Name: assembly and tracking of microbial community development within a microwell array platform
Uploaded: 2017-06-06T15:30:00
Description: Watch this Scientific Journal Video about Assembly and Tracking of Microbial Community Development within a Microwell Array Platform at JoVE.com

Microwell arrays were fabricated and characterized at the Center for Nanophase Materials Sciences User Facilities Division, Office of Basic Energy Sciences, U.S. Department of Energy. Financial support for this work was provided through the Oak Ridge National Laboratory Director&#39;s Research and Development Fund. The authors would also like to thank the J. Mougous Laboratory (University of Washington, Seattle, WA) for the supply of P. aeruginosa strains used in these studies.

1. Silicon Microwell-array Fabrication
<ol>
	<li>Parylene coating
	<ol>
		<li>Deposit between 1-1.5 &#181;m of parylene N on silicon wafers using a commercially available parylene coating system according to the manufacturer&#39;s specifications and instructions (settings: vaporizer set point = 160 &#176;C; furnace set point = 650 &#176;C). 
		NOTE: Approximately 6 g of parylene N loaded into a chamber yields coatings 1-1.5 &#181;m thick.</li>
	</ol>
	</li>
	<li>Photolithography
	...

<ol>
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S., Vlamakis, H., Kim, P., Khan, M., Kolter, R., Aizenberg, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+flagella+explore+microscale+hummocks+and+hollows+to+increase+adhesion.">Bacterial flagella explore microscale hummocks and hollows to increase adhesion.</a> Proc Natl Acad Sci USA. 110 (14), 5624-5629 (2013).</li><li>Zhou, J., Liu, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stochastic+Assembly+Leads+to+Alternative+Communities+with+Distinct+Functions+in+a+Bioreactor+Microbial+Community.">Stochastic Assembly Leads to Alternative Communities with Distinct Functions in a Bioreactor Microbial Community.</a> MBio. 4 (2), 1-8 (2013).</li><li>van Vliet, S., Hol, F. J., Weenink, T., Galajda, P., Keymer, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+chemical+interactions+and+culture+history+on+the+colonization+of+structured+habitats+by+competing+bacterial+populations.">The effects of chemical interactions and culture history on the colonization of structured habitats by competing bacterial populations.</a> BMC Microbiol. 14 (1), 116 (2014).</li><li>Niepa, T. H. R., Hou, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbial+Nanoculture+as+an+Artificial+Microniche.">Microbial Nanoculture as an Artificial Microniche.</a> Sci Rep. 6, 30578 (2016).</li><li>Hansen, R. H., Timm, A. 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D., Singh, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Type+VI+Secretion+System+of+Pseudomonas+aeruginosa+Targets+a+Toxin+to+Bacteria.">A Type VI Secretion System of Pseudomonas aeruginosa Targets a Toxin to Bacteria.</a> Cell Host Microbe. 7 (1), 25-37 (2010).</li><li>LeRoux, M., Ja De Leon, ., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+single-cell+characterization+of+bacterial+interactions+reveals+type+VI+secretion+is+a+double-edged+sword.">Quantitative single-cell characterization of bacterial interactions reveals type VI secretion is a double-edged sword.</a> Proc Natl Acad Sci. 109 (48), 19804-19809 (2012).</li><li>Whitney, J. C., Beck, C. 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This article presented a microwell array device and experimental protocols designed to enable high-throughput and high-content live-cell imaging-based analysis of bacterial community development. While the focus of the demonstration here was to study the effects of contact-mediated Type VI secretion on community development, the arrays were designed to be flexible and accommodate the study of a broad range of microbial communities and microbe-microbe interactions. The work here focuses solely on the use of bacteria that ...

