This research was supported by NSF CAREER Award #1944791.

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RRH and AJV have filed a patent on this technology. The remaining authors declare no conflict of interest.

This manuscript demonstrates the use of photodegradable hydrogels for bacteria screening and isolation. The material and approach enable high-throughput culture, control over growth media and growth conditions, and clean and precise cell extraction in a straightforward and cost-effective manner. Extraction only requires a fluorescent microscope coupled with the patterned illumination tool and can be done in a sequential manner to isolate multiple cell targets. Each extraction takes 5-10 min to perform, and up to 30 targeted colonies have been removed from a single hydrogel. A key advantage of the approach is its adaptability to a variety of different assay formats, as demonstrated here with screening from both bulk hydrogels and microwell arrays. The separation process in both formats has been successfully used to isolate bacteria that display unique growth behavior for downstream genotyping after culture and microscopic observation, a critical capability for connecting cell genotype to phenotype. To date, genomic characterizations of bacteria extracted from these interfaces have included 16S amplicon sequencing to identify multi-species collections of bacteria from environmental microbiomes that generate emergent growth behavior18, and for whole-genome sequencing to successfully identify genetic mutations that cause rare growth profiles in cells present within mutant libraries19.
Using bulk hydrogels for cell screening and isolation is the most straightforward and simple format. Bulk photodegradable hydrogels form rapidly (25 min) after mixing the precursors over transparent glass coverslips to encapsulate cells in a 3-D cell culture matrix that is imaged with a standard upright or inverted fluorescence microscope. Thus, the method has the potential to be translational to common microbiology laboratories that do not have microfabrication resources or expertise. A drawback to this format is that cells are randomly oriented throughout the three-dimensional hydrogel. Therefore, cells can appear out of the focal plane when imaging with higher magnification objectives and extraction can be difficult if cell colonies are oriented too close to each other or if there is a vertical overlay of colonies. Depositing a thin hydrogel (&#60;13 &#956;m) as described is critical to mitigating this drawback. Exposure in broken cross light patterns (Figure 4B) is preferable here, as this pattern results in cells free of the hydrogel that have minimal exposure to UV light and can be readily recovered through plating.
In contrast, the microwell array format provides a more well-controlled interface, as bacteria cells are partitioned into discrete microwells that serve as small culture or co-cultures sites17,18,26. Microwell dimension, pitch, and density are precisely fabricated using standard photolithographic techniques. Compared to bulk hydrogels, bacteria can be extracted from microwell arrays with a higher degree of specificity and lower chance of cross-contamination, as the cells are only present at predefined locations, not randomly dispersed throughout the hydrogel. The concentration and ratios of bacteria cells in the seeding solution can also be varied to control the quantity and composition of the microwell inoculum through a seeding process that has been characterized in previous reports26, giving the user flexibility in the experimental design of the screen. The primary drawback of screening with the microwell array format is the added time and expertise required for microfabrication. Fabrication of microwells was estimated to cost ~$10 per array, which includes material costs and cleanroom expenses. In addition, microwells arrays are traditionally made from silicon, which can cause imaging difficulties since the substrates are non-transparent. Moreover, a high amount of light scattering from the silicon surface can limit imaging within the microwells and can decrease pattern resolution during hydrogel exposure with UV light from the patterned illumination tool (seen in Figure 8A,B). Similar microwells have been fabricated on transparent quartz substrates to address these types of limitations27; however, this fabrication is considerably more difficult. Exposure to ring patterns that illuminate the perimeter of the well is preferable here to release free cells from the wells while minimizing UV exposure.
The most common problem that occurs in either format is detachment of the hydrogel from the underlying substrate during culture due to hydrogel swelling. If this is an issue for bulk hydrogels, the presence, density, and uniformity of thiol groups in the chemical (MTPS) attachment layer across the surface of the base glass coverslip should be verified using an appropriate surface characterization technique (XPS, ATR-FTIR, AFM, etc.). Low densities of surface thiol groups due to inefficient surface functionalization can lead to a weak interaction between the substrate and the hydrogel. If a low level of surface thiolation occurs, the stability of the MTPS solution should be checked. Care should be taken in the initial cleaning of the glass slide to assure a clean surface prior to MTPS treatment, and care should be taken to ensure the use of anhydrous toluene during the MTPS surface reaction (Protocol Section 4). In the case of microwell arrays, surfaces are not thiolated, and hydrogels instead attach through the partial filling of the wells with the hydrogel, which anchors the hydrogel to the silicon substrate17. If hydrogel detachment is an issue in this system, more microwells or other microscale features can be etched into the array to further anchor the hydrogel to the substrate to promote stronger attachment.
A limitation of the technique in either format is the limited stability of the hydrogels in the presence of bacteria. It has been noted that some bacteria, such as A. tumefaciens, can degrade the hydrogel over the course of 5-7 days17,19, which limits the experiment time. Current investigation of the mechanisms of bacterial degradation is underway; it is hypothesized that the ester groups present from the diacrylate monomers are subject to bacteria-mediated hydrolysis and/or enzymatic degradation, as noted in other systems17. Developing more stable hydrogel chemistries will extend the time that bacteria can remain in the hydrogel and will extend the screen to microorganisms with slower growth rates. A second limitation is that in both formats, cell recovery and extraction occur in an open environment, resulting in relatively high extraction volumes (30-100 &#956;L), which can be susceptible to outside contamination. Thus, care must be given to ensure enough cells are present from the target colonies while minimizing the extraction solution volume. To obtain enough cells for plating and recovery or for extraction of DNA material, it was observed that in bulk hydrogels, cells must be cultured long enough to reach colony diameters of at least 10 &#181;m. To lower the required volume for cell extraction, it was observed that using a microliter syringe and tubing (Figure 2) was more efficient than pipetting. The tubing allowed the isolates to be drawn from the release point more accurately, requiring less solution volume and lowering the chance of contamination.
Future work involves understanding the effect of hydrogel mechanical properties on cell growth, as mechanical features of these hydrogels are well-controlled by the selection of appropriate PEG-based&#160;monomer precursors of various molecular weights28, and mechanical interactions likely play an important role in bacteria behavior29. As the hydrogel materials can be readily incorporated into a variety of different systems and devices, further development is also focused on the integration of this material into microfluidic systems. This would reduce the extraction solution to femtoliter to picoliter volumes, compared to traditional 30-100 &#956;L volumes currently required in the open collection format. Smaller solution volumes would greatly reduce potential contamination and move the approach towards single-cell isolation and characterization.

