This research was supported by Advanced Research Projects Agency - Energy (ARPA-E) Award No. DE-AR0000313.

1. Establishment of Suspension Cell Cultures
<ol>
	<li>Prepare liquid BY-2 media by adding 4.43 g Linsmaier &#38; Skoog Basal media, 30 g of sucrose, 200 mg KH2PO4, and 200 &#181;g of 2,4-dichlorophenoxyacetic acid (2,4-D) to 900 ml of distilled water and pH to 5.8 with 0.1 M KOH. After adjusting pH, adjust final volume to 1,000 ml with distilled water and autoclave. Media can be stored up to 2 weeks at 4 &#176;C.</li>
	<li>Inoculate a 250 ml Erlenmeyer flask with 100 ml of liquid BY-2 media and a single piece of BY-2 callus (&#62;1 cm in diameter) grown on solid BY-2 media (liquid BY-2 media with the addition of 1% agar) and seal with aluminum foil. Incubate culture at 28-30 &#176;C with shaking for 5 days. 
	NOTE: BY-2 callus is maintained on solid media for long term storage, as liquid cultures will rapidly overgrow. Culture volume can be adjusted as necessary, typically a 100 ml culture will be capable of loading thirty-three 6-well plates.</li>
	<li>Transfer 2 ml of log phase BY-2 culture to 98 ml of fresh BY-2 media and incubate for 5-7 days at 28-30 &#176;C with shaking. 
	NOTE: Sub-culturing of established liquid cultures can be carried out regularly using 1:100 dilutions of 5-7 day old cultures, rather than inoculation with callus.</li>
	<li>Mix the culture thoroughly, as cells will rapidly settle, and transfer 6 ml of the cell culture to a 15 ml conical bottom centrifuge tube and let the cells settle for at least 10 min. Adjust the packed cell volume to 50% of the total volume by removing supernatant.</li>
	<li>Shake tube by inverting and pipet 500 &#181;l into each well of a 6-well plate for digestion. Use wide-bore pipets to transfer the cells at this stage, as they are dense and will clog standard pipet tips.</li>
</ol>
2. Protoplast Isolation
<ol>
	<li>Turn on all components of the robotic system (Figure 1A) and open the plate mover task scheduling software. The task scheduling program integrates the microplate mover with the other equipment to enable transfer of plates between each piece of equipment.</li>
	<li>Prior to experimentation, define the technical specifications for labware (e.g., plates, lids) used in the protocol, as well as starting and ending positions for each piece of labware, in the plate mover software, as follows:
	<ol>
		<li>Click Setup from the main tool bar in the plate mover software and select the Manage Container Types command.</li>
		<li>If the container type is listed in the existing container type library, then select the appropriate container.</li>
		<li>If the container type is not listed in the existing container type library then click Add to specify a new container type.</li>
		<li>After adding a new container type, insert the dimensions of the new container (height, width, stacking dimensions, and gripper offset) into the appropriate tabs and click Save.
		<ol>
			<li>If the correct dimensions are not available from the manufacturer, then accurately determine all dimensions to the nearest millimeter using calipers. Errors in measuring the container dimensions will result in failure of the plate mover.</li>
		</ol>
		</li>
		<li>Repeat steps 2.2.2 through 2.2.4.1 for all container types that the plate mover will encounter. After completion of this step for all labware, it is necessary to define the start and end location for each container (step 2.2.6). 
		NOTE: For this protocol the labware includes the 6-well plate, the deep-well plate, and the 96-well fluorescent screening plate.</li>
		<li>To define the start and end position of each container, click the Start/End tab in a specific protocol. First, select the start position of each container. Next, check the box for Lidded/Unlidded at both the start and end position. Repeat for all containers. 
		NOTE: If the container will be left on another piece of equipment (instead of the plate mover) the end location can be left blank.</li>
	</ol>
	</li>
	<li>At this stage manually load all plates into the starting position for the entire workflow. Load the 96-well fluorescent screening plates into Hotel 2, 6-well plates with BY-2 cells into Hotel 3, a 96 deep-well plate that will be used for transformation onto plate heater/chiller nest 2, and a 50 ml conical tube containing the enzyme solution (see Supplemental File, Section 1.2.2) onto the multi-mode dispenser.
	<ol>
		<li>If transformation will be carried out in the protocol, pre-load the 96 deep-well plate with 10 &#181;l of plasmid DNA per well (1 &#181;g/&#181;l, A260/280 &#62; 1.8) containing the OFP reporter construct and incubate on the plate heater/chiller at 4 &#176;C. Each well will be used for a single transformation, thus the number of wells filled will depend on the experimental setup.</li>
	</ol>
	</li>
