Name: high-throughput identification of resistance to pseudomonas syringae pv. tomato in tomato using seedling flood assay
Uploaded: 2020-03-10T14:00:00
Description: Watch this Scientific Journal Video about High-Throughput Identification of Resistance to Pseudomonas syringae pv. Tomato in Tomato using Seedling Flood Assay at JoVE.com

We thank Jamie Calma for testing the effect of media volume on disease or resistance outcomes. We thank Dr. Ma&#235;l Baudin and Dr. Karl J. Scheiber from the Lewis Lab for providing constructive comments and suggestions on the manuscript. Research on plant immunity in the Lewis laboratory was supported by the USDA ARS 2030-21000-046-00D and 2030-21000-050-00D (JDL), and the NSF Directorate for Biological Sciences IOS-1557661 (JDL).

1. Preparation and use of biosafety cabinet
<ol>
	<li>Wipe down biosafety cabinet with 70% ethanol.</li>
	<li>Close the sash and turn on the ultraviolet light in the biosafety cabinet for 15 min.</li>
	<li>After 15 min, turn off the ultraviolet light in the biosafety cabinet. Lift the sash and turn on the blower for 15 min.</li>
	<li>Wipe all items to be used in the biosafety cabinet with 70% ethanol prior to putting the items into the sterilized cabinet.</li>
	<li>Clean gloves or bare hands with 70% ethanol before wor...

<ol>
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A protocol for flood inoculation with PstDC3000 or Pst19 optimized to detect resistance to these bacterial strains in tomato seedlings is described. There are several critical parameters for optimal results in the seedling resistance assay, including bacterial concentration and surfactant concentration, which were empirically determined22. For PstDC3000, the optical density was optimized to achieve complete survival on a resistant cultivar containing the Pto/Prf...

An erratum was issued for:&#160;<a href="https://www.jove.com/t/60805">High-Throughput Identification of Resistance to Pseudomonas syringae pv. Tomato in Tomato using Seedling Flood Assay</a>. The Introduction, Protocol, Representative Results and Discussion&#160;sections were&#160;updated.

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

Pseudomonas syringae is a Gram-negative pathogenic bacterium that infects a wide range of plant hosts. Bacteria enter the host plant through the stomata or physical wounds and proliferate in the apoplast1. Plants have evolved a two-tiered immune response to protect against infection by bacterial pathogens. The first level occurs at the plant cell surface, where pattern recognition receptors on the plant cell membrane perceive highly conserved pathogen-associated molecular patterns (PAMPs) in a process called PAMP-triggered immunity (PTI)2. During this process, the host plant upregulates defense response pathways, including deposition of callose to the cell wall, closure of stomata, production of reactive oxygen species, and induction of pathogenesis-related genes.
Bacteria can overcome PTI by utilizing a type III secretion system to deliver proteins, called effectors, directly into the plant cell3. Effector proteins commonly target components of PTI and promote pathogen virulence4. The second tier of plant immunity occurs within the plant cell upon recognition of the effector proteins. This recognition is dependent on resistance genes, which encode nucleotide-binding site leucine-rich repeat containing receptors (NLRs). NLRs are capable of either recognizing effectors directly or recognizing their activity on a virulence target or decoy5. They then trigger a secondary immune response in a process called effector-triggered immunity (ETI), which is often associated with a hypersensitive response (HR), a form of localized cell death at the site of infection6. In contrast to gene-for-gene resistance associated with ETI, plants can exhibit quantitative partial resistance, which is dependent on the contribution of multiple genes7.
P. syringae pv. tomato (Pst) is the causal agent of bacterial speck on tomato and is a persistent agricultural problem. Predominant strains in the field have typically been Pst race 0 strains that express either or both of the type III effectors AvrPto and AvrPtoB. DC3000 (PstDC3000) is a representative race 0 strain and a model pathogen that can cause bacterial speck in tomato. To combat bacterial speck disease, breeders introgressed the Pto [P. syringae pv. tomato]/Prf [Pto resistance and fenthion sensitivity] gene cluster from the wild tomato species Solanum pimpinellifolium into modern cultivars8,9. The Pto gene encodes a serine-threonine protein kinase that, together with the Prf NLR, confer resistance to PstDC3000 via recognition of the effectors AvrPto and AvrPtoB10,11,12,13,14. However, this resistance is ineffective against emerging race 1 strains, allowing for their rapid and aggressive spread in recent years15,16. Race 1 strains evade recognition by the Pto/Prf cluster, because AvrPto is either lost or mutated in these strains, and AvrPtoB appears to accumulate minimally15,17,18.
Wild tomato populations are important reservoirs of natural variation for Pst resistance and have previously been used to identify potential resistance loci19,20,21. However, current screens for pathogen resistance utilize 4&#8211;5-week-old adult plants20,21. Therefore, they are limited by growth time, growth chamber space, and relatively small sample sizes. To address the limitations of conventional approaches, we developed a high-throughput tomato P. syringae resistance assay using 10-day-old tomato seedlings22. This approach offers several advantages over using adult plants: namely, shorter growth time, reduced space requirements, and higher throughput. Furthermore, we have demonstrated that this approach faithfully recapitulates disease resistance phenotypes observed in adult plants22.
In the seedling flood assay described in this protocol, tomato seedlings are grown on Petri dishes of sterile Murashige and Skoog (MS) media for 10 days and then are flooded with an inoculum containing the bacteria of interest and a surfactant. Following flooding, seedlings can be quantitatively evaluated for disease resistance via bacterial growth assays. Additionally, seedling survival or death can act as a discrete resistance or disease phenotype 7&#8211;14 days after flooding. This approach offers a high-throughput alternative for screening large numbers of wild tomato accessions for resistance to Pst race 1 strains, such as Pst strain 19 (Pst19), and can easily be adapted to other bacterial strains of interest.