Isolation of cells with unique behaviors from a complex and heterogeneous environment is fundamental for obtaining genetic information in biology1. In microbiology, the selection and isolation of rare or unique microbes after observation becomes important in many applications that require a connection between genomic information and observable phenotypic information. These applications include selecting phenotypically rare strains from mutant libraries2, selecting keystone microorganisms from complex microbial communities3,4, and selecting phenotypically rare but important bacteria from isogenic populations. Isolation of viable but nonculturable cells (VBNC) from a bacteria population is an important example of the latter, where cells with the VBNC phenotype are often hidden in bacteria populations at 1:102 to 1:105&#160;ratios5,6.&#160;Due to the widespread difficulties in bacteria isolation, much remains unknown about many phenotypically rare microorganisms. These limitations emphasize the need for cell isolation techniques to first identify the target cell or cells from a mixture and then retrieve and isolate them for downstream molecular analysis7.
Some of the most commonly established methods of cell isolation include flow cytometry and fluorescent activated cell sorting (FACS)8, immunomagnetic separation9,10, and microfluidics11. While these isolation methods have high value, they also have drawbacks that limit their use. For example, FACS can provide routine microbial isolation at the single-cell level for follow-up genomic analysis3 but is often limited by its availability and expense, as well as downstream contamination issues11. Microfluidic-based approaches such as microfluidic flow cytometry have obtained much attention, which, compared to conventional flow cytometry, allows for a significant reduction in the sample volume required12. However, separation and retrieval of an individual or small collections of cells from microfluidic devices is often a challenging issue that typically requires a more complex setup and device design13. Many microfluidic-based approaches genetically characterize cells before they are input and observed in a device14, limiting the number of unique species observed when performing a functional screen. Given these limitations, further innovation of both methods and materials that are practical for cell screening and isolation is required for widespread use in many laboratories.
This paper presents a new, materials-based approach for bacteria screening and isolation. The method uses photodegradable hydrogels for cell encapsulation, culture, microscopic observation, and on-demand release and recovery of targeted bacteria with unique phenotypes. Hydrogels are designed to contain 10 nm mesh size, where each crosslink contains o-nitrobenzyl groups15. The material encapsulates or traps cells for observation while enabling the diffusion of nutrients and waste products to and from cells for culture. Exposing the material to a patterned 365 nm UV light source through an upright microscope enables local degradation of the hydrogel through photocleavage of o-nitrobenzyl groups16,17. Degradation triggers the selective release of cells for recovery for downstream analysis, including genomic and, potentially, proteomic and transcriptomic analysis. The experimental setup and protocol are relatively simple, inexpensive, and translational to microbiology laboratories. It requires only cell encapsulation through hydrogel formation, observation of captured cells with an upright brightfield and fluorescence microscope, and the illumination of cells of interest with a patterned UV light source for retrieval.
A key advantage of this materials-based approach to screening is its adaptability to different screening formats. In its most basic format, the material can be used for screening by encapsulating a heterogeneous cell collection in bulk hydrogels. Cells are then observed for the desired phenotype, and individual cells of interest are removed for genomic characterization. In more elaborate formats, the material can also be integrated into lab-on-a-chip devices to provide precise cell release and retrieval from desired areas of the device. Both formats are described here, and both have enabled recent novel microbial screening and selection applications17,18,19. The method is demonstrated here with model Gram-negative organisms (Escherichia coli, Agrobacterium tumefaciens) and a model Gram-positive organism (Bacillus subtilis) and has been readily extended to a variety of other bacteria.

