The authors would like to thank Prof. Sze-Bi Hsu and Ms. Zhenzhen for useful discussions and help in the theoretical analysis and numerical simulation. Y. T. Y. would like to acknowledge funding support from the Ministry of Science and Technology under grant numbers MOST 103-2220-E-007-026 and MOST 104-2220-E-007-011, and from the National Tsing Hua University under grant numbers 103N2042E1, 104N2042E1, and 105N518CE1.

1. Montagem e pré-teste do dispositivo Morbidostat <ol><li> Assembleia da Morbidostat <ol><li> Soco 3 furos na tampa do frasco de cultura com uma agulha de seringa 18 G. Corte três peças de tubagem de polietileno ~ 7 cm de comprimento. Inserir essas três pedaços de tubos de polietileno na tampa. </li><li> Use fita para envolver a borda da tampa para servir como o elenco para a mistura de polidimetilsiloxano (PDMS). Dissolvem-se 5 g de componente A e 0,5 g de componente B de o PDMS em um recipiente de plástico de 150 ml por agitação manualmente com um palito. Carregar a mistura em uma seringa de 10 ml. <ol><li> Despeje a mistura de PDMS na tampa com a seringa. Asse toda a tampa para curar o PDMS durante 8 horas na estufa a 70 ° C. </li></ol></li><li> Soldar o ânodo LED com o fio de calibre 24 e catodo LED com o resistor de carbono com um ferro de solda. Solde um resistor de 680 Ω de carbono com um fio de 24 g. Isolar a ligação do fio usando fita isolante. </li><li> assimlder no colector do fotodetector com o G fio 24 e o emissor da célula fotoeléctrica com uma resistência de 100 kQ carbono. Isolar a ligação do fio usando fita isolante. </li><li> Coloque o diodo emissor de luz no suporte de cultura do frasco. Seguindo o esquema de circuitos na Suplementar Figura 2, conecte o diodo emissor de luz e ligá-lo com uma fonte de alimentação de 5 V. Verifique se o LED funciona usando uma câmera digital que pode detectar IR (por exemplo, uma câmera de telefone celular). </li><li> Colocar o fotodetector no suporte do frasco de cultura. Seguindo o esquema de circuitos na Suplementar Figura 2, conecte o fotodetector e ligá-lo com uma fonte de alimentação de 5 V. Ligue o fio de ambos os lados das resistências de fotodetectores para medir a tensão através da placa de controle eletrônico. </li><li> Cole um íman sobre o veio da ventoinha de arrefecimento, o qual serve como unidade de agitador magnético. Note-se que o alignmENT do eixo do ventilador para o frasco de cultura, e o espaçamento entre o frasco de cultura e o íman é muito crítica para a função adequada da unidade de agitação magnética. </li><li> Ligue cada pedaço de tubo de polietileno do frasco de cultura para a parte correspondente de tubo de silicone para os micro-bombas, garrafas médias e frasco de resíduos. Ligar a bomba durante 1 hora e medir o volume total a ser bombeado a partir da garrafa de forma para o frasco de cultura para determinar a taxa de bombagem. Note-se que a taxa de bombeamento típico deve ser ~ 4 ml / h, o que corresponde à taxa de diluição de D = 0,33 h -1 para a 12 ml de volume do frasco de cultura de trabalho. </li><li> Coloque todo o conjunto em uma incubadora shaker médio porte (34 cm x 34 cm x 43,5 cm). </li></ol></li><li> O pré-teste do Morbidostat <ol><li> Set tensão de alimentação do ventilador de fresco a 5 V para iniciar o seu motor para testar a barra de agitação magnética. Note-se que a ação de agitação pode ser inspeccionados visualmente. A tensão que conduzirs o motor do ventilador de arrefecimento é precisamente controlado por modulação de largura de pulso (PWM). </li><li> Preparar uma série de tipo selvagem E. Amostras de E. coli com as densidades ópticas a 600 nm, tipicamente variando 0,07-0,3 para a calibração. Coloque um teste E. amostra coli no frasco de cultura e registar a tensão fotodetector. <ol><li> Ponto ~ 100 uL da mesma amostra no leitor de placas e gravar a densidade óptica. Use a tensão fotodetector em função dos dados de densidade óptica para traçar a curva de calibração. Note-se que tipicamente, uma unidade de variação na densidade óptica corresponde a 7,6 V ~ mudança no fotodetector com o esquema de polarização utilizado aqui. </li></ol></li></ol></li></ol> 2. A execução do Morbidostat <ol><li> Preparação Antes de começar a experiência diária <ol><li> Prepara-se o frasco de cultura com três ultra-resistentes a substâncias químicas tubos Tygon com diâmetro interior de 1/32 polegadas e diâmetro externo de 3/32 de polegada para entradas para formar o dispositivo como na secção1.1 e na Figura Suplementar 1. </li><li> Preparar o meio M9 mínimo (0,2% de glicose) e trimetoprim (TMP) de drogas contendo meio. Esteriliza-se o meio, o meio de garrafa, a garrafa forma de droga, o frasco de resíduos, e o frasco de cultura num autoclave a 121 ° C. NOTA: O TMP é uma solução de reserva que é utilizado para alcançar as concentrações mais tarde no Quadro 1 e a droga TMP contendo meio não é autoclavado. </li></ol></li><li> Executando o Morbidostat <ol><li> No primeiro dia do experimento, descongelar 1 ml de tipo selvagem congelado E. coli MG1655 durante 5 min à temperatura ambiente e a transferência de 120 ul das células para o frasco de cultura contendo ~ 12 mL de meio de crescimento M9. Após o primeiro dia, descongelar 1 ml de E. congelado coli células a partir do dia anterior durante 5 min e transferência de 120 uL de células para um novo frasco de cultura contendo ~ 12 mL de meio de crescimento M9. </li><li> Ligue a incubadora shaker e definir a tempe<html> ratura de 30 ° C sem agitação. </li><li> Inicie a operação morbidostat girando sobre o micro-bomba, a unidade de agitação magnética, e a medição da densidade óptica. Nota: Durante a operação, um algoritmo de limiar de realimentação é utilizado para controlar a injecção de drogas. Quando a densidade óptica medida exceder um limite