accommodate microscale samples13,17 with widely-varying Young's moduli (range of 10 MPa to 5 GPa). As our interests are in the application of PVAc-NC as a biologically-adaptable neural probe substrate, a tool capable of mechanical characterization of samples at the microscale was necessary. This tool was adapted to provide humidity and temperature control, which minimized sample drying and cooling17. As a result, the mechanical characteristics of the explanted sample closely reflect those of the sample just prior to explantation.
The overall goal of this method is to quantitatively assess the in vivo mechanical properties, specifically the Young's modulus, of stimuli-responsive, mechanically-adaptive polymer-based materials. This is accomplished by first establishing the environmental conditions that will minimize a change in sample mechanical properties after explantation without contributing to a reduction in stiffness independent of that resulting from implantation. Samples are then prepared for implantation, handling, and testing (Figure 1A). Each sample is implanted into the cerebral cortex of rats, which is represented here as an explanted rat brain, for a specified duration (Figure 1B). At this point, the sample is explanted and immediately loaded into the microtensile tester, and then subjected to tensile testing (Figure 1C). Subsequent data analysis provides insight into the mechanical behavior of these innovative materials in the environment of the cerebral cortex.

Environmentally-controlled Microtensile Testing of Mechanically-adaptive Polymer Nanocomposites for ex vivo Characterization

A method is discussed by which the in vivo mechanical behavior of stimuli-responsive materials is monitored as a function of time. Samples are tested ex vivo using a microtensile tester with environmental controls to simulate the physiological environment. This work further promotes understanding the in vivo behavior of our material.

Teste de microtração ambientalmente controlado mecanicamente adaptativas nanocompósitos poliméricos para<em&gt; Ex vivo</em&gt; Caracterização

Implantable microdevices are gaining significant attention for several biomedical applications1-4. Such devices have been made from a range of materials, ...

A Versatile Automated Platform for Micro-scale Cell Stimulation Experiments

Study of cells in culture (in vitro analysis) has provided important insight into complex biological systems. Conventional methods and equipment for in vitro analysis are well suited to study of large numbers of cells (&ge;105) in milliliter-scale volumes (&ge;0.1 ml). However, there are many instances in which it is necessary or desirable to scale down culture size to reduce consumption of the cells of interest and/or reagents required for their culture, stimulation, or processing. Unfortunately, conventional approaches do not support precise and reproducible manipulation of micro-scale cultures, and the microfluidics-based automated systems currently available are too complex and specialized for routine use by most laboratories. To address this problem, we have developed a simple and versatile technology platform for automated culture, stimulation, and recovery of small populations of cells (100 - 2,000 cells) in micro-scale volumes (1 - 20 &mu;l). The platform consists of a set of fibronectin-coated microcapillaries ("cell perfusion chambers"), within which micro-scale cultures are established, maintained, and stimulated; a digital microfluidics (DMF) device outfitted with "transfer" microcapillaries ("central hub"), which routes cells and reagents to and from the perfusion chambers; a high-precision syringe pump, which powers transport of materials between the perfusion chambers and the central hub; and an electronic interface that provides control over transport of materials, which is coordinated and automated via pre-determined scripts. As an example, we used the platform to facilitate study of transcriptional responses elicited in immune cells upon challenge with bacteria. Use of the platform enabled us to reduce consumption of cells and reagents, minimize experiment-to-experiment variability, and re-direct hands-on labor. Given the advantages that it confers, as well as its accessibility and versatility, our platform should find use in a wide variety of laboratories and applications, and prove especially useful in facilitating analysis of cells and stimuli that are available in only limited quantities.

We have developed an automated cell culture and interrogation platform for micro-scale cell stimulation experiments. The platform offers simple, versatile, and precise control in cultivating and stimulating small populations of cells, and recovering lysates for molecular analyses. The platform is well suited to studies that use precious cells and/or reagents.