<table><tbody><tr><td>(3-Mercaptopropyl)triethoxysilane</td><td>Sigma-Aldrich</td><td>175617-25G</td><td>&gt; 95%</td></tr><tr><td>Alconox detergent powder</td><td>Alconox</td><td>1104</td><td></td></tr><tr><td>Ammonium sulfate</td><td>Fisher Chemical</td><td>A702-500</td><td>Certified ACS Granular</td></tr><tr><td>Autoclave SK300C</td><td>Yamato Scientific</td><td>18016</td><td></td></tr><tr><td>Bacillus subtilis 1A1135</td><td>Bacillus Genetic Stock Center</td><td>1A1135</td><td></td></tr><tr><td>Brightfield upright microscope</td><td>Olympus Corporation</td><td>BX51</td><td></td></tr><tr><td>Calcium chloride, anhydrous</td><td>Fisher Chemical</td><td>C614-500</td><td>For Desiccators Pellets, 4-20 Mesh</td></tr><tr><td>Centrifuge 5702</td><td>Eppendorf</td><td>5702</td><td></td></tr><tr><td>Citric acid monohydrate</td><td>Sigma-Aldrich</td><td>C1909-500G</td><td>ACS reagent, &gt; 99.0%</td></tr><tr><td>D-Glucose (Dextrose)</td><td>VWR Amresco Life Science</td><td>0188-1KG</td><td></td></tr><tr><td>Dneasy Blood &amp;amp; Tissue Kit</td><td>Qiagen</td><td>69504</td><td>DNA purification kit</td></tr><tr><td>DOWSIL 184 silicone elastomer base</td><td>Dow Silicones Corporation</td><td></td><td>Storage temperature: -30-60 &amp;deg;C</td></tr><tr><td>DOWSIL 184 silicone elastomer curing agent</td><td>Dow Silicones Corporation</td><td></td><td>Storage temperature: &amp;lt; 32 C</td></tr><tr><td>EPOCH2 microplate spectrophotometer</td><td>BioTek Instruments</td><td>EPOCH2</td><td></td></tr><tr><td>Escherichia coli ATCC 25922</td><td>Thermo Fisher Scientific</td><td>R19020</td><td></td></tr><tr><td>Ethanol, anhydrous</td><td>Fisher Chemical</td><td>459844</td><td></td></tr><tr><td>Fisherbrand microscope cover glass</td><td>Fisher Scientific</td><td>12540A</td><td></td></tr><tr><td>Fisherfinest premium microscope slides plain</td><td>Fisher Scientific</td><td>12-544-4</td><td></td></tr><tr><td>Hydrogen peroxide solution</td><td>Sigma-Aldrich</td><td>216763-500ML</td><td>Contains inhibitor, 30 wt% in H2O</td></tr><tr><td>Incu-Shaker Mini</td><td>Benchmark</td><td>E5-0014-01</td><td></td></tr><tr><td>Isopropanol</td><td>Sigma-Aldrich</td><td>190764</td><td></td></tr><tr><td>Magnesium sulfate, 7-hydrate</td><td>Macron Fine Chemicals</td><td>6066-04</td><td>Avantor Performance Materials, Inc.</td></tr><tr><td>Methanol</td><td>Sigma-Aldrich</td><td>179337-4L</td><td>ACS reagent, &gt; 99.8%</td></tr><tr><td>NanoDrop One spectrophotometer</td><td>Thermo Scientific</td><td>ND-ONEC-W</td><td></td></tr><tr><td>Nitrogen, compressed</td><td>Matheson</td><td>UN1066</td><td></td></tr><tr><td>Oxygen plasma cleaner</td><td>Harrick Plasma</td><td>PDC-001-HP</td><td></td></tr><tr><td>Pentaerythritol tetra(mercaptoethyl) polyoxyethylene (4 arm-PEG)</td><td>NOF America Corporation</td><td>PTE-100SH</td><td>Sunbright-PTE-100SH</td></tr><tr><td>Phosphate buffered saline (PBS), 10X</td><td>VWR Amresco Life Science</td><td>K813-500ML</td><td>Store between 15 &amp;deg;C&amp;ndash;30 &amp;deg;C</td></tr><tr><td>Polydimethyl siloxane (PDMS) Slygard 184</td><td>Dow Corning</td><td>4019862</td><td></td></tr><tr><td>Polygon400</td><td>Mightex</td><td>DSI-D-000</td><td></td></tr><tr><td>Premium microscope slides</td><td>Fisher Scientific</td><td>12-544-4</td><td>25 x 75 x 1 mm</td></tr><tr><td>Sodium bicarbonate</td><td>Sigma-Aldrich</td><td>S5761-500G</td><td></td></tr><tr><td>Sodium chloride</td><td>Sigma Life Science</td><td>S5886-500G</td><td>Bioreagent, suitable for cell culture</td></tr><tr><td>Sodium hydroxide</td><td>Sigma-Aldrich</td><td>S8045-500G</td><td>BioXtra, &gt; 98%, pellets (anhydrous)</td></tr><tr><td>Sodium phosphate monobasic dihydrate</td><td>Sigma-Aldrich</td><td>71505-250G</td><td>BioUltra, for molecular biology, &gt; 99.0% (T)</td></tr><tr><td>Stainless steel thickness gage</td><td>Precision Brand Products</td><td>77739</td><td></td></tr><tr><td>Sulfuric acid</td><td>Sigma-Aldrich</td><td>320501-2.5L</td><td></td></tr><tr><td>Toluene, anhydrous, 99.8%</td><td>Sigma-Aldrich</td><td>244511-1L</td><td>Anhydrous, &gt; 99.8%</td></tr><tr><td>Trichloro (1H,1H,2H,2H perfluorooctyl) silane (TPS), 97%</td><td>Sigma-Aldrich</td><td>448931-10G</td><td></td></tr><tr><td>Tryptic soy broth</td><td>Sigma-Aldrich</td><td>22092-500G</td><td>For microbiology</td></tr><tr><td>Super Nuova Digital Hot Plate Stirrer</td><td>Thermo Scientific</td><td>S131825</td><td></td></tr><tr><td>Ultrasonic sonicator</td><td>Fischer Scientific</td><td>FS-110H</td><td></td></tr></tbody></table>

photodegradable hydrogel interfaces for bacteria screening, selection, and isolation

Biologists have long attempted to understand the relationship between phenotype and genotype. To better understand this connection, it is crucial to develop practical technologies that couple microscopic cell screening with cell isolation at high purity for downstream genetic analysis. Here, the use of photodegradable poly(ethylene glycol) hydrogels for screening and isolation of bacteria with unique growth phenotypes from heterogeneous cell populations is described. The method relies on encapsulating or entrapping cells with the hydrogel, followed by culture, microscopic screening, then use of a high-resolution light patterning tool for spatiotemporal control of hydrogel degradation and release of selected cells into a solution for retrieval. Applying different light patterns allows for control over the morphology of the extracted cell, and patterns such as rings or crosses can be used to retrieve cells with minimal direct UV light exposure to mitigate DNA damage to the isolates. Moreover, the light patterning tool delivers an adjustable light dose to achieve various degradation and cell release rates. It allows for degradation at high resolution, enabling cell retrieval with micron-scale spatial precision. Here, the use of this material to screen and retrieve bacteria from both bulk hydrogels and microfabricated lab-on-a-chip devices is demonstrated. The method is inexpensive, simple, and can be used for common and emerging applications in microbiology, including isolation of bacterial strains with rare growth profiles from mutant libraries and isolation of bacterial consortia with emergent phenotypes for genomic characterizations.