Microbial communities are shaped by both deterministic factors, such as the structure of the environment, and stochastic processes, which are associated with cell death, division, protein concentration, number of organelles, and mutation1. Within the natural environment, it can be nearly impossible to parse the individual impact of these influences on community composition and activity. Obscured by natural structures and buried within a chemical and biological milieu, identifying community members and further resolving their spatiotemporal distribution within the natural environment is extremely challenging. Nonetheless, recent efforts have underscored the importance of spatial organization on community function and point towards the need to account for both member abundance and organization in ongoing studies2,3,4.
It is clear that the local chemical environment (i.e.&#160;the availability of nutrients and secondary metabolites), the physical structure (e.g., soil architecture, plant roots, ocean particles, or the intestinal microvilli), the presence or absence of oxygen, and the introduction of pathogenic species all affect the composition, architecture, and function of microbial communities5,6,7,8,9,10,11. Nonetheless, traditional techniques for cultures that neglect to capture these factors continue to prevail. Community composition (e.g., the presence of co-dependent species), physical attachment, signaling molecule concentration, and direct cell-cell contact are all important factors for shaping a microbial community and can be lost in conventional culture conditions. These properties are difficult to replicate in a bulk liquid culture or on an agar plate. The availability of microfluidic, micropatterning, and nanofabrication techniques that allow for the replication of key physical and chemical features of natural environments has, however, enabled many researchers to build bacterial communities to study their interactions12,13,14 and to develop synthetic environments that mimic natural conditions4,15,16,17,18,19,20.
This protocol describes a method to fabricate a microwell array device and provides detailed experimental procedures that can be used to functionalize the wells in the array and to grow bacteria, both as single-species colonies and in multi-member communities. This work also demonstrates how bacteria modified to produce fluorescent reporter proteins can be used to monitor bacterial growth within wells over time. A similar array was presented previously and showed that it is possible to track the growth of single-species colonies of Pseudomonas aeruginosa (P. aeruginosa)&#160;in microwells. By modulating well size and seeding density, the starting conditions of thousands of growth experiments can be varied in parallel to determine how the initial inoculation conditions affect the ability of the bacteria to grow21. The current work uses a slightly modified version of the microwell array that builds on the previous work by enabling the simultaneous comparison of multiple arrays and by using a more robust experimental protocol. The array used in this work contains multiple subarrays, or array ensembles, containing wells of different sizes, ranging from 15 - 100 &#181;m in diameter, that are arranged at three different pitches (i.e.&#160;2x, 3x, and 4x the well diameter). The arrays are etched into silicon, and the growth of the bacteria seeded in the silicon arrays is enabled by sealing the arrays with a coverslip that has been coated with a medium-infused agarose gel. P. aeruginosa mutants designed to study the Type VI secretion system are used in this demonstration.
The results presented here build toward the ultimate goal of analyzing multimember communities within microwell arrays, enabling researchers to monitor the abundance and organization of bacteria in situ while controlling and probing the chemical environment. This should ultimately provide insights into the &#34;rules&#34; that govern community development and succession.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Parylene N</td>
			<td>Specialty Coating Systems</td>
			<td>CAS NO.:1633-22-3</td>
		</tr>
		<tr>
			<td>Parylene coater</td>
			<td>Specialty Coating Systems</td>
			<td>Labcoter 2 Parylene Deposition Unit PDS2010</td>
		</tr>
		<tr>
			<td>Silicon Wafer</td>
			<td>WRS Materials</td>
			<td>100mm diameter, 500-550&#956;m thickness, Prime, 10-20 resistivity, N/Phos&#60;100&#62;,</td>
		</tr>
		<tr>
			<td>adhesion promoter</td>
			<td>Shin-Etsu Microsci</td>
			<td>MicroPrime P20 adhesion promoter</td>
		</tr>
		<tr>
			<td>postive tone photoresist</td>
			<td>Rohm and Haas Electronics Materials LLC (Owned by Dow)</td>
			<td>Microposit S1818 Positive Photoresist (code 10018357)</td>
		</tr>
		<tr>
			<td>Quintel Contact Aligner</td>
			<td>Neutronix Quintel Corp</td>
			<td>NXQ 7500 Mask Aligner</td>
		</tr>
		<tr>
			<td>Reactive Ion Etching Tool</td>
			<td>Oxford Instruments</td>
			<td>Plasmalab System 100 Reactive Ion Etcher</td>
		</tr>
		<tr>
			<td>R2A Broth</td>
			<td>TEKnova</td>
			<td>R0005</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Sigma</td>
			<td>A9647</td>
		</tr>
		<tr>
			<td>Multimode Plate Reader</td>
			<td>Perkin Elmer</td>
			<td>Enspire, 2300-0000</td>
		</tr>
		<tr>
			<td>Fluorescent Microscope</td>
			<td>Nikon</td>
			<td>Eclipse Ti-U</td>
		</tr>
		<tr>
			<td>Automated Stage</td>
			<td>Prior</td>
			<td>ProScan III</td>
		</tr>
		<tr>
			<td>CCD camera</td>
			<td>Nikon</td>
			<td>DS-QiMc</td>
		</tr>
		<tr>
			<td>Stage-top environmental control chamber</td>
			<td>In Vivo Scientific</td>
			<td>STEV ECU-HOC</td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline</td>
			<td>ThermoFisher Scientific</td>
			<td>14190144</td>
		</tr>
		<tr>
			<td>UltraPure Agarose</td>
			<td>ThermoFisher Scientific</td>
			<td>16500500</td>
		</tr>
		<tr>
			<td>25 x 75 mm No. 1.5 coverslip</td>
			<td>Nexterion</td>
			<td>High performance #1.5H coverslips</td>
		</tr>
		<tr>
			<td>Fluorescence Reference Slides</td>
			<td>Ted Pella</td>
			<td>2273</td>
		</tr>
		<tr>
			<td>Physical Stylus Profilometer</td>
			<td>KLA Tencor</td>
			<td>P-6</td>
		</tr>
		<tr>
			<td>lab wipes</td>
			<td>Kimberly Clark</td>
			<td>Kimipe KIMTECH SCIENCE Brand, 34155</td>
		</tr>
		<tr>
			<td>commercial software</td>
			<td>Nikon</td>
			<td>NIS Elements</td>
		</tr>
		<tr>
			<td>Zeiss 710 Confocal Microscope</td>
			<td>Zeiss</td>
			<td></td>
		</tr>
		<tr>
			<td>filter cubes</td>
			<td>Nikon</td>
			<td>Nikon FITC (96311), Nikon Texas Red(96313)</td>
		</tr>
	</tbody>
</table>