pré-definido, a bomba de injecção de drogas seria ligado e a bomba médio seria desligado. Caso contrário, a bomba de injecção de drogas permanece desligado ea bomba médio está ligado. </li><li> Monitorar a tensão ao longo do tempo para assegurar que não é o crescimento microbiano. Nota: Devido à congelação e descongelação ciclo, E. coli células apresentam um atraso de ~ 4 horas durante o crescimento de cada dia. </li><li> Depois de ~ 23 h, parar a micro-bomba desligando a tensão de alimentação e retire o frasco de cultura. Adiciona-se 15% de glicerol no frasco de cultura e congelar a amostra a -80 ° C durante o armazenamento. </li><li> Repita os passos 2.2.1 a 2.2.5. </li></ol></li></ol>le &quot;&gt; 3. Antibiótico Susceptibilidade Medição em 96 Bem Format NOTA: Executar a medição da taxa de crescimento em um leitor de placas com um formato de 96 poços para determinar o nível de resistência a antibióticos. <ol><li> Descongelar a amostra congelada diária à temperatura ambiente durante 5 min e transferir 15 ul da célula para o frasco de cultura contendo ~ 15 mL de meio M9. Permitir-lhes crescer até a densidade óptica a 600 nm atingir ~ 0,1. Dividir as 15 ml em frascos de 1 ml e adicionar o fármaco TMP nesta etapa para se obter as concentrações de droga desejadas, como mostrado na Tabela 1. </li><li> Tome 200 ul da amostra a partir do passo 3.1 e local para cada poço na placa de leitor e permitir-lhes crescer durante 12 horas 5. A densidade óptica no comprimento de onda de 600 nm é registado automaticamente durante a medição. NOTA: Para cada ponto de dados, as medições triplicadas são registrados e em média para garantir a consistência dos dados. Os dados sobre a densidade óptica em função do tempo pode be utilizado para obter a taxa de crescimento. </li><li> Plotar os dados da taxa de crescimento em comparação com a concentração da droga para obter IC50. Note-se que IC50 é definido como a concentração de fármaco à qual a taxa de crescimento é de cinquenta por cento da taxa de crescimento máxima 5. </li></ol> 4. única célula Morfologia e Antibiótico Susceptibilidade Medição em microcanais <ol><li> Realizar a fabricação dos dispositivos microfluídicos via litografia macia multicamadas de PDMS conforme já descrito 15. <ol><li> Use fotolitografia para fazer o molde fotorresistente para os níveis de controlo e de fluxo. Note-se que para a camada de controlo, utilizar fotorresistente negativa (~ 15? M de altura) e fotorresistente positivo (~ 8 uM altura) para produzir um molde sobre uma bolacha de silício nu. </li><li> Preparar misturas de PDMS com rácios de reticulação de 5: 1 e 20: 1 para a camada de controlo e camada de fluxo, respectivamente. Coloque as misturas em um exsicador sob vácuo para remover quaisquer bolhas, Durante 1 h. </li><li> Exponha o controlo moldes camada fotossensível em bolacha de silício para trimetilclorosilano vapor (TMCS). Adicione a camada de espessura ~ 6 milímetros de controlo, lançando-se a mistura de PDMS (com razão de reticulação 5: 1) sobre a bolacha de silício sobre o molde de controle camada foto-resistente. Cozer a camada de controlo durante 40 minutos a 80 ° C durante 1 hora no forno. </li><li> Adicione a camada de fluxo de rotação por revestimento de uma mistura de PDMS (com reticulação proporção de 20: 1, velocidade de rotação 3000 rpm durante 60 seg) em um molde fotorresistente camada de escoamento. Cozer a camada de fluxo durante 30 min a 80 ° C durante 1 hora no forno. </li><li> Use uma lâmina de barbear para cortar a camada de controle para o tamanho do chip desejado (tipicamente 3 cm x 2 cm) e manualmente retire a camada de controle. Coloque a camada de controlo no topo da camada de escoamento e alinhá-los sob um estereomicroscópio. Curar a montagem alinhada a 80 ° C na noite forno. Em seguida, mergulhe o conjunto em 250 ml de água deionizada em um recipiente de plástico durante 5 horas para dissolver o TMCS residual. </li><li> Subsequentemente, perfurar os orifícios de entrada com 20 g de agulhas e ligar todo o elastómero a uma lâmina de vidro revestida com uma camada de PDMS em branco (PDMS reticulação proporção de 5: 1, velocidade de rotação 2000 rpm durante 30 seg) por tratamento com plasma de ar de 40 seg a 1,2 Torr de pressão de ar. </li><li> Realizar ampla cozimento durante 36 h a 80 ° C para reduzir os efeitos citotóxicos de PDMS. Note-se que esta fase de cozedura é crítica para minimizar a toxicidade para as células bacterianas. </li></ol></li><li> No Experimento Crescimento Chip <ol><li> Antes de carregar no dispositivo de microfluidos, lavá-la com água desionizada durante 1 hora e meio M9 durante 1 h. </li><li> Descongelar a amostra congelada diária à temperatura ambiente durante 5 min e inocular um novo tubo de ensaio com meio fresco M9. Colocar a amostra num incubador com agitação a 37 ° C durante a noite. </li><li> Diluir o microbiana amostra de 10 vezes com meio M9 e carregá-lo para dentro do dispositivo microfluídico pressionando o recipiente da amostra microbiana a 5 psi. Em seguida, carregar a droga TMPmédio no chip pressionando o recipiente do meio de droga em 5 psi. Executar a mistura entre o micróbio e fármaco por actuação da válvula de micromecânica entre as duas câmaras no chip de microfluidos durante 15 min. </li><li> Colocar o chip de microfluidos na incubadora microscópio montado no microscópio invertido. Adquirem a imagem de células na câmara de crescimento a cada HR durante 8 horas com a câmara CCD montada sobre o microscópio invertido. </li><li> Use o script programa personalizado para calcular o número de células por meio de um algoritmo de limite sobre os dados de imagem de células 18. Note-se que os dados do número de células são calculados normalmente mais de três câmaras microfluídicos. </li></ol></li></ol>

