Name: Plasmid Stability Analysis with Open-Source Droplet Microfluidics (Video) | JoVE Journal
Uploaded: 2024-12-27T15:00:00.000+00:00
Duration: 7 min 43 s
Description: Explore open-source droplet microfluidics for plasmid stability analysis in bacteria using fluorescence microscopy. This method enables high-throughput quantification of plasmid retention in single-cell microcolonies.

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This work is part of funded projects granted to T.W. from ANID FONDECYT Regular 1241621 and the Chang Zuckerberg Initiative project &#8216;Latin American Hub for Bioimaging Through Open Hardware&#8217;. T.W. is also grateful for funding from CIFAR, as Azrieli Global Scholar in the CIFAR MacMillan Multiscale Human program.

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<li>Wang, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Construction+of+novel+pJRD215-derived+plasmids+using+chloramphenicol+acetyltransferase+%28cat%29+gene+as+a+selection+marker+for+Acidithiobacillus+caldus%5BTitle%5D)+AND+%22PLoS+ONE%22%5BJournal%5D)">Construction of novel pJRD215-derived plasmids using chloramphenicol acetyltransferase (cat) gene as a selection marker for Acidithiobacillus caldus.</a> PLoS ONE. 12 (8), e0183307(2017).</li>
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<li>Vitalis, C., Wenzel, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Leveraging+interactions+in+microfluidic+droplets+for+enhanced+biotechnology+screens%5BTitle%5D)+AND+%22Current+Opinion+in+Biotechnology%22%5BJournal%5D)">Leveraging interactions in microfluidic droplets for enhanced biotechnology screens.</a> Current Opinion in Biotechnology. 82, 102966(2023).</li>
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<li>Abate, A. R., Weitz, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Faster+multiple+emulsification+with+drop+splitting%5BTitle%5D)+AND+%22Lab+Chip%22%5BJournal%5D)">Faster multiple emulsification with drop splitting.</a> Lab Chip. 11 (11), 1911-1915 (2011).</li>
<li>Lau, B. T. C., Malkus, P., Paulsson, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((New+quantitative+methods+for+measuring+plasmid+loss+rates+reveal+unexpected+stability%5BTitle%5D)+AND+%22Plasmid%22%5BJournal%5D)">New quantitative methods for measuring plasmid loss rates reveal unexpected stability.</a> Plasmid. 70 (3), 353-361 (2013).</li>
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The authors declare that no competing financial interests or personal relationships could have influenced the work reported in this paper.