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			<td>3M Tape Micropore 1/2&#34; x 10 YD CS 240 (1.25 cm x 9.1 m)</td>
			<td>VWR International</td>
			<td>56222-182</td>
			<td></td>
		</tr>
		<tr>
			<td>3mm borosilicate glass beads</td>
			<td>Friedrich &#38; Dimmock</td>
			<td>GB3000B</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacto peptone</td>
			<td>BD</td>
			<td>211677</td>
			<td></td>
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		<tr>
			<td>Bacto agar</td>
			<td>BD</td>
			<td>214010</td>
			<td></td>
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			<td>Biophotometer Plus</td>
			<td>Eppendorf</td>
			<td>E952000006</td>
			<td></td>
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			<td>Biosafety cabinet, class II type A2</td>
			<td></td>
			<td></td>
			<td></td>
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			<td>BRAND Disposable Plastic Cuvettes, Polystyrene</td>
			<td>VWR International</td>
			<td>47744-642</td>
			<td></td>
		</tr>
		<tr>
			<td>Chenille Kraft Flat Wood Toothpicks</td>
			<td>VWR International</td>
			<td>500029-808</td>
			<td></td>
		</tr>
		<tr>
			<td>cycloheximide</td>
			<td>Research Products International</td>
			<td>C81040-5.0</td>
			<td></td>
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		<tr>
			<td>Dibasic potassium phosphate anhydrous, ACS grade</td>
			<td>Fisher Scientific</td>
			<td>P288-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethylformamide</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting microscope (Magnification of at least 10x)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol - 190 Proof</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon polystyrene 96 well microplates, flat-bottom</td>
			<td>Fisher Scientific</td>
			<td>08-772-3</td>
			<td></td>
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			<td>Glass Alcohol Burner Wick</td>
			<td>Fisher Scientific</td>
			<td>S41898A / No. W-125</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Alcohol Burners</td>
			<td>Fisher Scientific</td>
			<td>S41898 / No. BO125</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol ACS reagent</td>
			<td>VWR International</td>
			<td>EMGX0185-5</td>
			<td></td>
		</tr>
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			<td>Kimberly-Clark&#8482; Kimtech Science&#8482; Kimwipes&#8482; Delicate Task Wipers</td>
			<td>Fisher Scientific</td>
			<td>06-666-A</td>
			<td></td>
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			<td>Magnesium chloride, ACS grade</td>
			<td>VWR International</td>
			<td>97061-356</td>
			<td></td>
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			<td>Magnesium sulfate heptahydrate, ACS grade</td>
			<td>VWR International</td>
			<td>97062-130</td>
			<td></td>
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			<td>Microcentrifuge tubes, 1.5 mL</td>
			<td></td>
			<td></td>
			<td></td>
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			<td>Microcentrifuge tubes, 2.2 mL</td>
			<td></td>
			<td></td>
			<td></td>
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			<td>Mini Beadbeater-96, 115 volt</td>
			<td>Bio Spec Products Inc.</td>
			<td>1001</td>
			<td></td>
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		<tr>
			<td>Murashige &#38; Skoog, Basal Salts</td>
			<td>Caisson Laboratories, Inc.</td>
			<td>MSP01-50LT</td>
			<td></td>
		</tr>
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			<td>Pipet-Lite XLS LTS 8-CH Pipet 20-200uL</td>
			<td>Rainin</td>
			<td>L8-200XLS</td>
			<td></td>
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			<td>Pipet-Lite XLS LTS 8-CH Pipet 2-20uL</td>
			<td>Rainin</td>
			<td>L8-20XLS</td>
			<td></td>
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			<td>Polystyrene 100mm x 25mm sterile petri dish</td>
			<td>VWR International</td>
			<td>89107-632</td>
			<td></td>
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			<td>Polystyrene 150mm x 15mm sterile petri dish</td>
			<td>Fisher Scientific</td>
			<td>FB08-757-14</td>
			<td></td>
		</tr>
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			<td>Polystyrene 150x15mm sterile petri dish</td>
			<td>Fisher Scientific</td>
			<td>08-757-148</td>
			<td></td>
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			<td>Pure Bright Germicidal Ultra Bleach 5.7% Available Chlorine (defined as 100% bleach)</td>
			<td>Staples</td>
			<td>1013131</td>
			<td></td>
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			<td>Rifampicin</td>
			<td>Gold Biotechnology</td>
			<td>R-120-25</td>
			<td></td>
		</tr>
		<tr>
			<td>Silwet L-77 (non-ionic organosilicone surfactant co-polymer C13H34O4Si3 surfactant)</td>
			<td>Fisher Scientific</td>
			<td>NCO138454</td>
			<td></td>
		</tr>
		<tr>
			<td>Tips LTS 20 &#956;L 960/10 GPS-L10</td>
			<td>Rainin</td>
			<td>17005091</td>
			<td></td>
		</tr>
		<tr>
			<td>Tips LTS 250 &#956;L 960/10 GPS-L250</td>
			<td>Rainin</td>
			<td>17005093</td>
			<td></td>
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		<tr>
			<td>VWR dissecting forceps fine tip, 4.5&#34;</td>
			<td>VWR International</td>
			<td>82027-386</td>
			<td></td>
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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.