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The authors have nothing to disclose.

Um dispositivo morbidostat baixa pegada de componentes de baixo custo é demonstrada. Os aumentos no nível de resistência a drogas registadas pelo dispositivo são consistentes com os dos relatórios anteriores 5. Concebido para estudos evolutivos de resistência a drogas, o dispositivo é potencialmente aplicável a muitas outras experiências. Primeiro, um banco de dados abrangente de mutações induzidas pela droga pode ser estabelecido para um grande conjunto de antibióticos clinicamente relevantes. Po...

Desde a introdução da primeira penicilina antibiótico, a resistência aos antibióticos microbiana tornou-se um problema de saúde global 1. Embora a aquisição de resistência a antibióticos pode ser retrospectivamente estudados in vivo, as condições destas experiências muitas vezes não são controlados ao longo de toda a evolução 2. Alternativamente, evolução adaptativa laboratório pode revelar a evolução molecular de uma determinada espécie microbiana sob stresses ambientais ou pressão de seleção de um medicamento antibiótico 3. Recentemente, muitas experiências evolutivas bem controlados de resistência aos medicamentos antibióticos elucidaram o surgimento de resistência aos medicamentos antibióticos. Por exemplo, o grupo de Austin demonstrado surgimento rápido num ambiente compartimentado microfluidos adequadamente manipulado 4. O morbidostat recentemente desenvolvido induz a mutações sistemáticas sob droga pressão de seleção 5,6. O morbidostat, uma selec microbianação dispositivo que ajusta continuamente a concentração de antibiótico para manter uma população praticamente constante, é um grande avanço a partir do teste de flutuação utilizado em microbiologia 7,8. No teste de flutuação, uma droga antibiótica é injectada a alta concentração, e os sobreviventes mutantes são rastreadas e contadas. Em vez disso, os micróbios em um morbidostat são constantemente desafiados e adquirir múltiplas mutações. O morbidostat opera de forma semelhante ao quimiostato, cultura de um dispositivo inventado por Novick e Szliard em 1950 que mantém uma população constante através do fornecimento de nutrientes continuamente enquanto que a diluição da população microbiana 9. Desde a sua introdução, o quemostato tem sido avançado e melhorado. quimiostatos microfluídicos em curso alcançaram capacidades nanolitros e de uma única célula. No entanto, estes dispositivos não são adequados para experiências evolução adaptativa, que necessitam de uma população de células grandes com muitos eventos de mutação 10,11. Recentemente, mini-quimiostatos com volumes de trabalho de ~ 10 ml também foram desenvolvidos para preencher a lacuna entre biorreatores em escala litro eo microfluídico quemostato 12,13. Aqui apresentamos a concepção e utilização de um baixo custo, morbidostat automatizado para um estudo de resistência aos medicamentos antibióticos. O módulo proposto pode ser empregue num incubador agitador de laboratório com microbiologia requisito mínimo de hardware. O firmware open-source também é facilmente adaptado para aplicações específicas de evolução adaptativa, tais como engenharia metabólica 3. Finalmente, o morbidostat é integrado em uma plataforma microfluídica multiplexado para testes de sensibilidade aos antibióticos 14.