To investigate the ability of UV light to trigger controlled hydrogel degradation for cell release, hydrogels were first encapsulated over thiolated coverslips without bacteria present. Each hydrogel was exposed to three replicate circle patterns of light at different intensities and exposure times. The percent gel degradation was calculated after UV light exposure at each light intensity, and the exposure time was then quantified by coupling pendant thiol groups with a fluorescein-5-maelimide dye for fluorescence imaging19,24. A representative example of how these two parameters affect hydrogel degradation is shown in Figure 3. As evident, patterned light provided by the patterned illumination tool provides spatial-temporal control of hydrogel degradation at a resolution that can enable the release of only a small number of cells.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/63048/63048fig03.jpg" /> 
Figure 3: Control over hydrogel degradation. UV light dose and resulting hydrogel degradation rate are tunable via the patterned illumination tool. (Inset)&#160;Two different light intensities were chosen for patterned hydrogel degradation. After 365 nm UV light exposure, hydrogels were labeled with fluorescein-5-maleimide for fluorescence imaging. Reprinted (adapted) with permission from Fattahi et al.19 Copyright 2020 American Chemical Society.&#8203;&#160;<a href="https://www.jove.com/files/ftp_upload/63048/63048fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
For cell extraction, different light patterns were used to investigate cell release (Figure 4). Here, Agrobacterium bacteria cells were encapsulated into bulk hydrogels over thiolated glass coverslips, then cultured into microscale colonies. Hydrogels were then inspected in brightfield microscopy, and targeted microcolonies were exposed to varied UV light patterns. It was observed that different exposure patterns influenced the morphology of the released cells. This is potentially beneficial for various applications. For instance, exposing a ring pattern around the target colony results in the release of the entire colony still encapsulated in a protective PEG hydrogel and without direct UV light exposure (Figure 4A), which may preserve cells and provide easy downstream purification. In contrast, by exposing part or all of the colony to UV light, cells can be extracted either as aggregated cell clusters (Figure 4B) or as free, individual cells (Figure 4C).
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/63048/63048fig04.jpg" /> 
Figure 4: Control over the morphology of the extracted cells. (A) Use of a ring pattern to release the entire cell colony, protected in a PEG matrix. (B)&#160;Use of a broken cross pattern for cell release in aggregates. (C) Use of a cross pattern to release individual cells. Reprinted (adapted) with permission from Fattahi et al.19&#160;&#8203;&#160;<a href="https://www.jove.com/files/ftp_upload/63048/63048fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Critical in the encapsulation protocol is both the cell seeding density and the thickness of the hydrogel, as both of these parameters can influence the number of cells incorporated in the hydrogel for observation. To demonstrate, A. tumefaciens cells samples were encapsulated into hydrogels of two different thicknesses using thin spacers (12.7 &#181;m) or thick spacers (40 &#181;m) cultured, and&#160;imaged following the established protocols. Thinner hydrogels resulted in a microcolony density of 90 colonies/mm2 throughout the hydrogel, where minimal colony overlap was observed (Figure 5A). In contrast, hydrogel thicknesses greater than 12.7 &#181;m resulted in the formation of overlapping colonies in the vertical direction (Figure 5B), which may result in the extraction of multiple colonies. Overlapping colonies can cause cross-contamination during extraction due to the two-dimensional nature of the light pattern. For example, a top colony can be targeted, while an underlying colony also is extracted with it (Figure 5C). Therefore, using 12.7 &#181;m spacers is recommended for hydrogel preparation.
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/63048/63048fig05.jpg" /> 
Figure 5: Hydrogel thickness affects the extraction purity. (A) By utilizing spacers with a thickness of 12.7 &#181;m for hydrogel formation, colonies are formed within one 10x focal plane. (B) Overlay of colonies can be observed at 10x magnification if spacers with greater thicknesses than 12.7 &#181;m are used. (C) Cross-contamination can occur with an overlay of colonies during cell release: (i) a ring pattern is used to release a targeted cell colony, (ii) the targeted cell colony becomes detached from the hydrogel, and (iii) a second, underlying colony is observed during the light exposure beneath the targeted colony. This colony is also removed, resulting in cross-contamination. Reprinted (adapted) with permission from Fattahi et al.19&#160;Copyright 2020 American Chemical Society.&#8203;&#160;<a href="https://www.jove.com/files/ftp_upload/63048/63048fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Given the potential damage to bacteria with UV light, the effect of varied UV light micropatterns on cell viability was further studied using model, Gram-positive bacteria (B. subtilis) and model, Gram-negative bacteria (E. coli). Each was encapsulated within bulk hydrogels and cultured into microscale colonies according to standard protocols, verifying their compatibility with the hydrogel. Targeted microcolonies of equivalent sizes (26 &#177; 1 &#956;m diameter) were then exposed to a constant light dose (168 mJ/mm2), either in the form of circle patterns exposing entire microcolonies to UV light or cross-patterns that degrade only hydrogel edges to minimize light exposure to cells. Cells were then recovered and plated to quantify the CFU/mL recovered from each colony. No significant difference in cell recovery level was found (Figure 6A). To further investigate the purity of the extracted cells, DNA was extracted from E. coli samples and analyzed using a UV-Vis spectrophotometer. For both patterns, DNA quality levels fall within a A260/A280 range between 1.8 and 2.0 (Figure 6B), which is in the ideal range for genomic sequencing25. This demonstrates that the UV patterns used for release under the described conditions have minimal effect on the quantity of viable cells recovered from the bulk hydrogels or on genomic DNA quality after extraction.
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/63048/63048fig06.jpg" /> 