A plataforma automatizada versátil para experiências de estimulação Micro escala celulares

Study of cells in culture (in vitro analysis) has provided important insight into complex biological systems. Conventional methods and equipment for in ...

Real Time Monitoring of Intracellular Bile Acid Dynamics Using a Genetically Encoded FRET-based Bile Acid Sensor

F&#246;rster Resonance Energy Transfer (FRET) has become a powerful tool for monitoring protein folding, interaction and localization in single cells. Biosensors relying on the principle of FRET have enabled real-time visualization of subcellular signaling events in live cells with high temporal and spatial resolution. Here, we describe the application of a genetically encoded Bile Acid Sensor (BAS) that consists of two fluorophores fused to the farnesoid X receptor ligand binding domain (FXR-LBD), thereby forming a bile acid sensor that can be activated by a large number of bile acids species and other (synthetic) FXR ligands. This sensor can be targeted to different cellular compartments including the nucleus (NucleoBAS) and cytosol (CytoBAS) to measure bile acid concentrations locally. It allows rapid and simple quantitation of cellular bile acid influx, efflux and subcellular distribution of endogenous bile acids without the need for labeling with fluorescent tags or radionuclei. Furthermore, the BAS FRET sensors can be useful for monitoring FXR ligand binding. Finally, we show that this FRET biosensor can be combined with imaging of other spectrally distinct fluorophores. This allows for combined analysis of intracellular bile acid dynamics and i) localization and/or abundance of proteins of interest, or ii) intracellular signaling in a single cell.

We provide a detailed protocol to study bile acid dynamics in living cells using a genetically encoded BAS FRET sensor. This Bile Acid Sensor represents a unique tool to study (regulation of) bile acid transport and FXR activation in a wide range of cell types.

Monitoramento em Tempo Real de intracelular Bile Ácido Dynamics usando um sensor Ácido Bile baseado em FRET codificados geneticamente

F&#246;rster Resonance Energy Transfer (FRET) has become a powerful tool for monitoring protein folding, interaction and localization in single cells. ...

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate (LAL) Assays

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause inflammation, fever, hypo- or hypertension, and, in extreme cases, can lead to tissue and organ damage that may become fatal. The amounts of endotoxin in pharmaceutical products, therefore, are strictly regulated. Among the methods available for endotoxin detection and quantification, the Limulus Amoebocyte Lysate (LAL) assay is commonly used worldwide. While any pharmaceutical product can interfere with the LAL assay, nano-formulations represent a particular challenge due to their complexity. The purpose of this paper is to provide a practical guide to researchers inexperienced in estimating endotoxins in engineered nanomaterials and nanoparticle-formulated drugs. Herein, practical recommendations for performing three LAL formats including turbidity, chromogenic and gel-clot assays are discussed. These assays can be used to determine endotoxin contamination in nanotechnology-based drug products, vaccines, and adjuvants.

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate LAL Assays

Detection of endotoxins in engineered nanomaterials represents one of the grand challenges in the field of nanomedicine. Here, we present a case study that describes the framework composed of three different LAL formats to estimate potential endotoxin contamination in nanoparticles.

Deteção da endotoxina em Nano-formulações usando Limulus Amoebocyte Lysate (LAL) ensaios

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause ...

Transient Expression in Red Beet of a Biopharmaceutical Candidate Vaccine for Type-1 Diabetes

Plant molecular farming is the use of plants to produce molecules of interest. In this perspective, plants may be used both as bioreactors for the production and subsequent purification of the final product and for the direct oral delivery of heterologous proteins when using edible plant species. In this work, we present the development of a candidate oral vaccine against Type 1 Diabetes (T1D) in edible plant systems using deconstructed plant virus-based recombinant DNA technology, delivered with vacuum infiltration. Our results show that a red beet is a suitable host for the transient expression of a human derived autoantigen associated to T1D, considered to be a promising candidate as a T1D vaccine. Leaves producing the autoantigen were thoroughly characterized for their resistance to gastric digestion, for the presence of residual bacterial charge and for their secondary metabolic profile, giving an overview of the process production for the potential use of plants for direct oral delivery of a heterologous protein. Our analysis showed almost complete degradation of the freeze-dried candidate oral vaccine following a simulated gastric digestion, suggesting that an encapsulation strategy in the manufacture of the plant-derived GAD vaccine is required.