A gel microdroplet-based method is demonstrated to effectively identify and quantify colonies with and without plasmid-encoded genetic expression of fluorescent proteins, such as sfGFP. Colonies that do not sufficiently express the plasmid product are identified using a fluorescent DNA stain (here, Propidium Iodide) that stains all colonies and features a different emission wavelength. This integration of droplet microfluidics, gelling, and fluorescence microscopy, utilizing open-source technology, enables executing an advanced workflow in many research settings11,13. The successful generation of gel-microdroplets enables advanced molecular biology single-cell workflows, including cell lysis, single genome amplification, metabolic cell interaction screens, media exchange, and more8,9. These advantages are used in this protocol to grow, stain, and analyze microcolonies in a more scalable fashion than in traditional plate-based assays.
Critical steps 
The encapsulation process is a critical and delicate part of the protocol. Precise control of ingredient concentrations, flow rates, and pressures is required to generate uniform microgels within a specific size range and control the average number of cells per droplet. Furthermore, maintaining the concentration and temperature of the cell-agarose mix prevents clumping or premature gelation. The temperature control of the liquid agarose-cell suspension in a pipette tip is a particularly advantageous implementation of our open-source hardware microfluidics workstation that provides much easier and more robust microgel generation compared with efforts to control the temperature of syringe pumps and tubing. Since cells are mixed with the agarose growth medium before encapsulation and cultivation, the agarose microgels have to be generated quickly in order to avoid major cell concentration changes. For this purpose, a droplet-splitting microfluidic chip design inspired by Abate et al. was optimized18.
Modifications and troubleshooting
Several calibrations and modifications were necessary to refine the original protocol. The encapsulation of agarose is much more challenging than regular water-in-oil droplets, requiring the design of a system to maintain the agarose in a liquid state while ensuring the aqueous phase flow achieves a homogeneous particle size range. Changes in agarose viscosity due to gelation affect the flow rate, leading to larger particle sizes. The microscopy requires a careful selection of&#160;filters and light sources to ensure non-overlapping excitation and emission signals for clear differentiation. Initially, DAPI was chosen for staining bacteria, but its emission signal overlapped with sfGFP, causing sfGFP to be detected in the blue detection channel. We switched to PI because its emission is well-separated from sfGFP at long wavelengths (red light).
While plasmid loss was quantified using the proposed method, the sfGFP plasmid used was unexpectedly stable, displaying hardly any instances of plasmid loss in the first generation of cells cultivated without antibiotics, even under stress conditions such as pH9 media and incubation at 40 &#176;C. This observation is consistent with the findings of other research groups1,19. The plasmid stability limited the demonstration of the method&#39;s full quantification capabilities for initial cell culture generations, but it did demonstrate that the method is sensitive enough to detect even small differences in plasmid retention. The observation of high-plasmid stability in early generations has an important implication for droplet microfluidic screens using a negative selection assay, such as target bacteria inhibition. It means that the plasmid loss of selection targets is a low source of false-positive selection results. As droplet microfluidic screens typically exceed other high-throughput screens, such as pipetting robot workflows, by orders of magnitudes in throughput, these rare events need to be assessed and taken into account.
Limitations
Despite its advantages, there are limitations to the presented method. Microfluidic device fabrication requires expertise and meticulous attention to detail, as well as tight experimental control of flow rates to ensure deterministic encapsulation efficiency. These aspects may require optimization for different experimental setups. While this method relies on fluorescence microscopy for signal detection, necessitating access to suitable imaging equipment, this equipment can be fabricated using open-source hardware, making it more accessible. Furthermore, microgels can be processed in commercial flow cytometry with large nozzles, further improving accessibility and experimental throughput. Droplet sorters can also be used for this cytometric analysis.
Moreover, while the method is designed to detect fluorescent signals from plasmids, stains, or other markers, it is limited to cells that can be fluorescently labeled, which may not apply to all bacterial strains or experimental conditions. However, the method can be adapted to incorporate other types of microscopies, such as phase-contrast or brightfield microscopy, allowing for phenotyping applications beyond fluorescence. Additionally, it can be combined with spectroscopic techniques like FTIR or Raman spectroscopy, expanding its capabilities to analyze chemical compositions and structural information of the encapsulated cells. These adaptations broaden the range of its applicability, making it a versatile tool for diverse research settings.
Significance and applications
Traditional assays for plasmid loss19 do not allow a good quantification of the ratio of cells that lost their expression, information that can be very important in experimental method design and various biological applications. Usually, colony types are enumerated in agar plate assays, where well-defined isolated colonies can be obtained, as demonstrated in Figure 4. However, overlapping colonies are challenging to identify with confidence; in our hands, we do not always obtain an optimal colony density, and many plates are necessary to obtain good statistics of low-frequency plasmid loss events. The proposed method offers a more robust approach to accurately quantify fluorescent signals coming from isolated colonies with a higher number of colonies than the agar plate analog methods because, in microdroplets, colonies develop separately, are smaller, and are easy to load into imaging chambers, enabling microscopy or flow-cytometry based quantification of large colony numbers. This can significantly improve the statistical representation of the method and allow integration into other gel-microdroplet workflows.
The usage of open-source hardware11,20 allows researchers to customize the microfluidic workstation design and precisely adjust flow rat; therefore, particle size supports various cell types and experimental conditions. This flexibility extends to potentially incorporating other microscopy types, such as phase-contrast or spectroscopy, broadening the method&#39;s applicability. The method&#39;s capacity to evaluate plasmid stability under various conditions is crucial for applications requiring plasmid retention without antibiotic selection, under particular stress conditions, or various culture generations. The versatility and adaptability of the presented method make it valuable for diverse research applications in fields including synthetic biology, environmental monitoring, and clinical diagnostics2.