Figure 6: Impact of different light exposure patterns on cell viability and DNA quality of bacteria released from bulk hydrogels. (A) Cell recovery levels for both E. coli and B. subtilis after extraction using cross patterns and circle patterns. For this experiment, extraction was done from spherical colonies with the same diameter (26 &#181;m &#177; 1 &#181;m) to ensure the number of released cells from each colony was equivalent. The extracted solutions were then plated to calculate the CFU/mL acquired from each pattern. Statistical analysis showed no significant difference in CFU/mL obtained from cross and circle patterns for both E. coli and B. subtilis (P-value &#62; 0.05, n= 6 for both strains). (B) Spectrophotometric quantification of DNA quality for isolated E. coli cells using cross and circle patterns. Here, statistical analysis did not show a significant difference in DNA quality for the patterns used (P-value &#62; 0.05, n = 6) (C) Brightfield images of colonies with equal diameters exposed to cross and circle patterns. <a href="https://www.jove.com/files/ftp_upload/63048/63048fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Microwell arrays provide an alternative, lab-on-a-chip screening interface that offers more controlled screening features compared to bulk hydrogels. For example, microwell arrays enable the seeding of bacteria into discrete culture sites where the number of cells in the inoculum can be controlled. Geometric features of microwells such as well depth and diameter are also controlled through standard microfabrication methods. With these benefits, microwells have been useful for studying bacteria growth under spatial confinement26, and most recently for the discovery of symbiotic and antagonistic interactions between different bacterial species when confined together at the microscale18. Cellular extraction from wells for genomic analysis such as 16S amplicon sequencing is critical for these applications. Using the same hydrogel material, UV light can be exposed over a well containing cells of interest, either as circle or ring patterns. The latter ensures hydrogel degradation only at the microwell perimeter to prevent direct irradiation of cells. To demonstrate this, A. tumefaciens cells expressing mCherry were seeded into wells, the hydrogel was then attached to the microwell array. Cells were cultured and then irradiated with either circle or ring patterns. The membrane was then stained with fluorescein-5-maleimide dye. Two-color fluorescence images revealed that both the membrane and the cells within the wells are removed for both irradiation patterns17. Unlike the bulk hydrogel format, cell extraction here has only been observed in the shape of cell clusters18.
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/63048/63048fig07.jpg" /> 
Figure 7: Representative confocal microscopy images showing light pattern impact on cell isolation from microwell arrays. (A) Microwells with diameters of 40 &#181;m containing bacteria (red) after seeding and culture. (B) Light exposure using circle and ring patterns (blue). (C) Decreased red fluorescence demonstrates that cells are extracted from irradiated wells. (D) Two-color fluorescence image of membranes and bacteria after irradiation, indicating removal of both the hydrogel (green) and bacteria (red) from target wells. (E) Z-stack, two-color fluorescence image of target wells. The red line in (D) denotes the xz plane imaged in (E), and the green line in (E) denotes the xy plane imaged in (D). Samples in images (C-E) were washed for removal of released cells, then fixed and imaged. Scale bar = 40 &#181;m. Reprinted (adapted) with permission from van der Vlies et al.17. Copyright 2019 American Chemical Soceity.&#160;<a href="https://www.jove.com/files/ftp_upload/63048/63048fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
To quantify bacteria cell viability and DNA quality after extraction in this format, B. subtilis and E. coli cells were seeded, cultured, and then released from microwell arrays using circle and ring patterns (Figure 8A, B). Released cells were then plated on ATGN agar plates, and the DNA quality of the extracted cells was quantified. To ensure that a consistent number of cells was present during each extraction, microwells with similar fluorescent intensities (~6000 A.U.) and therefore a similar number of cells were targeted for release. The number of viable cells extracted using a circle pattern was not significantly different from the number of viable cells extracted using ring pattern for either bacteria&#160;(Figure 8C). Also, the DNA quality levels were not significantly different between the circle and ring patterns for either bacteria (Figure 8D). Hence, similar to findings in bulk hydrogels, the application of UV light at the intensity and duration specified here had a negligible impact on the viability and DNA quality of cells extracted from the microwell arrays. These findings demonstrate that viable bacteria cells can be selectively retrieved from microwells with minimal damage for downstream analysis.
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/63048/63048fig08.jpg" /> 
Figure 8: Impact of different light exposure patterns on cell viability and DNA quality of bacteria released from microwell arrays. (A,B) For both E. coli and B. subtilis, circle patterns and ring patterns were used for cell extraction from 10 &#181;m microwells. Circle pattern with a diameter of 10 &#181;m and ring pattern with an inner diameter of 10 &#181;m and outer diameter of 20 &#181;m were used in this experiment for cell extraction. Microwells with the same diameters were used to ensure that the number of released cells from each microwell was the same. (C) The extracted solutions were then plated to calculate the CFU/mL acquired from each exposure pattern. Statistical analysis showed no significant difference in CFU/mL obtained from circle and ring pattern for both E. coli and B. subtilis (P-value &#62; 0.05, n = 6 for both strains). (D) Spectrophotometry was used to measure the DNA quality of both E. coli and B. subtilis cells using circle and ring patterns. Here, statistical analysis did not show any significant difference in the DNA quality for the patterns used (P-value &#62; 0.05, n = 6 for both strains). <a href="https://www.jove.com/files/ftp_upload/63048/63048fig08large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary Figure 1: Design and fabrication of microwell arrays. (A) Standard microfabrication techniques were applied to fabricate microwell arrays on silicon wafers. (B) Each substrate consisted of 7 x 7 arrays of 10&#181;m diameter wells with 20 &#181;m depth and 30 &#181;m pitch. (C) Each array consisted of 225 microwells. This figure has been modified from Barua et al.18. Copyright 2021 Frontiers Media.&#160;<a href="https://www.jove.com/files/ftp_upload/63048/Supplementary Figure 1.pdf" target="_blank">Please click here to download this File.</a>