	<li>Load all supplies and plates on the automated liquid handling platform in the designated locations and define the position of each item in the automation control software (Figure 1B).
	<ol>
		<li>Load a 96-well plate preloaded with 200 &#181;l of 40% PEG in MMg (0.4 M mannitol, 100 mM MgCl2, 4 mM MES, pH 5.7) in column 1, 200 &#181;l of propidium iodide (PI, 1 &#181;g/ml) in column 2, and 200 &#181;l of ethanol in column 3 (nest 2), and a box of pipette tips in nest 8.</li>
		<li>To define the location of the labware in the automated liquid handling platform&#39;s software, select Tools from the menu and click Labware Editor.</li>
		<li>Select the type of labware from the pulldown menu or define a new labware type using the New Labware button.</li>
		<li>Define the position of each piece of labware selected by selecting the Main Protocol tab and clicking Configure Labware. Ensure that each piece of labware placed on the automated liquid handling platform is defined prior to running the protocol.</li>
		<li>To trigger the automated protocols, click the Profile Explorer window and select the name of the protocol to be run (Protoplast Isolation, Cell Count, and PEG-mediated Transformation).</li>
		<li>Click Run to initialize all devices that will be used in the protocol.</li>
		<li>Finally, in the Work Explorer window, click Add Work Unit to verify all containers/labware in the system and begin the automated protocol. 
		NOTE: Full descriptions of the automated protocols are contained in the Supplemental File.</li>
	</ol>
	</li>
</ol>
3. Establish Standard Curve for Cell Counts
<ol>
	<li>Manually concentrate protoplasts at a concentration of 1 x 106 protoplasts/ml in a volume of 1 ml. Centrifuge protoplasts at 1,000 x g for 10 min, and remove supernatant.</li>
	<li>Manually add 900 &#181;l of 70% ethanol (maintained at 4 &#176;C) to the protoplast pellet and re-suspend the pellet. Incubate for 10 min on ice to fix cells.</li>
	<li>Add 100 &#181;l of PI (1 &#181;g/ml) to the protoplasts to label the nuclei and allow detection by the plate reader.</li>
	<li>Load 80 &#181;l of liquid BY-2 media into each column and row of the 96-well fluorescent screening plate starting at column 2.</li>
	<li>In column 1 of a 96-well fluorescent screening plate, add 70 &#181;l of protoplasts and 50 &#181;l BY-2 media to each well in the column.</li>
	<li>Place the plate in nest 6 of the automated liquid handling platform, and run a 2-fold serial dilution.
	<ol>
		<li>To conduct a serial dilution on the automated liquid handling platform, click Launch Serial Dilution Wizard and adjust the transfer volume (60 &#181;l), number of mixes and volume (2, 70 &#181;l), initial wells (Column 1, Rows A-F), dilution wells (Columns 2-11, Rows A-F), and aspirate and dispense properties (0.5 mm from well bottom).</li>
		<li>After setting the parameters, save and run the protocol. 
		NOTE: Since all protoplasts are labeled with PI (due to fixation of cells), the fluorescence is proportionate to the protoplast concentration.</li>
		<li>After the serial dilution is completed, remove the plate from nest 9 and insert into the plate reader and run the standard curve protocol in the plate reader software. 
		NOTE: A standard curve will then be generated by comparing the fluorescence from PI (excitation 536 nm/emission 620 nm) in each well with the known protoplast concentration.</li>
	</ol>
	</li>
	<li>Upon completion of the plate reader protocol, remove the plate and collect any transgenic cells for autoclaving or other de-vitalization procedures as defined in biosafety protocols&#39;.</li>
</ol>
4. Microscopic Analysis of Transformation
<ol>
	<li>Remove plate with transformed protoplasts (the product of Automated Protocol 3, Supplemental File) from Hotel 1 of the robotic system.</li>
	<li>Turn on inverted microscope, camera, and fluorescent lamp.</li>
	<li>Select the 10X objective for initial focusing on the protoplasts, turn on the halogen lamp, and close the shutter for the fluorescent lamp.</li>
	<li>Load plate onto microscopic system and focus on the protoplasts using brightfield.</li>
	<li>After focusing on protoplasts, turn off the halogen bulb and open the shutter for the fluorescent lamp.</li>
	<li>Select the CY3/TRITC filter set for visualization of the pporRFP protein expressed by the transgenic protoplasts.</li>
	<li>Scan each well to determine the number of protoplasts expressing the pporRFP fluorescent marker.</li>
	<li>Calculate the transformation efficiency as the total number of protoplasts expressing the fluorescent marker the total number of protoplasts.</li>
	<li>Collect any plates containing transgenic cells in approved biosafety bags for autoclaving or other de-vitalization procedures as defined in biosafety protocols&#39;.</li>
</ol>

The authors declare that they have no competing financial interest.