Here, we present a protocol to produce an oral vaccine candidate against Type 1 diabetes in an edible plant.

Expressão transiente em beterraba vermelha de uma vacina candidata biofarmacêutica para Diabetes tipo-1

Plant molecular farming is the use of plants to produce molecules of interest. In this perspective, plants may be used both as bioreactors for the ...

A Multilayer Microfluidic Platform for the Conduction of Prolonged Cell-Free Gene Expression

The limitations of cell-based synthetic biology are becoming increasingly apparent as researchers aim to develop larger and more complex synthetic genetic regulatory circuits. The analysis of synthetic genetic regulatory networks in vivo is time consuming and suffers from a lack of environmental control, with exogenous synthetic components interacting with host processes resulting in undesired behavior. To overcome these issues, cell-free characterization of novel circuitry is becoming more prevalent. In vitro transcription and translation (IVTT) mixtures allow the regulation of the experimental environment and can be optimized for each unique system. The protocols presented here detail the fabrication of a multilayer microfluidic device that can be utilized to sustain IVTT reactions for prolonged durations. In contrast to batch reactions, where resources are depleted over time and (by-) products accumulate, the use of microfluidic devices allows the replenishment of resources as well as the removal of reaction products. In this manner, the cellular environment is emulated by maintaining an out-of-equilibrium environment in which the dynamic behavior of gene circuits can be investigated over extended periods of time. To fully exploit the multilayer microfluidic device, hardware and software have been integrated to automate the IVTT reactions. By combining IVTT reactions with the microfluidic platform presented here, it becomes possible to comprehensively analyze complex network behaviors, furthering our understanding of the mechanisms that regulate cellular processes.

The fabrication process of a PDMS-based, multilayer, microfluidic device that allows in vitro transcription and translation (IVTT) reactions to be performed over prolonged periods is described. Furthermore, a comprehensive overview of the hardware and software required to automate and maintain these reactions for prolonged durations is provided.

Uma plataforma Microfluídico multicamada para a condução de expressão gênica sem células prolongada

The limitations of cell-based synthetic biology are becoming increasingly apparent as researchers aim to develop larger and more complex synthetic genetic ...

In Vesiculo Synthesis of Peptide Membrane Precursors for Autonomous Vesicle Growth

Compartmentalization of biochemical reactions is a central aspect of synthetic cells. For this purpose, peptide-based reaction compartments serve as an attractive alternative to liposomes or fatty acid-based vesicles. Externally or within the vesicles, peptides can be easily expressed and simplify the synthesis of membrane precursors. Provided here is a protocol for the creation of vesicles with diameters of ~200 nm based on the amphiphilic elastin-like polypeptides (ELP) utilizing dehydration-rehydration from glass beads. Also presented are protocols for bacterial ELP expression and purification via inverse temperature cycling, as well as their covalent functionalization with fluorescent dyes. Furthermore, this report describes a protocol to enable the transcription of RNA aptamer dBroccoli inside ELP vesicles as a less complex example for a biochemical reaction. Finally, a protocol is provided, which allows in vesiculo expression of fluorescent proteins and the membrane peptide, whereas synthesis of the latter results in vesicle growth.

Presented here are protocols for the creation of peptide-based small unilamellar vesicles capable of growth. To facilitate in vesiculo production of the membrane peptide, these vesicles are equipped with a transcription-translation system and the peptide-encoding plasmid.

Na síntese de Vesiculo de precursores de membrana peptídica para crescimento de vesículas autônomas

Compartmentalization of biochemical reactions is a central aspect of synthetic cells. For this purpose, peptide-based reaction compartments serve as an ...