Watch this Scientific Journal Video about Photodegradable Hydrogel Interfaces for Bacteria Screening, Selection, and Isolation at JoVE.com

Photodegradable Hydrogel Interfaces for Bacteria Screening, Selection, and Isolation

The use of photodegradable hydrogels to isolate bacterial cells by utilizing a high-resolution light pattering tool is reported. Essential experimental procedures, results, and advantages of the process are reviewed. The method enables rapid and inexpensive isolation of targeted bacteria showing rare or unique functions from heterogeneous communities or populations.

Biologists have long attempted to understand the relationship between phenotype and genotype. To better understand this connection, it is crucial to ...

photodegradable-hydrogel-interfaces-for-bacteria-screening-selection

Research

JoVE Journal

Bioengineering

2.8K Views.  Kansas State University. Dit protocol is ontworpen om cellen snel te isoleren van screeninginterfaces voor genomische karakterisering en deze mogelijkheid is belangrijk omdat het macroscopisch waarneembare celkenmerken kan combineren met genomische informatie. De beoogde bacteriële cellen uit de screeningsinterfaces kunnen met behulp van deze techniek op een operationeel eenvoudige manier met een hoge ruimtelijke positie worden geïsoleerd. De hydrogels kunnen worden geïmplementeerd in andere screeninginterfaces.

Video: Photodegradable Hydrogel Interfaces for Bacteria Screening, Selection, and Isolation

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 ...

Engineering Adherent Bacteria by Creating a Single Synthetic Curli Operon

The method described here consists in redesigning E. coli adherence properties by assembling the minimum number of curli genes under the control of a strong and metal-overinducible promoter, and in visualizing and quantifying the resulting gain of bacterial adherence. This method applies appropriate engineering principles of abstraction and standardization of synthetic biology, and results in the BBa_K540000 Biobrick (Best new Biobrick device, engineered, iGEM 2011).
The first step consists in the design of the synthetic operon devoted to curli overproduction in response to metal, and therefore in increasing the adherence abilities of the wild type strain. The original curli operon was modified in silico in order to optimize transcriptional and translational signals and escape the "natural" regulation of curli. This approach allowed to test with success our current understanding of curli production. Moreover, simplifying the curli regulation by switching the endogenous complex promoter (more than 10 transcriptional regulators identified) to a simple metal-regulated promoter makes adherence much easier to control.
The second step includes qualitative and quantitative assessment of adherence abilities by implementation of simple methods. These methods are applicable to a large range of adherent bacteria regardless of biological structures involved in biofilm formation. Adherence test in 24-well polystyrene plates provides a quick preliminary visualization of the bacterial biofilm after crystal violet staining. This qualitative test can be sharpened by the quantification of the percentage of adherence. Such a method is very simple but more accurate than only crystal violet staining as described previously 1 with both a good repeatability and reproducibility. Visualization of GFP-tagged bacteria on glass slides by fluorescence or laser confocal microscopy allows to strengthen the results obtained with the 24-well plate test by direct observation of the phenomenon.

The design of a synthetic operon encoding both the secretory apparatus and the structural monomers of curli fibers is described. Overproduction of these amyloids and adherent polymers allows a measurable gain of adherence of the E. coli chassis1. Easy ways to visualize and quantify adherence are explained.

The  method described here consists in redesigning E. coli adherence properties by assembling the minimum number of  curli genes under the control of a ...

Stress-induced Antibiotic Susceptibility Testing on a Chip

We have developed a rapid microfluidic method for antibiotic susceptibility testing in a stress-based environment. Fluid is passed at high speeds over bacteria immobilized on the bottom of a microfluidic channel. In the presence of stress and antibiotic, susceptible strains of bacteria die rapidly. However, resistant bacteria survive these stressful conditions. The hypothesis behind this method is new: stress activation of biochemical pathways, which are targets of antibiotics, can accelerate antibiotic susceptibility testing. As compared to standard antibiotic susceptibility testing methods, the rate-limiting step -&#160;bacterial growth -&#160;is omitted during antibiotic application. The technical implementation of the method is in a combination of standard techniques and innovative approaches. The standard parts of the method include bacterial culture protocols, defining microfluidic channels in polydimethylsiloxane&#160;(PDMS), cell viability monitoring with fluorescence, and batch image processing for bacteria counting. Innovative parts of the method are in the use of culture media flow for mechanical stress application, use of enzymes to damage but not kill the bacteria, and use of microarray substrates for bacterial attachment. The developed platform can be used in antibiotic and nonantibiotic related drug development and testing. As compared to the standard bacterial suspension experiments, the effect of the drug can be turned on and off repeatedly over controlled time periods. Repetitive observation of the same bacterial population is possible over the course of the same experiment.

We have developed a microfluidic platform for rapid antibiotic susceptibility testing. Fluid is passed at high speeds over bacteria immobilized on the bottom of a microfluidic channel. In the presence of stress and antibiotic, susceptible strains of bacteria die rapidly. However, resistant bacteria can survive these stressful conditions.

We have developed a rapid microfluidic method for antibiotic susceptibility testing in a stress-based environment. Fluid is passed at high speeds over ...

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 ...