Detection of PtoR-mediated immunity in cultivars and isogenic lines using the seedling resistance assay 
Figure 5 shows representative results for Moneymaker-PtoR and Moneymaker-PtoS cultivars 7&#8211;10 days after flooding with PstDC3000. Prior to infection, 10-day-old seedlings displayed fully emerged and expanded cotyledons and emerging first true leaves. The seedlings were flooded with 10 mM MgCl2 + 0.015% surfactant as...

Watch this Scientific Journal Video about High-Throughput Identification of Resistance to Pseudomonas syringae pv. Tomato in Tomato using Seedling Flood Assay at JoVE.com

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.

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. ...

This high throughput protocol allows rapid screening of tomato seedlings from wild accessions to the bacterial pathogen Pseudomonas syringae. The seedling flood assay minimizes plant growth time and growth chamber needs, allows rapid turnover of plants, and allows large sample sizes to be tested. This assay is a powerful tool that can be used to screen for resistance in wild accessions and other lines with complex genetic backgrounds.The protocol provides specific directions for flood innoculation of two Pseudomonas syringae strains. However, it is versatile and can be modified to detect host resistance to other bacterial pathogens. Demonstrating the procedure will be Yana Hasan, a research specialist in my laboratory.Begin by growing the tomato seedlings. Place tomato seeds in a 2.2 milliliter microcentrifuge tube and add two milliliters of 50%bleach solution. Rock the tube for 25 minutes then remove the bleach solution with a pipette.Add two milliliters of ultra pure water and wash the seeds by inverting the tube five times. Aspirate the liquid from the tube, and repeat the wash four more times. After the final wash, add two milliliters of ultra-pure water and pour the seeds into a sterile Petri dish.Sterilize forceps by flaming them in ethanol then use them to transfer five to seven seeds onto a 100 by 25 milliliter plate with 0.5 XMS plus 0.8 percent agar media. Seal the edges of the plate with surgical tape. Ensure that the plates are stacked flat and face up.Stratify the sterilized seeds at four degrees Celsius in the dark for at least three days to synchronize germination. After three days, vertically orient the plates so that the roots will grow down along the surface of the plate and the line of seeds is oriented horizontally. Transfer the seeds to a growth chamber set to 22 degrees Celsius with a 16-hour light, 8-hour dark cycle.Grow the seedlings for ten days in the chamber, at which point they will typically display fully emerged and expanded cotyledons and emerging first true leaves. Streak fresh bacteria onto appropriate KB agar with a flat, sterile toothpick. For PstT1, incubate the plate at 28 degrees Celsius for 48 hours prior to using the bacteria in the flood experiment.To prepare the inoculum, aseptically resuspend the bacteria in sterile, ten millimolar magnesium chloride to an optical density of 0.1. Then make two serial dilutions to obtain a working concentration with an OD600 of 0075. Also prepare a one to ten dilution of non-ionic organosilicone surfactant copolymer in ten millimolar magnesium chloride.Vortex it and add it to the bacteria, swirling the tube to mix. Transfer the plates with the ten-day-old seedlings from the growth chamber to the biosafety cabinet and remove the surgical tape. Then transfer six milliliters of inoculum to each plate.Gently push the seedlings into the inoculum with a pipette