The protocol described above has been successfully validated for protoplast isolation, enumeration, and transformation using the BY-2 tobacco suspension cell culture; however, the protocol could easily be extended to any plant suspension culture. At present, protoplast isolation and transformation has been achieved in numerous plants, including maize (Zea mays)10, carrot (Daucus carota)32, poplar (Populus euphratica)33, grape (Vitis vinifera)34</su...

In recent years there has been significant impetus placed on the design of transgenic crops to overcome various diseases1, endow herbicide resistance2, confer drought3,4 and salt tolerance5, prevent herbivory6, increase biomass yield7, and decrease cell wall recalcitrance8. This trend has been aided by the development of new molecular tools for generating transgenic plants, including genome-editing using CRISPR and TALENs9, and gene silencing through dsRNA10, miRNA11, and siRNA12. While these technologies have simplified the generation of transgenic plants, they have also created a bottleneck, where the sheer number of transgenic plants generated cannot be screened using traditional systems that rely on plant regeneration. Related to this bottleneck, while silencing and genome-editing constructs can be rapidly inserted into plants, many of the targeted traits fail to produce the desired effect, which is often not discovered until plants are analyzed in the greenhouse. In this work, we have developed a method for rapid, automated, high-throughput screening of plant protoplasts, specifically to address the current bottleneck in early screening of large numbers of genome-editing and gene silencing targets.
The use of protoplasts, as opposed to intact plant cells, has several advantages for the development of an automated platform. First, protoplasts are isolated after digestion of the plant cell wall, and with this barrier no longer present, transformation efficiency is increased13. In intact plant cells there are only two well established methods for transformation, biolistics14 and Agrobacterium-mediated transformation15. Neither of these methods can be easily translated to liquid handling platforms, as biolistics requires specialized equipment for transformation, whereas Agrobacterium-mediated transformation requires co-culture and subsequent removal of the bacteria. Neither are amenable for high throughput methods. In the case of protoplasts, transformation is routinely conducted using polyethylene glycol (PEG)-mediated transfection16, which only requires several solution exchanges, and is ideally suited for liquid handling platforms. Second, protoplasts, by definition, are single-cell cultures, and thus the problems associated with clumping and chain formation in plant cell cultures, are not observed in protoplasts. In terms of rapid screening using a plate-based spectrophotometer, clumping of cells, or cells in multiple planes will lead to difficulty in acquiring consistent measurements. Since protoplasts are also denser than their culture media, they sediment to the bottom of wells, forming a monolayer, which is conducive for plate-based spectrophotometry. Finally, while plant cell suspension cultures are primarily derived from callus17, protoplasts can be harvested from a number of plant tissues, leading to the ability to identify tissue-specific expression. For example, the ability to analyze root- or leaf-specific expression of a gene can be very important to phenotype prediction. For these reasons, the protocols developed in this work were validated using protoplasts isolated from the widely-used tobacco (Nicotiana tabacum L.) &#39;Bright Yellow&#39; 2 (BY-2) suspension culture.
The BY-2 suspension culture has been described as the &#34;HeLa&#34; cell of higher plants, owing to its ubiquitous use in molecular analysis of plant cells18. Recently, BY-2 cells have been used to study the effects of plant stressors19-22, intracellular protein localization23,24, and basic cell biology25-27 demonstrating the broad utility of these cultures in plant biology. An additional advantage of BY-2 cultures is the ability to synchronize the cultures with aphidicolin, which can lead to enhanced reproducibility for gene expression studies28. Furthermore, methods have been developed for the extraction of BY-2 protoplasts using low-cost enzymes29,30, as enzymes traditionally used for generating protoplasts are cost prohibitive for high-throughput systems. As such, the protocol described below has been validated using the BY-2 suspension culture, but it should be amendable to any plant cell suspension culture. Proof-of-concept experiments are performed using an orange fluorescence protein (OFP) reporter gene (pporRFP) from the hard coral Porites porites31 under the control of the CAMV 35S promoter.