High-Throughput Identification of Resistance to Pseudomonas syringae pv. Tomato in Tomato using Seedling Flood Assay

Tomato is an agronomically important crop that can be infected by Pseudomonas syringae, a Gram-negative bacterium, resulting in bacterial speck disease. The tomato-P. syringae pv. tomato pathosystem is widely used to dissect the genetic basis of plant innate responses and disease resistance. While disease was successfully managed for many decades through the introduction of the Pto/Prf gene cluster from Solanum pimpinellifolium into cultivated tomato, race 1 strains of P. syringae have evolved to overcome resistance conferred by the Pto/Prf gene cluster and occur worldwide.
Wild tomato species are important reservoirs of natural diversity in pathogen recognition, because they evolved in diverse environments with different pathogen pressures. In typical screens for disease resistance in wild tomato, adult plants are used, which can limit the number of plants that can be screened due to their extended growth time and greater growth space requirements. We developed a method to screen 10-day-old tomato seedlings for resistance, which minimizes plant growth time and growth chamber space, allows a rapid turnover of plants, and allows large sample sizes to be tested. Seedling outcomes of survival or death can be treated as discrete phenotypes or on a resistance scale defined by amount of new growth in surviving seedlings after flooding. This method has been optimized to screen 10-day-old tomato seedlings for resistance to two P. syringae strains and can easily be adapted to other P. syringae strains.

High-Throughput Identification of Resistance to Pseudomonas syringae pv. Tomato in Tomato using Seedling Flood Assay

The seedling flood assay facilitates rapid screening of wild tomato accessions for the resistance to the Pseudomonas syringae bacterium. This assay, used in conjunction with the seedling bacterial growth assay, can assist in further characterizing the underlying resistance to the bacterium, and can be used to screen mapping populations to determine the genetic basis of resistance.

Identificação de alta duração da resistência a Pseudomonas syringae pv. Tomate no Tomate usando Ensaio de Inundação de Mudas

Tomato is an agronomically important crop that can be infected by Pseudomonas syringae, a Gram-negative bacterium, resulting in bacterial speck disease. ...

Layer Microdissection of Tricuspid Valve Leaflets for Biaxial Mechanical Characterization and Microstructural Quantification

The tricuspid valve (TV) regulates the unidirectional flow of unoxygenated blood from the right atrium to the right ventricle. The TV consists of three leaflets, each with unique mechanical behaviors. These variations among the three TV leaflets can be further understood by examining their four anatomical layers, which are the atrialis (A), spongiosa (S), fibrosa (F), and ventricularis (V). While these layers are present in all three TV leaflets, there are differences in their thicknesses and microstructural constituents that further influence their respective mechanical behaviors. 
This protocol includes four steps to elucidate the layer-specific differences: (i) characterize the mechanical and collagen fiber architectural behaviors of the intact TV leaflet, (ii) separate the composite layers (A/S and F/V) of the TV leaflet, (iii) carry out the same characterizations for the composite layers, and (iv) perform post-hoc histology assessment. This experimental framework uniquely allows the direct comparison of the intact TV tissue to each of its composite layers. As a result, detailed information regarding the microstructure and biomechanical function of the TV leaflets can be collected with this protocol. Such information can potentially be used to develop TV computational models that seek to provide guidance for the clinical treatment of TV disease.

This protocol describes the biaxial mechanical characterization, polarized spatial frequency domain imaging-based collagen quantification, and microdissection of tricuspid valve leaflets. The provided method elucidates how the leaflet layers contribute to the holistic leaflet behaviors.

Microdisseção de camada de folhetos de válvulas tricúsplicas para caracterização mecânica biaxial e quantificação microestrutural

The tricuspid valve (TV) regulates the unidirectional flow of unoxygenated blood from the right atrium to the right ventricle. The TV consists of three ...