Plasmids are essential genetic elements in prokaryotic cells, significantly contributing to microbial evolution through lateral DNA transfer and rapid adaptation to environmental changes1,2. These extrachromosomal DNA molecules carry genes that provide advantageous traits, such as antibiotic resistance, metabolic functions, and virulence factors, making them valuable for microbiology research, synthetic biology, and evolution studies2. However, plasmid maintenance in cell populations is challenging due to the metabolic burden of replication and segregation, often resulting in plasmid loss without selection pressure3. Additionally, stable inheritance requires mechanisms like toxin-antitoxin and partitioning systems, adding complexity to plasmid maintenance. Assessing plasmid stability under varying conditions is crucial for both fundamental research and practical applications that utilize plasmids as a primary research element4,5. Most current methods for assessing plasmid stability have significant limitations: Flow cytometry-based methods provide indirect, population-level data, require expensive equipment, and lack direct visualization of colonies6. Bulk transcriptomics and proteomics methods are costly, provide only average cellular responses, and cannot directly quantify plasmid retention in individual colonies6. Traditional methods like serial dilution and passaging are simple&#160;but time-consuming and lack precision and representability7. Overall, quantitatively inferring or projecting the number of colonies that retain a specific functional plasmid over time or selective pressures remains challenging.
To address these challenges, a novel microfluidic workflow utilizing open hardware research instruments is presented to quantify fluorescent signals in multiple isolated colonies of bacteria, using Escherichia coli as a model. This method allows for high-throughput and precise analysis of plasmid retention over various conditions or selective pressures. Single-cell resolution analysis provides a precise method to manipulate isolated colonies, yielding sensitive data on plasmid quantification that can help to assess the retention and loss rates4.
Microfluidics, particularly droplet microfluidics, has emerged as a powerful tool for encapsulating and manipulating individual cells in controlled environments8. Specifically, microgel droplets can encapsulate single cells for high-throughput and precise analysis without the need for maintaining droplets suspended in oil9, allowing a&#160;controlled study of plasmid dynamics in a defined microenvironment. Following encapsulation of cell suspensions directly from a pipette tip10, fluorescence techniques can be used to monitor the growth of microcolonies within droplets, enabling detailed analysis of plasmid retention and segregation under different selection pressures3.
The advantages of this method over traditional bulk culture techniques include increased precision, reduced variability, and the ability to conduct high-throughput analyses. Open-source microfluidics technology overcomes the limitations of expensive proprietary systems, such as issues of accessibility, adaptability, and maintenance, which often hinder research progress11,12,13. By demonstrating how to apply the advanced experimental workflow of plasmid retention analysis in microgels with open-source instrumentation, an accessible and reliable method is provided for research in plasmid biology, synthetic biology applications, and microfluidic droplet analysis techniques.
In summary, this article presents an accessible method for quantitatively assessing plasmid retention in E. coli with high statistical power. This method&#39;s capabilities make it a valuable tool for advancing the understanding of plasmid biology and improving synthetic biology applications.