tip and start a timer for three minutes. Hold one plate in each hand and tilt the front of the plate down to submerge the cotyledons and leaves of the seedlings. Swish the inoculum side to side five to seven times, then tip the plates back to cover the roots with the inoculum.Tilt the plates down again and repeat the cycle for a total of three minutes. Pour the inoculum off the plates, set them down on a flat surface, and pour off any residual inoculum for a second time. Re-wrap the plates and place them back in the growth chamber.Moneymaker-PtoR and Moneymaker-PtoS cultivars were flooded with PSTDC3000 and phenotyped seven to ten days after flooding. Moneymaker-PtoR seedlings carry the PtoPRF gene cluster and were resistant to the PSTDC3000. While near-isogenic Moneymaker-PtoS seedlings, which cannot recognize the PSTDC3000 effectors AVRPto or AVRPto-B died quickly, within seven days after flooding.Ten day old seedlings were flooded with PstT1 and phenotyped at least ten days after flooding. Susceptible accessions were dead, had brown apical meristems and lacked new growth. In contrast, resistant seedlings displayed a high level of new green growth and survived infection with PstT1.Bacterial growth assays were carried out to quantitatively confirm PstT1 resistance and Solanum NeoRici LA1329. There was a 1.7 log difference in bacterial growth between resistant and susceptible LA1329 and a 1.6 log difference between resistant LA1329 and Moneymaker-PtoS, which correlated with the phenotypic results. When performing this assay, the most important things to remember are to pay close attention to aseptic technique throughout the protocol and ensure that all seedlings on the plate are thoroughly flooded.Be sure to autoclave infected plant tissue and bacteria and dispose of materials in accordance with appropriate regulations.

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Research

JoVE Journal

Bioengineering

Cell-based Calcium Assay for Medium to High Throughput Screening of TRP Channel Functions using FlexStation 3

The Molecular Devices' FlexStation 3 is a benchtop multi-mode microplate reader capable of automated fluorescence measurement in multi-well plates. It is ideal for medium- to high-throughput screens in academic settings. It has an integrated fluid transfer module equipped with a multi-channel pipetter and the machine reads one column at a time to monitor fluorescence changes of a variety of fluorescent reagents. For example, FlexStation 3 has been used to study the function of Ca2+-permeable ion channels and G-protein coupled receptors by measuring the changes of intracellular free Ca2+ levels. Transient receptor potential (TRP) channels are a large family of nonselective cation channels that play important roles in many physiological and pathophysiological functions. Most of the TRP channels are calcium permeable and induce calcium influx upon activation. In this video, we demonstrate the application of FlexStation 3 to study the pharmacological profile of the TRPA1 channel, a molecular sensor for numerous noxious stimuli. HEK293 cells transiently or stably expressing human TRPA1 channels, grown in 96-well plates, are loaded with a Ca2+-sensitive fluorescent dye, Fluo-4, and real-time fluorescence changes in these cells are measured before and during the application of a TRPA1 agonist using the FLEX mode of the FlexStation 3. The effect of a putative TRPA1 antagonist was also examined. Data are transferred from the SoftMax Pro software to construct concentration-response relationships of TRPA1 activators and inhibitors.