<table><tbody><tr><td>Environmental Shaker Incubator</td><td>BioSan</td><td>ES-20</td><td></td></tr><tr><td>Arduino Leonardo board</td><td>Arduino</td><td>Leonardo</td><td></td></tr><tr><td>680 Ohm Carbon Resistor</td><td>Digikey</td><td></td><td>Bias resistor for LED</td></tr><tr><td>100k Ohm Carbon resistor</td><td>Digikey</td><td></td><td>Bias resistor for phototransistor</td></tr><tr><td>940 nm light emitting diode</td><td>Bright LED Electronic</td><td>BIR-BM13E4G-2</td><td>Optical density measurement</td></tr><tr><td>940 nm phototransistor</td><td>Kodenshi&amp;nbsp;</td><td>ST-2L2B</td><td>Optical density measurement</td></tr><tr><td>Darlington pair IC Toshiba</td><td>Mouser</td><td>ULN2803APG</td><td>this IC drives micropumps and magnetic stirring unit</td></tr><tr><td>5 V DC brushless fan</td><td>ADDA</td><td>AD0405LX-G70</td><td>spec: 5 V supply voltage and 80&amp;nbsp;mA; available www.jameco.com</td></tr><tr><td>Piezoelectric micropump</td><td>CurieJet</td><td>PS15I-FT-5L</td><td>Pressure &gt; 3&amp;nbsp;kPa; Flow rate &gt;5 ml/min</td></tr><tr><td>Tygon 3350 Tuning</td><td>Saint Gobain</td><td>ABW00001</td><td>ID: 1/32&quot; OD: 3/32&quot; L:50&#039;&amp;nbsp;</td></tr><tr><td>Magnetic Stir bar</td><td>COWIE</td><td></td><td>tapered shape dim: 10&amp;nbsp;mm x 4 mm</td></tr><tr><td>Glass scintillation 20 ml vial</td><td>DGS</td><td></td><td>Pyrex glass 28 mm (diameter) x 61 mm (height)</td></tr><tr><td>Culture vial holder</td><td></td><td></td><td>Custom made from Polyformaldehyde&amp;nbsp;</td></tr><tr><td>Silicone</td><td>Dow Corning</td><td>Sylgald 184</td><td>used to seal the glass vial</td></tr><tr><td>Medium bottle</td><td>VWR</td><td>66022-065</td><td></td></tr><tr><td>Difco M9 minimal salt 5x</td><td>BD</td><td></td><td>Medium</td></tr><tr><td>Cadamino Acid</td><td>BD</td><td></td><td>Medium</td></tr><tr><td>glucose</td><td>Sigma</td><td></td><td></td></tr><tr><td>Agar Bateriological</td><td>Oxoid</td><td></td><td>for agar plate</td></tr><tr><td>Luria Bertani medium</td><td></td><td></td><td></td></tr><tr><td>Inverted microscope</td><td>Leica Microsystems</td><td>Leica DMI-LED</td><td>used for microfluidic measurement Use 40X objective NA = 0.55</td></tr><tr><td>Microscope Incubator</td><td>Live Cell Instrument</td><td>CU-109</td><td>used for microfluidic measurement</td></tr><tr><td>Solenoidal valves</td><td>Pneumadyne</td><td>S10MM-31-12-3</td><td>Normally open 1.3 Watt 12 Vdc</td></tr><tr><td>USB interface card</td><td>Hobby Engineering</td><td>USBIO24-R Digital I/O Module&amp;nbsp;</td><td>for microfluidics measurement</td></tr><tr><td>Air compressor</td><td>Rocker Scientific</td><td>ROCKER 440</td><td>Pressure source for microfluidcs; Max. Pressure 80 psi</td></tr><tr><td>Male luer-lock fittings to 1/8&quot; barb</td><td>ValuePlastics.com</td><td>MTLL230-1</td><td>used for microfluidic control</td></tr><tr><td>1/8&quot; barb to 10-32 threaded port</td><td>ValuePlastics.com</td><td>B-1</td><td>used for microfluidic control</td></tr><tr><td>Female luer-lock fittings to 10-32 threaded port</td><td>ValuePlastics.com</td><td>KFTL-1</td><td>used for microfluidic control</td></tr><tr><td>NPN darlington transistor 500 mA, 40&amp;nbsp;V (2N6427)</td><td>DigiKey.com</td><td>2N6427GOS-ND</td><td>used for microfluidic control</td></tr><tr><td>10 kOhm, carbon film resistor, 0.25 W</td><td>DigiKey.com</td><td>P10KBACT-ND</td><td>used for microfluidic control</td></tr><tr><td>Tantalum capacitor, 10&amp;nbsp;&amp;mu;F, 25 V, 10%</td><td>DigiKey.com</td><td>478-1841-ND</td><td>used for microfluidic control</td></tr><tr><td>Andor CCD camera</td><td>Andor</td><td>Zyla 4.2 Plus SCMOS</td><td>used for microfluidic on chip imaging</td></tr><tr><td>ELISA plate reader</td><td></td><td></td><td></td></tr><tr><td>two component Silicone&amp;nbsp;</td><td>Momentive</td><td>RTV 615</td><td>used for microfluidic chip fabrication</td></tr><tr><td>SU-8 photoresist</td><td>Micrchem</td><td>SU8 2015</td><td>used for microfluidic chip fabrication</td></tr><tr><td>AZ4620 photoresist</td><td>Clariant</td><td>AZ 4620</td><td>used for microfluidic chip fabrication</td></tr><tr><td>Plasma cleaner</td><td>Harrick Plasma</td><td>PDC 32G</td><td>used for microfluidic chip fabrication</td></tr><tr><td>20 Gauge Syringe Needle</td><td>BD</td><td></td><td>used for microfluidic chip fabrication</td></tr><tr><td>Labcycler</td><td>Sensoquest</td><td>Labcycler</td><td>PCR</td></tr><tr><td>DNA polymerase</td><td>Toyobo</td><td>KDO Plus</td><td>PCR amplification</td></tr><tr><td>Trimethoprim</td><td>Sigma</td><td></td><td></td></tr><tr><td>Plate reader</td><td>Biotek</td><td>Synergy H1 hybrid&amp;nbsp;</td><td>antibiotic resistane measurement</td></tr></tbody></table>

design and use of a low cost, automated morbidostat for adaptive evolution of bacteria under antibiotic drug selection