<table><tbody><tr><td>1H,1H,2H,2H-Perfluoro-1-octanol</td><td>Sigma-Aldrich</td><td>370533-25G</td><td>For breaking emulsion</td></tr><tr><td>70% ethanol</td><td></td><td></td><td>For cell permeabilization</td></tr><tr><td>Agar-Agar</td><td>Winkler</td><td>9002-18-0</td><td></td></tr><tr><td>Biopsy Punch</td><td></td><td></td><td>0.75 mm and 1.8 mm</td></tr><tr><td>Blue LED transilluminator</td><td>IO Rodeo</td><td></td><td></td></tr><tr><td>Culture tube</td><td></td><td></td><td>15 mL</td></tr><tr><td>Desiccator</td><td></td><td></td><td>With vacuum pump</td></tr><tr><td>Disposable cup</td><td></td><td></td><td>For mixing PDMS</td></tr><tr><td>Disposable fork</td><td></td><td></td><td>For mixing PDMS</td></tr><tr><td>E. coli TOP10 strain</td><td></td><td></td><td></td></tr><tr><td>FluoPi microscope</td><td>https://github.com/wenzel-lab/FluoPi</td><td></td><td>Green fluorescence imaging system for analyzing plates</td></tr><tr><td>Fluorinated Oil</td><td>3M</td><td></td><td>Novec 7500</td></tr><tr><td>Glass slide heater</td><td>https://github.com/wenzel-lab/modular-microfluidics-workstation-controller/tree/master/module-heating-and-stirring</td><td></td><td>For controlling the temperature at 40 &amp;deg;C of microfluidic chip</td></tr><tr><td>Glass Slides</td><td></td><td></td><td></td></tr><tr><td>Hotplate</td><td>Mechanic</td><td></td><td>For evaporating Aquapel</td></tr><tr><td>Image analysis software</td><td>Fiji/ImageJ</td><td></td><td>2.14.0/1.54f</td></tr><tr><td>Incubator</td><td>Mundo Lab</td><td></td><td>MLAB Scientific / For incubation of plates and microgels&amp;nbsp;</td></tr><tr><td>Isopropanol</td><td></td><td></td><td>For cleaning glass slides</td></tr><tr><td>Kanamycin</td><td></td><td></td><td>100 ug/mL concentration</td></tr><tr><td>L-shaped spreader</td><td></td><td></td><td>For spreading bacteria on agar plates</td></tr><tr><td>Master mold</td><td></td><td></td><td>Chip design on silicone or glass wafer</td></tr><tr><td>Microtubes</td><td></td><td></td><td>2 mL</td></tr><tr><td>NaCl solution</td><td></td><td></td><td>Sodium chloride 0.9% w/v</td></tr><tr><td>Open-source hardware strobe-enhanced microscopy stage</td><td>https://github.com/wenzel-lab/flow-microscopy-platform</td><td></td><td>For bright-field microscopy</td></tr><tr><td>Petri dish</td><td>Citotest</td><td>2303-1090</td><td>90 x 15 mm</td></tr><tr><td>Pipette tip heater</td><td>https://github.com/wenzel-lab/modular-microfluidics-workstation-controller/tree/master/module-heating-and-stirring</td><td></td><td>For controlling the temperature at 40 &amp;deg;C of pipette tip</td></tr><tr><td>Plasma Cleaner</td><td>Diener Electronic</td><td>117056</td><td>For bonding PDMS with a glass slide</td></tr><tr><td>Plasmid pCA_Odd1</td><td></td><td></td><td>Encodes sfGFP and kanamycin resistance</td></tr><tr><td>Polytetrafluoroethylene (PTFE) tubing</td><td>Adtech Polymer Engineering Ltd</td><td></td><td></td></tr><tr><td>Pre-mixed Luria Bertoni medium</td><td>US Biological Life Science</td><td>L1520</td><td></td></tr><tr><td>Propidium iodide (PI)</td><td></td><td></td><td>For staining</td></tr><tr><td>Raspberry Pi-based pressure and flow controller system</td><td>https://github.com/wenzel-lab/modular-microfluidics-workstation-controller</td><td></td><td>For controlling pressure and flow rates</td></tr><tr><td>Silicone elastomer base</td><td>Sylgard</td><td></td><td>PDMS kit - 184 Silicone Elastomer Kit</td></tr><tr><td>Silicone elastomer curing agent</td><td>Sylgard</td><td></td><td>PDMS kit - 184 Silicone Elastomer Kit</td></tr><tr><td>Spectrophotometer</td><td></td><td></td><td>For measuring absorbance</td></tr><tr><td>SQUID microscope</td><td>https://github.com/wenzel-lab/SQUID-bioimaging-platform</td><td></td><td>Multi-fluorescence imaging system for analyzing stained cells</td></tr><tr><td>Sterile loop</td><td></td><td></td><td>For picking a colony and streaking plating</td></tr><tr><td>Surfactant</td><td>Sphere Fluidics</td><td></td><td>Pico-Surf</td></tr><tr><td>Syringes</td><td>NIPRO</td><td></td><td>With filters and tubing</td></tr><tr><td>Temperature-controlled shaker</td><td>Mundo Lab</td><td></td><td>DLAB HCM100-Pro</td></tr><tr><td>Tweezer</td><td></td><td></td><td></td></tr><tr><td>Ultra-low gelling temperature agarose</td><td>Sigma-Aldrich</td><td>A2576-5G</td><td>For generating hydrogel beads</td></tr><tr><td>Water repelent solution (fluoroalkyl silane)</td><td>Aquapel</td><td></td><td>For treating microchannels of PDMS device</td></tr></tbody></table>