Microfluidic Co-culture of Epithelial Cells and Bacteria for Investigating Soluble Signal-mediated Interactions

The human gastrointestinal (GI) tract is a unique environment in which intestinal epithelial cells and non-pathogenic (commensal) bacteria coexist. It has been proposed that the microenvironment that the pathogen encounters in the commensal layer is important in determining the extent of colonization. Current culture methods for investigating pathogen colonization are not well suited for investigating this hypothesis as they do not enable co-culture of bacteria and epithelial cells in a manner that mimics the GI tract microenvironment. Here we describe a microfluidic co-culture model that enables independent culture of eukaryotic cells and bacteria, and testing the effect of the commensal microenvironment on pathogen colonization. The co-culture model is demonstrated by developing a commensal Escherichia coli biofilm among HeLa cells, followed by introduction of enterohemorrhagic E. coli (EHEC) into the commensal island, in a sequence that mimics the sequence of events in GI tract infection.

This protocol describes a microfluidic co-culture model for simultaneous and localized culture of epithelial cells and bacteria. This model can be used for investigating the role of different soluble molecular signals on pathogenesis as well as screen the effectiveness of putative probiotic bacterial strains.

The human gastrointestinal (GI) tract is a unique environment in which intestinal epithelial cells and non-pathogenic (commensal) bacteria coexist. It has ...

Biology

Microfluidic Acoustophoresis for Flowthrough Separation of Gram-Negative Bacteria using Aptamer Affinity Beads

This article describes the fabrication and operation of microfluidic acoustophoretic chips using a microfluidic acoustophoresis technique and aptamer-modified microbeads that can be used for the fast, efficient isolation of Gram-negative bacteria from a medium. This method enhances the separation efficiency using a mix of long, square microchannels. In this system, the sample and buffer are injected into the inlet port through a flow controller. For bead centering and sample separation, AC power is applied to the piezoelectric transducer via a function generator with a power amplifier to generate acoustic radiation force in the microchannel. There is a bifurcated channel at both the inlet and outlet, enabling simultaneous separation, purification, and concentration. The device has a recovery rate of &gt;98% and purity of 97.8% up to a 10x dose concentration. This study has demonstrated a recovery rate and purity higher than the existing methods for separating bacteria, suggesting that the device can separate bacteria efficiently.

This paper describesthe fabrication and operation of microfluidic acoustophoretic chips using the microfluidic acoustophoresis technique and aptamer-modified microbeads that can be used for fast, efficient isolation of Gram-negative bacteria from a medium.

This article describes the fabrication and operation of microfluidic acoustophoretic chips using a microfluidic acoustophoresis technique and ...

Engineering

Automated Hydrophobic Interaction Chromatography Column Selection for Use in Protein Purification

In contrast to other chromatographic methods for purifying proteins (e.g. gel filtration, affinity, and ion exchange), hydrophobic interaction chromatography (HIC) commonly requires experimental determination (referred to as screening or "scouting") in order to select the most suitable chromatographic medium for purifying a given protein 1. The method presented here describes an automated approach to scouting for an optimal HIC media to be used in protein purification.
HIC separates proteins and other biomolecules from a crude lysate based on differences in hydrophobicity. Similar to affinity chromatography (AC) and ion exchange chromatography (IEX), HIC is capable of concentrating the protein of interest as it progresses through the chromatographic process. Proteins best suited for purification by HIC include those with hydrophobic surface regions and able to withstand exposure to salt concentrations in excess of 2 M ammonium sulfate ((NH4)2SO4). HIC is often chosen as a purification method for proteins lacking an affinity tag, and thus unsuitable for AC, and when IEX fails to provide adequate purification. Hydrophobic moieties on the protein surface temporarily bind to a nonpolar ligand coupled to an inert, immobile matrix. The interaction between protein and ligand are highly dependent on the salt concentration of the buffer flowing through the chromatography column, with high ionic concentrations strengthening the protein-ligand interaction and making the protein immobile (i.e. bound inside the column) 2. As salt concentrations decrease, the protein-ligand interaction dissipates, the protein again becomes mobile and elutes from the column. Several HIC media are commercially available in pre-packed columns, each containing one of several hydrophobic ligands (e.g. S-butyl, butyl, octyl, and phenyl) cross-linked at varying densities to agarose beads of a specific diameter 3. Automated column scouting allows for an efficient approach for determining which HIC media should be employed for future, more exhaustive optimization experiments and protein purification runs 4.
The specific protein being purified here is recombinant green fluorescent protein (GFP); however, the approach may be adapted for purifying other proteins with one or more hydrophobic surface regions. GFP serves as a useful model protein, due to its stability, unique light absorbance peak at 397 nm, and fluorescence when exposed to UV light 5. Bacterial lysate containing wild type GFP was prepared in a high-salt buffer, loaded into a Bio-Rad DuoFlow medium pressure liquid chromatography system, and adsorbed to HiTrap HIC columns containing different HIC media. The protein was eluted from the columns and analyzed by in-line and post-run detection methods. Buffer blending, dynamic sample loop injection, sequential column selection, multi-wavelength analysis, and split fraction eluate collection increased the functionality of the system and reproducibility of the experimental approach.

An automated method for identifying suitable hydrophobic interaction chromatography (HIC) media to be used in the process of protein purification is presented. The method utilizes a medium-pressure liquid chromatography system including automated buffer blending, dynamic sample loop injection, sequential column selection, multi-wavelength analysis, and split fraction eluate collection.

In contrast to other chromatographic methods for purifying proteins (e.g. gel filtration, affinity, and ion exchange), hydrophobic interaction ...