<table><tbody><tr><td>Orbitor RS Microplate mover</td><td>Thermo Scientific</td><td></td><td></td></tr><tr><td>Bravo Liquid Handler</td><td>Agilent</td><td></td><td></td></tr><tr><td>Synergy H1 Multi-mode Reader</td><td>BioTek</td><td></td><td></td></tr><tr><td>MultiFlo FX Multi-mode Dispenser</td><td>BioTek</td><td></td><td></td></tr><tr><td>Teleshake</td><td>Inheco</td><td>3800048</td><td></td></tr><tr><td>CPAC Ultraflat Heater/cooler</td><td>Inheco</td><td>7000190</td><td></td></tr><tr><td>Vworks Automation Software</td><td>Agilent</td><td></td><td>Software used to control and write protocols for Agilent Bravo</td></tr><tr><td>Momentum Software</td><td>Thermo Scientific</td><td></td><td>Task scheduling software for controlling Orbiter RS</td></tr><tr><td>Liquid Handling Control 2.17 Software</td><td>Biotek</td><td></td><td>Software used to control and write protocols for MultiFlo FX</td></tr><tr><td>IX81 Inverted Microscope</td><td>Olympus</td><td></td><td></td></tr><tr><td>Zyla 3-Tap microscope camera</td><td>Andor</td><td></td><td></td></tr><tr><td>ET-CY3/TRITC Filter Set</td><td>Chroma Technology Corp</td><td>49004</td><td></td></tr><tr><td>Rohament CL</td><td>AB Enzymes</td><td>sample bottle</td><td>low-cost cellulase</td></tr><tr><td>Rohapect UF</td><td>AB Enzymes</td><td>sample bottle</td><td>low-cost pectinase</td></tr><tr><td>Rohapect 10L</td><td>AB Enzymes</td><td>sample bottle</td><td>low-cost pectinase/arabinase</td></tr><tr><td>Linsmaier &amp;amp; Skoog Basal Medium</td><td>Phytotechnology Laboratories</td><td>L689</td><td></td></tr><tr><td>2,4-dichlorophenoxyacetic acid</td><td>Phytotechnology Laboratories</td><td>D295</td><td></td></tr><tr><td>propidium iodide</td><td>Sigma Aldrich</td><td>P4170</td><td></td></tr><tr><td>Poly(ethylene glycol) 4000</td><td>Sigma Aldrich</td><td>95904-250G-F</td><td>Formerly Fluka PEG</td></tr><tr><td>Propidium Iodide</td><td>Fisher Scientific</td><td>25535-16-4</td><td>Acros Organics</td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma Aldrich</td><td>C7902-1KG</td><td></td></tr><tr><td>Sodium Acetate</td><td>Fisher Scientific</td><td>BP333-500</td><td></td></tr><tr><td>Mannitol</td><td>Sigma Aldrich</td><td>M1902-1KG</td><td></td></tr><tr><td>Sucrose</td><td>Fisher Scientific</td><td>S5-3</td><td></td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Fisher Scientific</td><td>AC424205000</td><td></td></tr><tr><td>KOH</td><td>Sigma Aldrich</td><td>P1767</td><td></td></tr><tr><td>Gelzan CM</td><td>Sigma Aldrich</td><td>G1910-250G</td><td></td></tr><tr><td>6-well plate</td><td>Thermo Scientific</td><td>103184</td><td></td></tr><tr><td>96-well 1.2 ml deep well plate</td><td>Thermo Scientific</td><td>AB-0564</td><td></td></tr><tr><td>96 well optical bottom plate</td><td>Thermo Scientific</td><td>165305</td><td></td></tr><tr><td>Finntip 1000 Wide bore Pipet tips</td><td>Thermo Scientific</td><td>9405 163</td><td></td></tr><tr><td>NaCl</td><td>Fisher Scientific</td><td>BP358-10</td><td></td></tr><tr><td>KCl</td><td>Sigma Aldrich</td><td>P4504-1KG</td><td></td></tr><tr><td>MES</td><td>Fisher Scientific</td><td>AC17259-5000</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Fisher Scientific</td><td>M33-500</td><td></td></tr></tbody></table>

a robotic platform for high-throughput protoplast isolation and transformation

Over the last decade there has been a resurgence in the use of plant protoplasts that range from model species to crop species, for analysis of signal transduction pathways, transcriptional regulatory networks, gene expression, genome-editing, and gene-silencing. Furthermore, significant progress has been made in the regeneration of plants from protoplasts, which has generated even more interest in the use of these systems for plant genomics. In this work, a protocol has been developed for automation of protoplast isolation and transformation from a &#39;Bright Yellow&#39; 2 (BY-2) tobacco suspension culture using a robotic platform. The transformation procedures were validated using an orange fluorescent protein (OFP) reporter gene (pporRFP) under the control of the Cauliflower mosaic virus 35S promoter (35S). OFP expression in protoplasts was confirmed by epifluorescence microscopy. Analyses also included protoplast production efficiency methods using propidium iodide. Finally, low-cost food-grade enzymes were used for the protoplast isolation procedure, circumventing the need for lab-grade enzymes that are cost-prohibitive in high-throughput automated protoplast isolation and analysis. Based on the protocol developed in this work, the complete procedure from protoplast isolation to transformation can be conducted in under 4 hr, without any input from the operator. While the protocol developed in this work was validated with the BY-2 cell culture, the procedures and methods should be translatable to any plant suspension culture/protoplast system, which should enable acceleration of crop genomics research.