Descreve-se um baixo custo, morbidostat configurável para a caracterização da via evolutiva da resistência aos antibióticos. O morbidostat é um dispositivo de cultura bacteriana que monitora continuamente o crescimento bacteriano e ajusta dinamicamente a concentração da droga para desafiar constantemente as bactérias à medida que evoluem para adquirir resistência aos medicamentos. O dispositivo dispõe de um volume de trabalho de ~ 10 mL e é totalmente automatizado e equipado com medição de densidade óptica e micro-bombas para médio e entrega de droga. Para validar a plataforma, medimos a aquisição gradual de resistência trimetoprim em Escherichia coli MG 1655, e integrada com o dispositivo de microfluidos de uma plataforma multiplexado para investigar a morfologia das células e sensibilidade aos antibióticos. A abordagem pode ser up-dimensionado para estudos laboratoriais de resistência aos medicamentos antibióticos, e é extensível para adaptativa evolução de melhorias de tensão em engenharia metabólica e outros experimentos de cultura bacteriana.

O morbidostat acima descrito está esquematizado na Figura 1. As operações morbidostat comuns, incluindo a evolução experimental, teste de susceptibilidade aos antibióticos e a morfologia das células de controlo, foram validados em uma E. coli MG1655 cultura exposta ao trimetoprim (TMP), um fármaco antibiótico comumente utilizado 5,6. TMP induz aumentos graduais muito distintas em resistência aos medicamentos, e as mutações são agrupados e...

Watch this Scientific Journal Video about Design and Use of a Low Cost, Automated Morbidostat for Adaptive Evolution of Bacteria Under Antibiotic Drug Selection at JoVE.com

Design and Use of a Low Cost, Automated Morbidostat for Adaptive Evolution of Bacteria Under Antibiotic Drug Selection

We describe a low cost, configurable morbidostat that enables the characterization of antibiotic drug resistance by dynamically adjusting the drug concentration. The device can be integrated with a multiplexed microfluidic platform. The approach can be scaled up for laboratory antibiotic drug resistance studies.

Concepção e utilização de um baixo custo, Automated Morbidostat para Adaptive Evolução das bactérias sob Antibiótico Seleção de Drogas

We describe a low cost, configurable morbidostat for characterizing the evolutionary pathway of antibiotic resistance. The morbidostat is a bacterial ...

design-use-low-cost-automated-morbidostat-for-adaptive-evolution

Research

JoVE Journal

Genetics

9.7K visualizações.  National Tsing Hua University. We describe a low cost, configurable morbidostat that enables the characterization of antibiotic drug resistance by dynamically adjusting the drug concentration. The device can be integrated with a multiplexed microfluidic platform. The approach can be scaled up for laboratory antibiotic drug resistance studies.

Vídeo: Design and Use of a Low Cost, Automated Morbidostat for Adaptive Evolution of Bacteria Under Antibiotic Drug Selection

Procedure for Adaptive Laboratory Evolution of Microorganisms Using a Chemostat

Natural evolution involves genetic diversity such as environmental change and a selection between small populations. Adaptive laboratory evolution (ALE) refers to the experimental situation in which evolution is observed using living organisms under controlled conditions and stressors; organisms are thereby artificially forced to make evolutionary changes. Microorganisms are subject to a variety of stressors in the environment and are capable of regulating certain stress-inducible proteins to increase their chances of survival. Naturally occurring spontaneous mutations bring about changes in a microorganism's genome that affect its chances of survival. Long-term exposure to chemostat culture provokes an accumulation of spontaneous mutations and renders the most adaptable strain dominant. Compared to the colony transfer and serial transfer methods, chemostat culture entails the highest number of cell divisions and, therefore, the highest number of diverse populations. Although chemostat culture for ALE requires more complicated culture devices, it is less labor intensive once the operation begins. Comparative genomic and transcriptome analyses of the adapted strain provide evolutionary clues as to how the stressors contribute to mutations that overcome the stress. The goal of the current paper is to bring about accelerated evolution of microorganisms under controlled laboratory conditions.

Here, we present a protocol to obtain adaptive laboratory evolution of microorganisms under conditions using chemostat culture. Also, genomic analysis of the evolved strain is discussed.

Processo para o Adaptive Laboratório Evolução de microrganismos utilizando um quimiostato

Natural evolution involves genetic diversity such as environmental change and a selection between small populations. Adaptive laboratory evolution (ALE) ...