This video provides a detailed protocol for studying the pharmacological profile of human TRPA1 channels using FlexStation 3. The protocol covers details of cell preparation, dye loading and operation of the microplate reader, FlexStation 3.

The Molecular Devices' FlexStation 3 is a benchtop multi-mode microplate reader capable of automated fluorescence measurement in multi-well plates.  It is ...

Polymer Microarrays for High Throughput Discovery of Biomaterials

The discovery of novel biomaterials that are optimized for a specific biological application is readily achieved using polymer microarrays, which allows a combinatorial library of materials to be screened in a parallel, high throughput format1. Herein is described the formation and characterization of a polymer microarray using an on-chip photopolymerization technique 2. This involves mixing monomers at varied ratios to produce a library of monomer solutions, transferring the solution to a glass slide format using a robotic printing device and curing with UV irradiation. This format is readily amenable to many biological assays, including stem cell attachment and proliferation, cell sorting and low bacterial adhesion, allowing the ready identification of 'hit' materials that fulfill a specific biological criterion3-5. Furthermore, the use of high throughput surface characterization (HTSC) allows the biological performance to be correlated with physio-chemical properties, hence elucidating the biological-material interaction6. HTSC makes use of water contact angle (WCA) measurements, atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (ToF-SIMS). In particular, ToF-SIMS provides a chemically rich analysis of the sample that can be used to correlate the cell response with a molecular moiety. In some cases, the biological performance can be predicted from the ToF-SIMS spectra, demonstrating the chemical dependence of a biological-material interaction, and informing the development of hit materials5,3.

A description of the formation of a polymer microarray using an on-chip photopolymerization technique. The high throughput surface characterization using atomic force microscopy, water contact angle measurements, X-ray photoelectron spectroscopy and time of flight secondary ion mass spectrometry and a cell attachment assay is also described.

The discovery of novel biomaterials that are optimized for a specific biological application is readily achieved using polymer microarrays, which allows a ...

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.

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

Use of a High-throughput In Vitro Microfluidic System to Develop Oral Multi-species Biofilms

There are few high-throughput in vitro systems which facilitate the development of multi-species biofilms that contain numerous species commonly detected within in vivo oral biofilms. Furthermore, a system that uses natural human saliva as the nutrient source, instead of artificial media, is particularly desirable in order to support the expression of cellular and biofilm-specific properties that mimic the in vivo communities. We describe a method for the development of multi-species oral biofilms that are comparable, with respect to species composition, to supragingival dental plaque, under conditions similar to the human oral cavity. Specifically, this methods article will describe how a commercially available microfluidic system can be adapted to facilitate the development of multi-species oral biofilms derived from and grown within pooled saliva. Furthermore, a description of how the system can be used in conjunction with a confocal laser scanning microscope to generate 3-D biofilm reconstructions for architectural and viability analyses will be presented. Given the broad diversity of microorganisms that grow within biofilms in the microfluidic system (including Streptococcus, Neisseria, Veillonella, Gemella, and Porphyromonas), a protocol will also be presented describing how to harvest the biofilm cells for further subculture&#160;or DNA extraction and analysis. The limits of both the microfluidic biofilm system and the current state-of-the-art data analyses will be addressed. Ultimately, it is envisioned that this article will provide a baseline technique that will improve the study of oral biofilms and aid in the development of additional technologies that can be integrated with the microfluidic platform.

Use of a High-throughput In Vitro Microfluidic System to Develop Oral Multi-species Biofilms

The goal of this methods paper is to describe the use of a microfluidic system for the development of multi-species biofilms that contain species typically identified in human supragingival dental plaque.&#160;Methods to describe biofilm architecture, biofilm viability, and an approach to harvest biofilm for culture-dependent or culture-independent analyses are highlighted.

There are few high-throughput in vitro systems which facilitate the development of multi-species biofilms that contain numerous species commonly detected ...