plasmid stability analysis with open-source droplet microfluidics

Plasmid Stability Analysis with Open-Source Droplet Microfluidics

Plasmids play a vital role in synthetic biology by enabling the introduction and expression of foreign genes in various organisms, thereby facilitating the construction of biological circuits and pathways within and between cell populations. For many applications, maintaining functional plasmids without antibiotic selection is critical. This study introduces an open-hardware-based microfluidic workflow for analyzing plasmid retention by culturing single cells in gel microdroplets and quantifying microcolonies using fluorescence microscopy. This approach allows for the parallel analysis of numerous droplets and microcolonies, providing greater statistical power compared to traditional plate counting and enabling the integration of the assay into other droplet microfluidic workflows. By using plasmids expressing fluorescent proteins alongside a non-specific fluorescent DNA stain, single colonies can be identified and differentiated based on plasmid loss or fluorescent marker expression. Notably, this advanced workflow, implemented with open-source hardware, offers precise flow control and temperature management of both the sample and the microfluidic chip. These features enhance the workflow&#39;s ease of use, robustness, and accessibility. While the study focuses on Escherichia coli as the experimental model, the method&#39;s true potential lies in its versatility. It can be adapted for various studies requiring fluorescence signal quantification from plasmids or stains, as well as for other applications. The adoption of open-source hardware broadens the potential for conducting high-throughput bioanalyses using accessible technology in diverse research settings.

Validation of cell encapsulation and microcolony formation
The cell encapsulation can be visually confirmed by performing brightfield microscopy on the gel microdroplets before breaking the emulsion and washing the microgels. A representative result of the emulsion at this step is shown in Figure 4.
<img alt="Microfluidic droplets with green fluorescence, microscopy image for cell encapsulation study." class="xfigimg" src="/files/ftp_upload/67659/67659fig4.jpg" data-altupdatedat="1788304762000" data-alt="Figure 4"> 
Figure 4: Section of a fluorescence microscopy overlay image. After overnight incubation, representative microcolonies of sfGFP expressing E. coli colonies inside of gel microdroplets. A microscope objective with 10x magnification and a 0.30 NA was used. Scale bar: 100 µm. <a href="https://www.jove.com/files/ftp_upload/67659/67659fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Image analysis outcomes
Once microgels are stained and brightfield, as well as fluorescence channels acquired at several positions, the colonies identified as negative in the original images can be visualized (see Figure 5A). Data extracted from all images of one experiment can be plotted to show the fluorescence ratio of various colonies, highlighting those that lost plasmid-encoded fluorescence (see Figure 5B). Results indicate that 100 colonies lost their plasmid or plasmid functionality from a total of 2785 analyzed microcolonies, corresponding to 3.6%.
<img alt="Cell analysis, fluorescence microscopy image and scatter plot; green vs. red fluorescence data." class="xfigimg" src="/files/ftp_upload/67659/67659fig5.jpg" data-altupdatedat="1788304762000" data-alt="Figure 5"> 
Figure 5: Quantification of negative microcolonies. (A) Section of a fluorescence microscopy overlay image. After oil removal and staining, representative colonies in microgels expressing sfGFP and two negative colonies showed the red fluorescence of the DNA stain (circled in black). Scale bar: 100 µm. (B) Scatterplot of the fluorescence values of individual microcolonies extracted from 16 multi-channel microscopy images. Colonies without any green fluorescence were counted as negative, as indicated in red in the graph. <a href="https://www.jove.com/files/ftp_upload/67659/67659fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Agar plate quantification
Images of the triplicate plates are shown in Figure 6, with non-fluorescent colonies indicated by white arrows. The first plate (Figure 6A) displayed a total of 213 colonies, of which 1 was not fluorescent. The second plate (Figure 6B) had a total of 49 colonies, with no non-fluorescent colonies. The third plate (Figure 6C) showed a total of 252 colonies, 6 of which were not fluorescent. These results correspond to a mean colony plasmid loss rate of 2.3%, with a large standard deviation of 3.2.
<img alt="Bacterial colony growth on agar plates; fluorescent analysis; experiment results comparing samples A, B, C." class="xfigimg" src="/files/ftp_upload/67659/67659fig6.jpg" data-altupdatedat="1788304762000" data-alt="Figure 6"> 
Figure 6: Identification of negative colonies on plates. (A–C) Fluorescent and non-fluorescent E. coli colonies on LB-agar plates (diameter: 90 mm, height: 15 mm). The inoculum, derived from E. coli with sfGFP from -80 °C stock, was streaked on day 1, cultured with antibiotic on day 2, and diluted 1:100 daily from day 3 to 6 to allow plasmid loss. Colonies were incubated at 37 °C for 24 h and imaged in a FluoPi chamber. Non-fluorescent colonies were enhanced with GIMP and indicated with white arrows. <a href="https://www.jove.com/files/ftp_upload/67659/67659fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

An accessible, open-source microfluidic workflow is presented for the parallelized analysis of plasmid retention in bacteria. By employing fluorescence microscopy to quantify plasmid presence in single-cell microcolonies within gel microdroplets, this method provides a precise, accessible, and scalable alternative to traditional plate counting.