Genética

Testing the Role of Multicopy Plasmids in the Evolution of Antibiotic Resistance

Multicopy plasmids are extremely abundant in prokaryotes but their role in bacterial evolution remains poorly understood. We recently showed that the increase in gene copy number per cell provided by multicopy plasmids could accelerate the evolution of plasmid-encoded genes. In this work, we present an experimental system to test the ability of multicopy plasmids to promote gene evolution. Using simple molecular biology methods, we constructed a model system where an antibiotic resistance gene can be inserted into Escherichia coli MG1655, either in the chromosome or on a multicopy plasmid. We use an experimental evolution approach to propagate the different strains under increasing concentrations of antibiotics and we measure survival of bacterial populations over time. The choice of the antibiotic molecule and the resistance gene is so that the gene can only confer resistance through the acquisition of mutations. This &#34;evolutionary rescue&#34; approach provides a simple method to test the potential of multicopy plasmids to promote the acquisition of antibiotic resistance. In the next step of the experimental system, the molecular bases of antibiotic resistance are characterized. To identify mutations responsible for the acquisition of antibiotic resistance we use deep DNA sequencing of samples obtained from whole populations and clones. Finally, to confirm the role of the mutations in the gene under study, we reconstruct them in the parental background and test the resistance phenotype of the resulting strains.

Here we present an experimental method to test the role of multicopy plasmids in the evolution of antibiotic resistance.

Testando o papel de plasmídeos multicópia na evolução da resistência aos antibióticos

Multicopy plasmids are extremely abundant in prokaryotes but their role in bacterial evolution remains poorly understood. We recently showed that the ...

Designing Automated, High-throughput, Continuous Cell Growth Experiments Using eVOLVER

Continuous culture methods enable cells to be grown under quantitatively controlled environmental conditions, and are thus broadly useful for measuring fitness phenotypes and improving our understanding of how genotypes are shaped by selection. Extensive recent efforts to develop and apply niche continuous culture devices have revealed the benefits of conducting new forms of cell culture control. This includes defining custom selection pressures and increasing throughput for studies ranging from long-term experimental evolution to genome-wide library selections and synthetic gene circuit characterization. The eVOLVER platform was recently developed to meet this growing demand: a continuous culture platform with a high degree of scalability, flexibility, and automation. eVOLVER provides a single standardizing platform that can be (re)-configured and scaled with minimal effort to perform many different types of high-throughput or multi-dimensional growth selection experiments. Here, a protocol is presented to provide users of the eVOLVER framework a description for configuring the system to conduct a custom, large-scale continuous growth experiment. Specifically, the protocol guides users on how to program the system to multiplex two selection pressures &#8212; temperature and osmolarity &#8212; across many eVOLVER vials in order to quantify fitness landscapes of Saccharomyces cerevisiae mutants at fine resolution. We show how the device can be configured both programmatically, through its open-source web-based software, and physically, by arranging fluidic and hardware layouts. The process of physically setting up the device, programming the culture routine, monitoring and interacting with the experiment in real-time over the internet, sampling vials for subsequent offline analysis, and post experiment data analysis are detailed. This should serve as a starting point for researchers across diverse disciplines to apply eVOLVER in the design of their own complex and high-throughput cell growth experiments to study and manipulate biological systems.

The eVOLVER framework enables high-throughput continuous microbial culture with high resolution and dynamic control over experimental parameters. This protocol demonstrates how to apply the system to conduct a complex fitness experiment, guiding users on programming automated control over many individual cultures, measuring, collecting, and interacting with experimental data in real-time.

Projetando experimentos de crescimento de células contínuas, de alta produtividade e automatizados usando o eVOLVER

Continuous culture methods enable cells to be grown under quantitatively controlled environmental conditions, and are thus broadly useful for measuring ...

Quantification of Plasmid-Mediated Antibiotic Resistance in an Experimental Evolution Approach

Plasmids play a major role in microbial ecology and evolution as vehicles of lateral gene transfer and reservoirs of accessory gene functions in microbial populations. This is especially the case under rapidly changing environments such as fluctuating antibiotics exposure. We recently showed that plasmids maintain antibiotic resistance genes in Escherichia coli without positive selection for the plasmid presence. Here we describe an experimental system that allows following both the plasmid genotype and phenotype in long-term evolution experiments. We use molecular techniques to design a model plasmid that is subsequently introduced to an experimental evolution batch system approach in an E. coli host. We follow the plasmid frequency over time by applying replica plating of the E. coli populations while quantifying the antibiotic resistance persistence. In addition, we monitor the conformation of plasmids in host cells by analyzing the extent of plasmid multimer formation by plasmid nicking and agarose gel electrophoresis. Such an approach allows us to visualize not only the genome size of evolving plasmids but also their topological conformation-a factor highly important for plasmid inheritance. Our system combines molecular strategies with traditional microbiology approaches and provides a set-up to follow plasmids in bacterial populations over a long time. The presented approach can be applied to study a wide range of mobile genetic elements in the future.

Our experimental approach provides a strategy to follow plasmid abundance and antibiotic resistance over time in bacterial populations.

Quantificação da resistência aos antibióticos mediada por plasmídeos em uma abordagem de evolução experimental

Plasmids play a major role in microbial ecology and evolution as vehicles of lateral gene transfer and reservoirs of accessory gene functions in microbial ...