A Multi-well Format Polyacrylamide-based Assay for Studying the Effect of Extracellular Matrix Stiffness on the Bacterial Infection of Adherent Cells

Extracellular matrix stiffness comprises one of the multiple environmental mechanical stimuli that are well known to influence cellular behavior, function, and fate in general. Although increasingly more adherent cell types&#39; responses to matrix stiffness have been characterized, how adherent cells&#39; susceptibility to bacterial infection depends on matrix stiffness is largely unknown, as is the effect of bacterial infection on the biomechanics of host cells. We hypothesize that the susceptibility of host endothelial cells to a bacterial infection depends on the stiffness of the matrix on which these cells reside, and that the infection of the host cells with bacteria will change their biomechanics. To test these two hypotheses, endothelial cells were used as model hosts and Listeria monocytogenes as a model pathogen. By developing a novel multi-well format assay, we show that the effect of matrix stiffness on infection of endothelial cells by L. monocytogenes can be quantitatively assessed through flow cytometry and immunostaining followed by microscopy. In addition, using traction force microscopy, the effect of L. monocytogenes infection on host endothelial cell biomechanics can be studied. The proposed method allows for the analysis of the effect of tissue-relevant mechanics on bacterial infection of adherent cells, which is a critical step towards understanding the biomechanical interactions between cells, their extracellular matrix, and pathogenic bacteria. This method is also applicable to a wide variety of other types of studies on cell biomechanics and response to substrate stiffness where it is important to be able to perform many replicates in parallel in each experiment.

We have developed a multi-well format polyacrylamide-based assay for probing the effect of extracellular matrix stiffness on bacterial infection of adherent cells. This assay is compatible with flow cytometry, immunostaining, and traction force microscopy, allowing for quantitative measurements of the biomechanical interactions between cells, their extracellular matrix, and pathogenic bacteria.

Extracellular matrix stiffness comprises one of the multiple environmental mechanical stimuli that are well known to influence cellular behavior, ...

Immunology and Infection

Generating Controlled, Dynamic Chemical Landscapes to Study Microbial Behavior

We demonstrate a method for the generation of controlled, dynamic chemical pulses&#8213;where localized chemoattractant becomes suddenly available at the microscale&#8213;to create micro-environments for microbial chemotaxis experiments. To create chemical pulses, we developed a system to introduce amino acid sources near-instantaneously by photolysis of caged amino acids within a polydimethylsiloxane (PDMS) microfluidic chamber containing a bacterial suspension. We applied this method to the chemotactic bacterium, Vibrio ordalii, which can actively climb these dynamic chemical gradients while being tracked by video microscopy. Amino acids, rendered biologically inert (&#39;caged&#39;) by chemical modification with a photoremovable protecting group, are uniformly present in the suspension but not available for consumption until their sudden release, which occurs at user-defined points in time and space by means of a near-UV-A focused LED beam. The number of molecules released in the pulse can be determined by a calibration relationship between exposure time and uncaging fraction, where the absorption spectrum after photolysis is characterized by using UV-Vis spectroscopy. A nanoporous polycarbonate (PCTE) membrane can be integrated into the microfluidic device to allow the continuous removal by flow of the uncaged compounds and the spent media. A strong, irreversible bond between the PCTE membrane and the PDMS microfluidic structure is achieved by coating the membrane with a solution of 3-aminopropyltriethoxysilane (APTES) followed by plasma activation of the surfaces to be bonded. A computer-controlled system can generate user-defined sequences of pulses at different locations and with different intensities, so as to create resource landscapes with prescribed spatial and temporal variability. In each chemical landscape, the dynamics of bacterial movement at the individual scale and their accumulation at the population level can be obtained, thereby allowing the quantification of chemotactic performance and its effects on bacterial aggregations in ecologically relevant environments.

A protocol for the generation of dynamic chemical landscapes by photolysis within microfluidic and millifluidic setups is presented. This methodology is suitable to study diverse biological processes, including the motile behavior, nutrient uptake, or adaptation to chemicals of microorganisms, both at the single cell and population level.

We demonstrate a method for the generation of controlled, dynamic chemical pulses&#8213;where localized chemoattractant becomes suddenly available at the ...

Kinetic Visualization of Single-Cell Interspecies Bacterial Interactions

Polymicrobial communities are ubiquitous in nature, yet studying their interactions at the single-cell level is difficult. Thus, a microscopy-based method has been developed for observing interspecies interactions between two bacterial pathogens. The use of this method to interrogate interactions between a motile Gram-negative pathogen, Pseudomonas aeruginosa and a non-motile Gram-positive pathogen, Staphylococcus aureus is demonstrated here. This protocol consists of co-inoculating each species between a coverslip and an agarose pad, which maintains the cells in a single plane and allows for visualization of bacterial behaviors in both space and time.
Furthermore, the time-lapse microscopy demonstrated here is ideal for visualizing the early interactions that take place between two or more bacterial species, including changes in bacterial species motility in monoculture and in coculture with other species. Due to the nature of the limited sample space in the microscopy setup, this protocol is less applicable for studying later interactions between bacterial species once cell populations are too high. However, there are several different applications of the protocol which include the use of staining for imaging live and dead bacterial cells, quantification of gene or protein expression through fluorescent reporters, and tracking bacterial cell movement in both single species and multispecies experiments.

This live-bacterial cell imaging protocol allows for visualization of interactions between multiple bacterial species at the single-cell level over time. Time-lapse imaging allows for observation of each bacterial species in monoculture or coculture to interrogate interspecies interactions in multispecies bacterial communities, including individual cell motility and viability.

Polymicrobial communities are ubiquitous in nature, yet studying their interactions at the single-cell level is difficult. Thus, a microscopy-based method ...

Photodegradable Hydrogel Interfaces for Bacteria Screening, Selection, and Isolation

Samenvatting

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