Using Tomoauto: A Protocol for High-throughput Automated Cryo-electron Tomography

Cryo-electron tomography (Cryo-ET) is a powerful three-dimensional (3-D) imaging technique for visualizing macromolecular complexes in their native context at a molecular level. The technique involves initially preserving the sample in its native state by rapidly freezing the specimen in vitreous ice, then collecting a series of micrographs from different angles at high magnification, and finally computationally reconstructing a 3-D density map. The frozen-hydrated specimen is extremely sensitive to the electron beam and so micrographs are collected at very low electron doses to limit the radiation damage. As a result, the raw cryo-tomogram has a very low signal to noise ratio characterized by an intrinsically noisy image. To better visualize subjects of interest, conventional imaging analysis and sub-tomogram averaging in which sub-tomograms of the subject are extracted from the initial tomogram and aligned and averaged are utilized to improve both contrast and resolution. Large datasets of tilt-series are essential to understanding and resolving the complexes at different states, conditions, or mutations as well as obtaining a large enough collection of sub-tomograms for averaging and classification. Collecting and processing this data can be a major obstacle preventing further analysis. Here we describe a high-throughput cryo-ET protocol based on a computer-controlled 300kV cryo-electron microscope, a direct detection device (DDD) camera and a highly effective, semi-automated image-processing pipeline software wrapper library tomoauto developed in-house. This protocol has been effectively utilized to visualize the intact type III secretion system (T3SS) in Shigella flexneri minicells. It can be applicable to any project suitable for cryo-ET.

We present a protocol on how to utilize high-throughput cryo-electron tomography to determine high resolution in situ structures of molecular machines. The protocol permits large amounts of data to be processed, avoids common bottlenecks and reduces resource downtime, allowing the user to focus on important biological questions.

Cryo-electron tomography (Cryo-ET) is a powerful three-dimensional (3-D) imaging technique for visualizing macromolecular complexes in their native ...

High-throughput Identification of Bacteria Repellent Polymers for Medical Devices

Medical devices are often associated with hospital-acquired infections, which place enormous strain on patients and the healthcare system as well as contributing to antimicrobial resistance. One possible avenue for the reduction of device-associated infections is the identification of bacteria-repellent polymer coatings for these devices, which would prevent bacterial binding at the initial attachment step. A method for the identification of such repellent polymers, based on the parallel screening of hundreds of polymers using a microarray, is described here. This high-throughput method resulted in the identification of a range of promising polymers that resisted binding of various clinically relevant bacterial species individually and also as multi-species communities. One polymer, PA13 (poly(methylmethacrylate-co-dimethylacrylamide)), demonstrated significant reduction in attachment of a number of hospital isolates when coated onto two commercially available central venous catheters. The method described could be applied to identify polymers for a wide range of applications in which modification of bacterial attachment is important.

A high-throughput microarray method for the identification of polymers which reduce bacterial surface binding on medical devices is described.

Medical devices are often associated with hospital-acquired infections, which place enormous strain on patients and the healthcare system as well as ...

Process Optimization using High Throughput Automated Micro-Bioreactors in Chinese Hamster Ovary Cell Cultivation

Optimization of bioprocesses to increase the yield of desired products is of importance in the biopharmaceutical industry. This can be achieved by strain selection and by developing bioprocess parameters. Shake flasks have been used for this purpose. They, however, lack the capability to control the process parameters such as pH and dissolved oxygen (DO). This limitation can be overcome with the help of an automated micro-bioreactor. These bioreactors mimic cultivation at a larger scale. One of the major advantages of this system is the integration of the Design of Experiment (DOE) in the software. This integration enables establishing a design where multiple process parameters can be varied simultaneously. The critical process parameters and optimum bioprocess conditions can be analyzed within the software. The focus of the work presented here is to introduce the user to the steps involved in process design in the software and incorporation of the DOE within the cultivation run.

Here, we present a detailed procedure to run a Design of Experiment in an automated micro-bioreactor followed by cell harvest and protein quantification using a Protein A column.

Optimization of bioprocesses to increase the yield of desired products is of importance in the biopharmaceutical industry. This can be achieved by strain ...