assembly and tracking of microbial community development within a microwell array platform

The development of microbial communities depends on a combination of complex deterministic and stochastic factors that can dramatically alter the spatial distribution and activities of community members. We have developed a microwell array platform that can be used to rapidly assemble and track thousands of bacterial communities in parallel. This protocol highlights the utility of the platform and describes its use for optically monitoring the development of simple, two-member communities within an ensemble of arrays within the platform. This demonstration uses two mutants of Pseudomonas aeruginosa, part of a series of mutants developed to study Type VI secretion pathogenicity. Chromosomal inserts of either mCherry or GFP genes facilitate the constitutive expression of fluorescent proteins with distinct emission wavelengths that can be used to monitor community member abundance and location within each microwell. This protocol describes a detailed method for assembling mixtures of bacteria into the wells of the array and using time-lapse fluorescence imaging and quantitative image analysis to measure the relative growth of each member population over time. The seeding and assembly of the microwell platform, the imaging procedures necessary for the quantitative analysis of microbial communities within the array, and the methods that can be used to reveal interactions between microbial species area all discussed.

The experimental platform presented here is designed for high-throughput and high-content studies of bacterial communities. The design enables thousands of communities, growing in wells of various sizes, to be analyzed simultaneously. With this microwell array design, the dependence of the final community composition on initial seeding densities, well size, and chemical environment can be determined. This work demonstrates the growth of a two-member community in the microwell array and pu...

Watch this Scientific Journal Video about Assembly and Tracking of Microbial Community Development within a Microwell Array Platform at JoVE.com

Assembly and Tracking of Microbial Community Development within a Microwell Array Platform

The development of microbial communities depends on a combination of factors, including environmental architecture, member abundance, traits, and interactions. This protocol describes a synthetic, microfabricated environment for the simultaneous tracking of thousands of communities contained in femtoliter wells, where key factors such as niche size and confinement can be approximated.

The development of microbial communities depends on a combination of complex deterministic and stochastic factors that can dramatically alter the spatial ...