In the current study, the doubling rate of BY-2 varied from 14-18 hr dependent on the temperature at which the cultures were incubated, consistent with previous reports of a mean cell cycle length of 15 hr. With this doubling rate, a 1:100 starting inoculum was used to initiate cultures, leading to cultures with a packed cell volume (PCV) of 50% in 5-7 days. In the current protocol, in which cultures were grown in 200 ml of media, a PCV of 100 ml was generated in 7 days, which provided en...

Watch this Scientific Journal Video about A Robotic Platform for High-throughput Protoplast Isolation and Transformation at JoVE.com

A Robotic Platform for High-throughput Protoplast Isolation and Transformation

A high-throughput, automated, tobacco protoplast production and transformation methodology is described. The robotic system enables massively parallel gene expression and discovery in the model BY-2 system that should be translatable to non-model crops.

Over the last decade there has been a resurgence in the use of plant protoplasts that range from model species to crop species, for analysis of signal ...

a-robotic-platform-for-high-throughput-protoplast-isolation

Universidad San Sebastian

Research

JoVE Journal

Genetics

14.2K Views.  University of Tennessee, Knoxville. A high-throughput, automated, tobacco protoplast production and transformation methodology is described. The robotic system enables massively parallel gene expression and discovery in the model BY-2 system that should be translatable to non-model crops.

Video: A Robotic Platform for High-throughput Protoplast Isolation and Transformation

High-Throughput Robotically Assisted Isolation of Temperature-sensitive Lethal Mutants in Chlamydomonas reinhardtii

Systematic identification and characterization of genetic perturbations have proven useful to decipher gene function and cellular pathways. However, the conventional approaches of permanent gene deletion cannot be applied to essential genes. We have pioneered a unique collection of ~70 temperature-sensitive (ts) lethal mutants for studying cell cycle regulation in the unicellular green algae Chlamydomonas reinhardtii1. These mutations identify essential genes, and the ts alleles can be conditionally inactivated by temperature shift, providing valuable tools to identify and analyze essential functions. Mutant collections are much more valuable if they are close to comprehensive, since scattershot collections can miss important components. However, this requires the efficient collection of a large number of mutants, especially in a wide-target screen. Here, we describe a robotics-based pipeline for generating ts lethal mutants and analyzing their phenotype in Chlamydomonas. This technique can be applied to any microorganism that grows on agar. We have collected over 3000 ts mutants, probably including mutations in most or all cell-essential pathways, including about 200 new candidate cell cycle mutations. Subsequent molecular and cellular characterization of these mutants should provide new insights in plant cell biology; a comprehensive mutant collection is an essential prerequisite to ensure coverage of a broad range of biological pathways. These methods are integrated with downstream genetics and bioinformatics procedures for efficient mapping and identification of the causative mutations that are beyond the scope of this manuscript.

High-Throughput Robotically Assisted Isolation of Temperature-sensitive Lethal Mutants in Chlamydomonas reinhardtii

Temperature-sensitive (ts) lethal mutants are valuable tools to identify and analyze essential functions. Here we describe methods to generate and classify ts lethal mutants in high throughput.

Systematic identification and characterization of genetic perturbations have proven useful to decipher gene function and cellular pathways. However, the ...

A High-throughput Automated Platform for the Development of Manufacturing Cell Lines for Protein Therapeutics

The fast-growing biopharmaceutical industry demands speedy development of highly efficient and reliable production systems to meet the increasing requirement for drug supplies. The generation of production cell lines has traditionally involved manual operations that are labor-intensive, low-throughput and vulnerable to human errors. We report here an integrated high-throughput and automated platform for development of manufacturing cell lines for the production of protein therapeutics.
The combination of BD FACS Aria Cell Sorter, CloneSelect Imager and TECAN Freedom EVO liquid handling system has enabled a high-throughput and more efficient cell line development process. In this operation, production host cells are first transfected with an expression vector carrying the gene of interest 1, followed by the treatment with a selection agent. The stably-transfected cells are then stained with fluorescence-labeled anti-human IgG antibody, and are subsequently subject to flow cytometry analysis 2-4. Highly productive cells are selected based on fluorescence intensity and are isolated by single-cell sorting on a BD FACSAria. Colony formation from single-cell stage was detected microscopically and a series of time-laps digital images are taken by CloneSelect Imager for the documentation of cell line history. After single clones have formed, these clones were screened for productivity by ELISA performed on a TECAN Freedom EVO liquid handling system. Approximately 2,000 - 10,000 clones can be screened per operation cycle with the current system setup.
This integrated approach has been used to generate high producing Chinese hamster ovary (CHO) cell lines for the production of therapeutic monoclonal antibody (mAb) as well as their fusion proteins. With the aid of different types of detecting probes, the method can be used for developing other protein therapeutics or be applied to other production host systems. Comparing to the traditional manual procedure, this automated platform demonstrated advantages of significantly increased capacity, ensured clonality, traceability in cell line history with electronic documentation and much reduced opportunity in operator error.