High Throughput Yeast Strain Phenotyping with Droplet-Based RNA Sequencing

The powerful tools available to edit yeast genomes have made this microbe a valuable platform for engineering. While it is now possible to construct libraries of millions of genetically distinct strains, screening for a desired phenotype remains a significant obstacle. With existing screening techniques, there is a tradeoff between information output and throughput, with high-throughput screening typically being performed on one product of interest. Therefore, we present an approach to accelerate strain screening by adapting single cell RNA sequencing to isogenic picoliter colonies of genetically engineered yeast strains. To address the unique challenges of performing RNA sequencing on yeast cells, we culture isogenic yeast colonies within hydrogels and spheroplast prior to performing RNA sequencing. The RNA sequencing data can be used to infer yeast phenotypes and sort out engineered pathways. The scalability of our method addresses a critical obstruction in microbial engineering.

A bottleneck in the &#8216;design-build-test&#8217; cycle of microbial engineering is the speed at which we can perform functional screens of strains. We describe a high-throughput method for strain screening applied to hundreds to thousands of yeast cells per experiment that utilizes droplet-based RNA sequencing.

The powerful tools available to edit yeast genomes have made this microbe a valuable platform for engineering. While it is now possible to construct ...

Generation of Dynamical Environmental Conditions using a High-Throughput Microfluidic Device

Mimicking in vivo environmental conditions is crucial for in vitro studies on complex life machinery. However, current techniques targeting live cells and organs are either highly expensive, like robotics, or lack nanoliter volume and millisecond time accuracy in liquid manipulation. We herein present the design and fabrication of a microfluidic system, which consists of 1,500 culture units, an array of enhanced peristaltic pumps and an on-site mixing modulus. To demonstrate the capacities of the microfluidic device, neural stem cell (NSC) spheres are maintained in the proposed system. We observed that when the NSC sphere is exposed to CXCL in day 1 and EGF in day 2, the round-shaped conformation is well maintained. Variation in the input order of 6 drugs causes morphological changes to the NSC sphere and the expression level representative marker for NSC stemness (i.e., Hes5 and Dcx). These results indicate that dynamic and complex environmental conditions have great effects on NSC differentiation and self-renewal, and the proposed microfluidic device is a suitable platform for high throughput studies on the complex life machinery.

We present a microfluidic system for high throughput studies on complex life machinery, which consists of 1500 culture units, an array of enhanced peristaltic pumps and an on-site mixing modulus. The microfluidic chip allows for the analysis of the highly complex and dynamic micro-environmental conditions in vivo.

Mimicking in vivo environmental conditions is crucial for in vitro studies on complex life machinery. However, current techniques targeting live cells and ...

Simplified, High-throughput Analysis of Single-cell Contractility using Micropatterned Elastomers

Cellular contractile force generation is a fundamental trait shared by virtually all cells. These contractile forces are crucial to proper development, function at both the cellular and tissue levels,and regulate the mechanical systems in the body. Numerous biological processes are force-dependent, including motility, adhesion, and division of single-cells, as well as contraction and relaxation of organs such as the heart, bladder, lungs, intestines, and uterus. Given its importance in maintaining proper physiological function, cellular contractility can also drive disease processes when exaggerated or disrupted. Asthma, hypertension, preterm labor, fibrotic scarring, and underactive bladder are all examples of mechanically driven disease processes that could potentially be alleviated with proper control of cellular contractile force. Here, we present a comprehensive protocol for utilizing a novel microplate-based contractility assay technology known as fluorescently labeled elastomeric contractible surfaces (FLECS), that provides simplified and intuitive analysis of single-cell contractility in a massively scaled manner. Herein, we provide a step-wise protocol for obtaining two six-point dose-response curves describing the effects of two contractile inhibitors on the contraction of primary human bladder smooth muscle cells in a simple procedure utilizing just a single FLECS assay microplate, to demonstrate proper technique to users of the method. Using FLECS Technology, all researchers with basic biological laboratories and fluorescent microscopy systems gain access to studying this fundamental but difficult-to-quantify functional cell phenotype, effectively lowering the entry barrier into the field of force biology and phenotypic screening of contractile cell force.

This work presents a flexible protocol for utilizing fluorescently labeled elastomeric contractible surfaces (FLECS) Technology in microwell format for simplified, hands-off quantification of single-cell contractile forces based on visualized displacements of fluorescent protein micropatterns.

Cellular contractile force generation is a fundamental trait shared by virtually all cells. These contractile forces are crucial to proper development, ...