Establishment and Optimization of a High Throughput Setup to Study Staphylococcus epidermidis and Mycobacterium marinum Infection as a Model for Drug Discovery

Zebrafish are becoming a valuable tool in the preclinical phase of drug discovery screenings as a whole animal model with high throughput screening possibilities. They can be used to bridge the gap between cell based assays at earlier stages and in vivo validation in mammalian models, reducing, in this way, the number of compounds passing through to testing on the much more expensive rodent models. In this light, in the present manuscript is described a new high throughput pipeline using zebrafish as in vivo model system for the study of Staphylococcus epidermidis and Mycobacterium marinum infection. This setup allows the generation and analysis of large number of synchronous embryos homogenously infected. Moreover the flexibility of the pipeline allows the user to easily implement other platforms to improve the resolution of the analysis when needed. The combination of the zebrafish together with innovative high throughput technologies opens the field of drug testing and discovery to new possibilities not only because of the strength of using a whole animal model but also because of the large number of transgenic lines available that can be used to decipher the mode of action of new compounds.

Establishment and Optimization of a High Throughput Setup to Study Staphylococcus epidermidis and Mycobacterium marinum Infection as a Model for Drug Discovery

This video article describes the high throughput pipeline that has been successfully established to infect and analyze large numbers of zebrafish embryos providing a new powerful tool for compound testing and drug discovery using a whole animal vertebrate organism.

Criação e Otimização de um Setup de Alto Rendimento para o Estudo<em&gt; Staphylococcus epidermidis</em&gt; E<em&gt; Mycobacterium marinum</em&gt; Infecção como um modelo para a Descoberta de Drogas

Zebrafish are becoming a valuable tool in the preclinical phase of drug discovery screenings as a whole animal model with high throughput screening ...

Imunologia e Infecção

Daily Transfers, Archiving Populations, and Measuring Fitness in the Long-Term Evolution Experiment with Escherichia coli

The Long-Term Evolution Experiment (LTEE) has followed twelve populations of Escherichia coli as they have adapted to a simple laboratory environment for more than 35 years and 77,000 bacterial generations. The setup and procedures used in the LTEE epitomize reliable and reproducible methods for studying microbial evolution. In this protocol, we first describe how the LTEE populations are transferred to fresh medium and cultured each day. Then, we describe how the LTEE populations are regularly checked for possible signs of contamination and archived to provide a permanent frozen "fossil record" for later study. Multiple safeguards included in these procedures are designed to prevent contamination, detect various problems when they occur, and recover from disruptions without appreciably setting back the progress of the experiment. One way that the overall tempo and character of evolutionary changes are monitored in the LTEE is by measuring the competitive fitness of populations and strains from the experiment. We describe how co-culture competition assays are conducted and provide both a spreadsheet and an R package (fitnessR) for calculating relative fitness from the results. Over the course of the LTEE, the behaviors of some populations have changed in interesting ways, and new technologies like whole-genome sequencing have provided additional avenues for investigating how the populations have evolved. We end by discussing how the original LTEE procedures have been updated to accommodate or take advantage of these changes. This protocol will be useful for researchers who use the LTEE as a model system for studying connections between evolution and genetics, molecular biology, systems biology, and ecology. More broadly, the LTEE provides a tried-and-true template for those who are beginning their own evolution experiments with new microbes, environments, and questions.

Daily Transfers, Archiving Populations, and Measuring Fitness in the Long-Term Evolution Experiment with Escherichia coli

This protocol describes how to maintain the Escherichia coli Long-Term Evolution Experiment (LTEE) by performing its daily transfers and periodic freeze-downs and how to conduct competition assays to measure fitness improvements in evolved bacteria. These procedures can serve as a template for researchers starting their own microbial evolution experiments.

Transferências diárias, arquivamento de populações e medição da aptidão no experimento de evolução de longo prazo com Escherichia coli

The Long-Term Evolution Experiment (LTEE) has followed twelve populations of Escherichia coli as they have adapted to a simple laboratory environment for ...

Mutagenesis and Functional Selection Protocols for Directed Evolution of Proteins in E. coli

The efficient generation of genetic diversity represents an invaluable molecular tool that can be used to label DNA synthesis, to create unique molecular signatures, or to evolve proteins in the laboratory. Here, we present a protocol that allows the generation of large (>1011) mutant libraries for a given target sequence. This method is based on replication of a ColE1 plasmid encoding the desired sequence by a low-fidelity variant of DNA polymerase I (LF-Pol I). The target plasmid is transformed into a mutator strain of E. coli and plated on solid media, yielding between 0.2 and 1 mutations/kb, depending on the location of the target gene. Higher mutation frequencies are achieved by iterating this process of mutagenesis. Compared to alternative methods of mutagenesis, our protocol stands out for its simplicity, as no cloning or PCR are involved. Thus, our method is ideal for mutational labeling of plasmids or other Pol I templates or to explore large sections of sequence space for the evolution of activities not present in the original target. The tight spatial control that PCR or randomized oligonucleotide-based methods offer can also be achieved through subsequent cloning of specific sections of the library. Here we provide protocols showing how to create a random mutant library and how to establish drug-based selections in E. coli to identify mutants exhibiting new biochemical activities.

Mutagenesis and Functional Selection Protocols for Directed Evolution of Proteins in E. coli

Here we demonstrate a simple protocol to create a random mutant library for a given target sequence. We show how this method, which is performed in vivo in Escherichia coli, can be coupled with functional selections to evolve new enzymatic activities.

Mutagênese e Funcional Protocolos de Seleção para a Evolução Dirigida de proteínas em E. coli</em

The efficient generation of genetic diversity represents an invaluable molecular tool that can be used to label DNA synthesis, to create unique molecular ...