A high-throughput, automated platform of manufacturing cell line development for producing protein therapeutics is described. Implementation of BD FACS Aria Cell Sorter, CloneSelect Imager and TECAN Freedom EVO liquid handling system has demonstrated significantly increased processing capacity in cell line development with improved cell line quality and high reproducibility.

The fast-growing biopharmaceutical industry demands speedy development of highly efficient and reliable production systems to meet the increasing ...

Medicine

Studying Protein Import into Chloroplasts Using Protoplasts

The chloroplast is an essential organelle that is responsible for various cellular processes in plants, such as photosynthesis and the production of many secondary metabolites and lipids. Chloroplasts require a large number of proteins for these various physiological processes. Over 95% of chloroplast proteins are nucleus-encoded and imported into chloroplasts from the cytosol after translation on cytosolic ribosomes. Thus, the proper import or targeting of these nucleus-encoded chloroplast proteins to chloroplasts is essential for the proper functioning of chloroplasts as well as the plant cell. Nucleus-encoded chloroplast proteins contain signal sequences for specific targeting to chloroplasts. Molecular machinery localized to the chloroplast or cytosol recognize these signals and carry out the import process. To investigate the mechanisms of protein import or targeting to chloroplasts in vivo, we developed a rapid, efficient protoplast-based method to analyze protein import into chloroplasts of Arabidopsis. In this method, we use protoplasts isolated from leaf tissues of Arabidopsis. Here, we provide a detailed protocol for using protoplasts to investigate the mechanism by which proteins are imported into chloroplasts.

Here we describe a protocol to express proteins into protoplasts by using PEG-mediated transformation method. The method provides easy expression of proteins of interest, and efficient investigation of protein localization and the import process for various experimental conditions in vivo.

The chloroplast is an essential organelle that is responsible for various cellular processes in plants, such as photosynthesis and the production of many ...

Biochemistry

Fluorescence Activated Cell Sorting of Plant Protoplasts

High-resolution, cell type-specific analysis of gene expression greatly enhances understanding of developmental regulation and responses to environmental stimuli in any multicellular organism. In situ hybridization and reporter gene visualization can to a limited extent be used to this end but for high resolution quantitative RT-PCR or high-throughput transcriptome-wide analysis the isolation of RNA from particular cell types is requisite. Cellular dissociation of tissue expressing a fluorescent protein marker in a specific cell type and subsequent Fluorescence Activated Cell Sorting (FACS) makes it possible to collect sufficient amounts of material for RNA extraction, cDNA synthesis/amplification and microarray analysis.
An extensive set of cell type-specific fluorescent reporter lines is available to the plant research community. In this case, two marker lines of the Arabidopsis thaliana root are used: PSCR::GFP (endodermis and quiescent center) and PWOX5::GFP (quiescent center). Large numbers (thousands) of seedlings are grown hydroponically or on agar plates and harvested to obtain enough root material for further analysis. Cellular dissociation of plant material is achieved by enzymatic digestion of the cell wall. This procedure makes use of high osmolarity-induced plasmolysis and commercially available cellulases, pectinases and hemicellulases to release protoplasts into solution.
FACS of GFP-positive cells makes use of the visualization of the green versus the red emission spectra of protoplasts excited by a 488 nm laser. GFP-positive protoplasts can be distinguished by their increased ratio of green to red emission. Protoplasts are typically sorted directly into RNA extraction buffer and stored for further processing at a later time.
This technique is revealed to be straightforward and practicable. Furthermore, it is shown that it can be used without difficulty to isolate sufficient numbers of cells for transcriptome analysis, even for very scarce cell types (e.g. quiescent center cells). Lastly, a growth setup for Arabidopsis seedlings is demonstrated that enables uncomplicated treatment of the plants prior to cell sorting (e.g. for the cell type-specific analysis of biotic or abiotic stress responses). Potential supplementary uses for FACS of plant protoplasts are discussed.

A method for isolating specific cell types from plant material is demonstrated. This technique employs transgenic marker lines expressing fluorescent proteins in particular cell types, cellular dissociation and Fluorescence Activated Cell Sorting. Additionally, a growth setup is established here that facilitates treatment of Arabidopsis thaliana seedlings prior to cell sorting.