Plasmids play a vital role in synthetic biology by enabling the introduction and expression of foreign genes in various organisms, thereby facilitating ...

plasmid-stability-analysis-with-open-source-droplet-microfluidics

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Droplet-based microfluidic platforms are promising candidates for high throughput experimentation since they are able to generate picoliter, self-compartmentalized vessels inexpensively at kHz rates. Through integration with fast, sensitive and high resolution fluorescence spectroscopic methods, the large amounts of information generated within these systems can be efficiently extracted, harnessed and utilized.

Fluorescence detection methods for microfluidic droplet platforms

We present a simple method to produce microfluidic devices capable of applying similar dynamic conditions to multiple distinct strains, without the need for a clean room or soft lithography.

A Microfluidic Device for Studying Multiple Distinct Strains

Source: Hemmerich, J., et al. Protocol for Optimization of Heterologous Protein Production Using Automated Microbioreactor Technology. J. Vis. Exp. (2017).This video demonstrates the use of an automated microbioreactor to monitor bacterial growth and fluorescent protein secretion in response to varying media formulations. A genetically modified bacterium expressing a secretory fluorescent protein is cultured under antibiotic selection and inoculated into nutrient-varied wells. Real-time optical...

Monitoring Bacterial Growth in Distinct Media Formulations with an Automated Microbioreactor

In this protocol the fabrication, setup and basic operation of a microfluidic picoliter bioreactor (PLBR) for single-cell analysis of prokaryotic microorganisms is introduced. Industrially relevant microorganisms were analyzed as proof of principle allowing insights into growth rate, morphology, and phenotypic heterogeneity over certain time periods, hardly possible with conventional methods.

Microfluidic Picoliter Bioreactor for Microbial Single-cell Analysis: Fabrication, System Setup, and Operation

We describe an endpoint digital assay for quantifying nucleic acids with a simplified (analog) readout. We measure bulk fluorescence of droplet-based digital assays using a standard qPCR machine rather than specialized instrumentation and confirm our results by microscopy.

Simple Bulk Readout of Digital Nucleic Acid Quantification Assays

This work provides a method for the fabrication of droplet-based microfluidic platforms and the application of polyacrylamide microspheres for microsphere-PCR amplification. The microsphere-PCR method makes it possible to obtain single-stranded DNA amplicons without separating double-stranded DNA.

A Droplet-Based Microfluidic Approach and Microsphere-PCR Amplification for Single-Stranded DNA Amplicons

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

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

The overall goal of the protocol is to prepare over one million ordered, uniform, stable, and biocompatible femtoliter droplets on a 1 cm2 planar substrate that can be used for cell-free protein synthesis.

A Femtoliter Droplet Array for Massively Parallel Protein Synthesis from Single DNA Molecules

The fabrication of a polydimethylsiloxane (PDMS)-based bilayer device for the production of combinatorial libraries in water-in-oil emulsions (plugs) is presented here. The necessary hardware and software required to automate plug production are detailed in the protocol, and the production of a quantitative library of fluorescent plugs is also demonstrated.

Bilayer Microfluidic Device for Combinatorial Plug Production

This protocol presents a standard high-throughput screening workflow for industrial microorganisms. It involves constructing a mutant library via ARTP mutagenesis, performing positive droplet screening with automated microfluidics, and subsequently verifying the results. The workflow’s reliability was validated by screening high-yield B. coagulans strains.

High-Throughput Screening of L-Lactic Acid-Hyperproducing Bacillus coagulans Mutants Using an Automated Droplet Microfluidic Platform

Explore open-source droplet microfluidics for plasmid stability analysis in bacteria using fluorescence microscopy. This method enables high-throughput quantification of plasmid retention in single-cell microcolonies.

Plasmid Stability Analysis with Open-Source Droplet Microfluidics

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