Biologia

The Use of Chemostats in Microbial Systems Biology

Cells regulate their rate of growth in response to signals from the external world. As the cell grows, diverse cellular processes must be coordinated including macromolecular synthesis, metabolism and ultimately, commitment to the cell division cycle. The chemostat, a method of experimentally controlling cell growth rate, provides a powerful means of systematically studying how growth rate impacts cellular processes - including gene expression and metabolism - and the regulatory networks that control the rate of cell growth. When maintained for hundreds of generations chemostats can be used to study adaptive evolution of microbes in environmental conditions that limit cell growth. We describe the principle of chemostat cultures, demonstrate their operation and provide examples of their various applications. Following a period of disuse after their introduction in the middle of the twentieth century, the convergence of genome-scale methodologies with a renewed interest in the regulation of cell growth and the molecular basis of adaptive evolution is stimulating a renaissance in the use of chemostats in biological research.

Cell growth rate is a regulated process and a primary determinant of cell physiology. Continuous culturing using chemostats enables extrinsic control of cell growth rate by nutrient limitation facilitating the study of molecular networks that control cell growth and how those networks evolve to optimize cell growth.

O Uso de quimiostatos em Microbial Biologia de Sistemas

Cells regulate their rate of growth in response to signals from the external world. As the cell grows, diverse cellular processes must be coordinated ...

Design and Use of Multiplexed Chemostat Arrays

Chemostats are continuous culture systems in which cells are grown in a tightly controlled, chemically constant environment where culture density is constrained by limiting specific nutrients.1,2 Data from chemostats are highly reproducible for the measurement of quantitative phenotypes as they provide a constant growth rate and environment at steady state. For these reasons, chemostats have become useful tools for fine-scale characterization of physiology through analysis of gene expression3-6 and other characteristics of cultures at steady-state equilibrium.7 Long-term experiments in chemostats can highlight specific trajectories that microbial populations adopt during adaptive evolution in a controlled environment. In fact, chemostats have been used for experimental evolution since their invention.8 A common result in evolution experiments is for each biological replicate to acquire a unique repertoire of mutations.9-13 This diversity suggests that there is much left to be discovered by performing evolution experiments with far greater throughput. 
We present here the design and operation of a relatively simple, low cost array of miniature chemostats&mdash;or ministats&mdash;and validate their use in determination of physiology and in evolution experiments with yeast. This approach entails growth of tens of chemostats run off a single multiplexed peristaltic pump. The cultures are maintained at a 20 ml working volume, which is practical for a variety of applications. It is our hope that increasing throughput, decreasing expense, and providing detailed building and operation instructions may also motivate research and industrial application of this design as a general platform for functionally characterizing large numbers of strains, species, and growth parameters, as well as genetic or drug libraries.

We developed and validated a small-footprint array of miniature chemostats built from readily available parts for low cost. Physiological and experimental evolution results were similar to larger volume chemostats. The ministat array provides a compact, inexpensive, and accessible platform for traditional chemostat experiments, functional genomics, and chemical screening applications.

Concepção e utilização de matrizes quemostato Multiplexados

Chemostats are continuous culture systems in which cells are grown in a tightly controlled, chemically constant environment where culture density is ...

An Automated Culture System for Use in Preclinical Testing of Host-Directed Therapies for Tuberculosis

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), was the most significant infectious disease killer globally until the advent of COVID-19. Mtb has evolved to persist in its intracellular environment, evade host defenses, and has developed resistance to many anti-tubercular drugs. One approach to solving resistance is identifying existing&#160;approved drugs that will boost the host immune response to Mtb. These drugs could then be repurposed as adjunctive host-directed therapies (HDT) to shorten treatment time and help overcome antibiotic resistance.
Quantification of intracellular Mtb growth in macrophages is a crucial aspect of assessing potential HDT. The gold standard for measuring Mtb growth is counting colony-forming units (CFU) on agar plates. This is a slow, labor-intensive assay that does not lend itself to rapid screening of drugs. In this protocol, an automated, broth-based culture system, which is more commonly used to detect Mtb in clinical specimens, has been adapted for preclinical screening of host-directed therapies. The capacity of the liquid culture assay system to investigate intracellular Mtb growth in macrophages treated with HDT was evaluated. The HDTs tested for their ability to inhibit Mtb growth were all-trans Retinoic acid (AtRA), both in solution and encapsulated in poly(lactic-co-glycolic acid) (PLGA) microparticles and the combination of interferon-gamma and linezolid. The advantages of this automated liquid culture-based technique over the CFU method include simplicity of setup, less labor-intensive preparation, and faster time to results (5-12 days compared to 21 days or more for agar plates).

Rapid and efficient quantification of intracellular M. tuberculosis growth is crucial for pursuing improved therapies against tuberculosis (TB). This protocol describes a broth-based colorimetric detection assay using an automated liquid culture system to quantify Mtb growth in macrophages treated with candidate host-directed therapies.

Um Sistema de Cultura Automatizado para Uso em Testes Pré-Clínicos de Terapias Dirigidas ao Hospedeiro para Tuberculose

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), was the most significant infectious disease killer globally until the advent ...

Concepção e utilização de um baixo custo, Automated Morbidostat para Adaptive Evolução das bactérias sob Antibiótico Seleção de Drogas

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