High-resolution, cell type-specific analysis of gene expression greatly enhances understanding of developmental regulation and responses to environmental ...

Biology

A Microfluidic Platform for High-throughput Single-cell Isolation and Culture

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet high-throughput, method to perform single-cell culture experiments. Here, we report a microfluidic chip-based strategy for high-efficiency single-cell isolation (~77%) and demonstrate its capability of performing long-term single-cell culture (up to 7 d) and cellular heterogeneity analysis using clonogenic assay. These applications were demonstrated with KT98 mouse neural stem cells, and A549 and MDA-MB-435 human cancer cells. High single-cell isolation efficiency and long-term culture capability are achieved by using different sizes of microwells on the top and bottom of the microfluidic channel. The small microwell array is designed for precisely isolating single-cells, and the large microwell array is used for single-cell clonal culture in the microfluidic chip. This microfluidic platform constitutes an attractive approach for single-cell culture applications, due to its flexibility of adjustable cell culture spaces for different culture strategies, without decreasing isolation efficiency.

Here, we present a protocol for isolating and culturing single cells with a microfluidic platform, which utilizes a new microwell design concept to allow for high-efficiency single cell isolation and long-term clonal culture.

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet ...

Bioengineering

Automated Robotic Liquid Handling Assembly of Modular DNA Devices

Recent advances in modular DNA assembly techniques have enabled synthetic biologists to test significantly more of the available &#34;design space&#34; represented by &#34;devices&#34; created as combinations of individual genetic components. However, manual assembly of such large numbers of devices is time-intensive, error-prone, and costly. The increasing sophistication and scale of synthetic biology research necessitates an efficient, reproducible way to accommodate large-scale, complex, and high throughput device construction.
Here, a DNA assembly protocol using the Type-IIS restriction endonuclease based Modular Cloning (MoClo) technique is automated on two liquid-handling robotic platforms. Automated liquid-handling robots require careful, often times tedious optimization of pipetting parameters for liquids of different viscosities (e.g. enzymes, DNA, water, buffers), as well as explicit programming to ensure correct aspiration and dispensing of DNA parts and reagents. This makes manual script writing for complex assemblies just as problematic as manual DNA assembly, and necessitates a software tool that can automate script generation. To this end, we have developed a web-based software tool, http://mocloassembly.com, for generating combinatorial DNA device libraries from basic DNA parts uploaded as Genbank files. We provide access to the tool, and an export file from our liquid handler software which includes optimized liquid classes, labware parameters, and deck layout. All DNA parts used are available through Addgene, and their digital maps can be accessed via the Boston University BDC ICE Registry. Together, these elements provide a foundation for other organizations to automate modular cloning experiments and similar protocols.
The automated DNA assembly workflow presented here enables the repeatable, automated, high-throughput production of DNA devices, and reduces the risk of human error arising from repetitive manual pipetting. Sequencing data show the automated DNA assembly reactions generated from this workflow are ~95% correct and require as little as 4% as much hands-on time, compared to manual reaction preparation.

Here, an automated workflow to perform modular DNA &#34;device&#34; assembly using a modular cloning DNA assembly method on liquid-handling robots is presented. The protocol uses an intuitive software tool for generating liquid handler picklists for combinatorial DNA device library generation, which we demonstrate using two liquid handling platforms.

Recent advances in modular DNA assembly techniques have enabled synthetic biologists to test significantly more of the available &#34;design space&#34; ...

Efficient Polyethylene Glycol (PEG) Mediated Transformation of the Moss Physcomitrella patens

A simple and efficient method to transform Physcomitrella pantens protoplasts is described. This method is adapted from protocols for Physocmitrella protonemal protoplast and Arabidopsis mesophyll protoplast transformation1. Due to its capacity to undergo efficient mitotic homologous recombination, Physcomitrella patens has emerged as an important model system in recent years2. This capacity allows high frequencies of gene targeting3-9, which is not seen in other model plants such as Arabidopsis. To take full advantage of this system, we need an effective and easy method to deliver DNA into moss cells. The most common ways to transform this moss are particle bombardment10 and PEG-mediated DNA uptake11. Although particle bombardment can produce a high transformation efficiency12, gene guns are not readily available to many laboratories and the protocol is difficult to standardize. On the other hand, PEG mediated transformation does not require specialized equipments, and can be performed in any laboratory with a sterile hood. Here, we show a simple and highly efficient method for transformation of moss protoplasts. This method can generate more than 120 transient transformants per microgram of DNA, which is an improvement from the most efficient protocol previously reported13. Because of its simplicity, efficiency, and reproducibility, this method can be applied to projects requiring large number of transformants as well as for routine transformation.