The overall goal of this method is high throughput parallel analysis of multi-member microbial communities in confined environments using silicon micro well arrays. The main advantage of this technique is that it allows for a high throughput highly parallel screening of finely localized microbial interactions that are significant to industry, medicine, and the environment. This method can help answer key questions in the biomedical and microbial ecology fields such as how do spacial constraints inherent at fine scales drive deterministic and stochastic parameters of community development and what are they key effects on microbial member abundance and organization?Begin with preparing the micro well arrays. First, cut individual micro well array chips from silicon wafers printed with multiple arrays using a diamond scribe. Each individual chip contains sub-arrays of wells with diameters ranging from five to 100 microns at three different spacing densities.On each chip, the complete motif of wells is also repeated four times. Next, make a humidified chamber using a pipette tip box and a PBS soaked wipe. Then apply a 150 microliter droplet of BSA solution to each chip and incubate the chips in the box for an hour at room temperature.Meanwhile prepare the bacteria. Spin down a culture and re-suspend it in 500 microliters of fresh R2A medium with two percent glycerol. Then measure the culture density at 600 nanometers and adjust the concentration to an optical density of 0.02.After the chip incubation, remove the BSA solution and rinse the chips three times with PBS. After the rinses, dry the chips with nitrogen gas and return them to the humidified chamber. Next, add 150 microliters of culture suspension to each dry chip and incubate the chips for an hour at four degrees Celsius so that the bacteria will have time to adhere to the wells, but the cell division of any possible contaminate is slow.To prepare a chip for imaging, first make an agarose-coated cover slip for each chip. Liquefy a small volume of agarose, about five milliliters is needed for each cover slip. Next, wash one side of a cover slip with ethanol and center it onto a glass slide with the washed side up.Then position two one millimeter thick PDMS spacers along the long edges of the cover slip and shift the cover slip so one millimeter hangs off the slide. Now, pour enough agarose onto the cover slip to completely cover it. Then use a second glass slide to flatten the agarose into a uniform thickness.Prepare several such slide assemblies in parallel. When the agarose begins to solidify, transfer the assemblies to four degrees Celsius. After 15 minutes of cooling, trim off the excess agarose around the cover slips.Then, put the assemblies in petri dishes and store them at four degrees Celsius. After the chips have incubated with the bacteria, dip the chips in ultra pure water one at a time for 10 seconds each. Then set the wet chips on their edges over a tissue until most of the liquid has drained off.Now, remove the bacteria that did not settle into the wells. Cut a piece of tape the length of the silicon chip and adhere it to the parylene coat on the silicon. Then peel off the tape to remove the parylene coat and immediately prepare to invert the chip.Now, place the chip onto a slide assembly such that the micro wells are in contact with the agarose. Be very careful not to move or shift the chip once in contact with the agarose. Now, transfer the complete assembly into the slide holder of a stagetop environmental control chamber and acquire time-lapse images over the next 24 hours at the desired interval and under 10x magnification.Process the image stacks using conventional, freely available software. First, import the image sequence. For averaging, load the correction or the dark field images.Then perform a background subtraction. Provide a radius value such as 135 pixels and select sliding paraboloid. Next, remove the average dark field image from the average illumination field image by selecting the two images and using the subtract operation.Now, apply an illumination correction as follows. Set the operation to divide, set i1 to the well image, set i2 to the correction image, set k1 to the correction image mean, and set k2 to zero. Then click Create New Window.To quantify the bacterial growth in the micro wells, first select the regions of interest with the micro array plug in. In the map menu, click reset grid and specify the rows, columns and well diameters. Then from the ROI shape menu, select circle.To adjust the ROI array to the image, the ROI array can be moved by pressing the ALT or ALT+SHIFT key and selecting the top left ROI with the mouse. To resize the ROIs, press the shift key while selecting the bottom right of the ROI array. To adjust the spacing between the ROIs, press the shift key while dragging the top or bottom side of the array.Once the ROI array fits over the wells of the image, click Measure RT.Then a table with all the measurements for each image or time point will be outputted and it can be copied into a spreadsheet for analysis. The growth of two strains of Pseudomonas aeruginosa was compared. One strain constitutively expresses toxic effector proteins associated with type six secretions.The other is susceptible to type six pathogenesis due to loss of function. Both express a different fluorescent protein. Co-cultures were studied longitudinally and in different well sizes.The bacteria were cultured individually and as a co-culture. 20 hours of growth was imaged in 30 minute intervals. After software corrections were performed on the imaging data, quantitative growth trajectories were plotted.In the co-culture, the data does not suggest too much of a change in growth. To advance the analysis, each trajectory was fitted to a modified logistic function with three parameters, maximum signal, maximum rate, and lag time. This analysis suggest that the co-culture of these species had a negligible effect on their overall growth and their variability seen in the growth curves is most likely due to environmental factors.Once mastered, the set up can be executed in just a few hours if it is performed properly. While attempting this procedure, it is important to have robustly growing cells to properly adjust the final cell OD values to have uniform auger layers in the chip assemblies and to be mindful during the parylene peel off step. Following this procedure, questions like how does gene expression within each community change as a function of community composition and organization, can be addressed by genetic material analysis.Don't forget that working with Pseudomonas aeruginosa can be potentially hazardous, and precautions such as gloves, jacket, goggles, and proper aseptic techniques should always be used while performing this procedure.

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