Efficient Polyethylene Glycol PEG Mediated Transformation of the Moss Physcomitrella patens

A simple and efficient method to transform Physcomitrella pantens protoplasts is described. This method is adapted from protocols for Physocmitrella protonemal protoplast and Arabidopsis mesophyll protoplast transformation1.

A simple and efficient method to transform Physcomitrella pantens protoplasts is described. This method is adapted from protocols for Physocmitrella ...

High-throughput Yeast Plasmid Overexpression Screen

The budding yeast, Saccharomyces cerevisiae, is a powerful model system for defining fundamental mechanisms of many important cellular processes, including those with direct relevance to human disease. Because of its short generation time and well-characterized genome, a major experimental advantage of the yeast model system is the ability to perform genetic screens to identify genes and pathways that are involved in a given process. Over the last thirty years such genetic screens have been used to elucidate the cell cycle, secretory pathway, and many more highly conserved aspects of eukaryotic cell biology 1-5. In the last few years, several genomewide libraries of yeast strains and plasmids have been generated 6-10. These collections now allow for the systematic interrogation of gene function using gain- and loss-of-function approaches 11-16. Here we provide a detailed protocol for the use of a high-throughput yeast transformation protocol with a liquid handling robot to perform a plasmid overexpression screen, using an arrayed library of 5,500 yeast plasmids. We have been using these screens to identify genetic modifiers of toxicity associated with the accumulation of aggregation-prone human neurodegenerative disease proteins. The methods presented here are readily adaptable to the study of other cellular phenotypes of interest.

Here we describe a plasmid overexpression screen in Saccharomyces cerevisiae, using an arrayed plasmid library and a high-throughput yeast transformation protocol with a liquid handling robot.

The budding yeast, Saccharomyces cerevisiae, is a powerful model system for defining fundamental mechanisms of many important cellular processes, ...

A Simple Method for Isolation of Soybean Protoplasts and Application to Transient Gene Expression Analyses

Soybean (Glycine max (L.) Merr.) is an important crop species and has become a legume model for the studies of genetic and biochemical pathways. Therefore, it is important to establish an efficient transient gene expression system in soybean. Here, we report a simple protocol for the preparation of soybean protoplasts and its application for transient functional analyses. We found that young unifoliate leaves from soybean seedlings resulted in large quantities of high quality protoplasts. By optimizing a PEG-calcium-mediated transformation method, we achieved high transformation efficiency using soybean unifoliate protoplasts. This system provides an efficient and versatile model for examination of complex regulatory and signaling mechanisms in live soybean cells and may help to better understand diverse cellular, developmental and physiological processes of legumes.

We developed a simple and efficient protocol for the preparation of large quantities of soybean protoplasts to study complex regulatory and signaling mechanisms in live cells.

Soybean (Glycine max (L.) Merr.) is an important crop species and has become a legume model for the studies of genetic and biochemical pathways. ...

Scalable Transfection of Maize Mesophyll Protoplasts

The transfection of maize mesophyll cells often involves digesting the plant cell walls to create protoplasts and then inserting DNA via electroporation or polyethylene glycol (PEG). Previous methods were developed to produce tens of thousands of transfected protoplasts at once. Here,&#160;we describe a straightforward method to isolate and transfect millions of leaf mesophyll protoplasts in maize (Zea mays L.). This streamlined process removes certain common protoplasting steps, such as washing in W5. Additionally, steps such as centrifugation, PEG-mediated transfection, and incubation have been modified to work with a greater number of protoplasts. The ability to express large libraries of plasmid constructs enables genome-scale experiments, such as massively parallel reporter assays in maize.

Here, we present a high-throughput protocol to transiently transfect millions of maize protoplasts for testing large libraries of plasmids in maize mesophyll cells.

The transfection of maize mesophyll cells often involves digesting the plant cell walls to create protoplasts and then inserting DNA via electroporation ...

A Robotic Platform for High-throughput Protoplast Isolation and Transformation

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High-Throughput Robotically Assisted Isolation of Temperature-sensitive Lethal Mutants in Chlamydomonas reinhardtii

A High-throughput Automated Platform for the Development of Manufacturing Cell Lines for Protein Therapeutics

Studying Protein Import into Chloroplasts Using Protoplasts

Fluorescence Activated Cell Sorting of Plant Protoplasts

A Microfluidic Platform for High-throughput Single-cell Isolation and Culture

Automated Robotic Liquid Handling Assembly of Modular DNA Devices

Efficient Polyethylene Glycol (PEG) Mediated Transformation of the Moss Physcomitrella patens

High-throughput Yeast Plasmid Overexpression Screen

A Simple Method for Isolation of Soybean Protoplasts and Application to Transient Gene Expression Analyses

Scalable Transfection of Maize Mesophyll Protoplasts