Name: design and implementation of an automated illuminating, culturing, and sampling system for microbial optogenetic applications
Uploaded: 2017-02-19T14:00:00
Description: Watch this Scientific Journal Video about Design and Implementation of an Automated Illuminating, Culturing, and Sampling System for Microbial Optogenetic Applications at JoVE.com

We would like to acknowledge Molly Lazar and Ver&#243;nica Delgado for assistance in testing the protocol, Kieran Sweeney for helpful discussions and editing, and Taylor Scott, My An-adirekkun, and Stephanie Geller for critical reading of the manuscript. Megan Nicole McClean, Ph.D. holds a Career Award at the Scientific Interface from the Burroughs Wellcome Fund.

<img alt="Figure 1" src="/files/ftp_upload/54894/54894fig1.jpg" />
Figure 1: The continuous culturing apparatus. This simplified diagram shows how the apparatus should be assembled when it is used to culture, illuminate, and measure optical properties of the microbes. <a href="http://cloudfront.jove.com/files/ftp_upload/54894/54894fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<p class="jove_content" fo:keep-togeth...

<ol>
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O., Taxis, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+LOV2+Domain-Based+Optogenetic+Tool+to+Control+Protein+Degradation+and+Cellular+Function.">A LOV2 Domain-Based Optogenetic Tool to Control Protein Degradation and Cellular Function.</a> Chemistry &amp; Biology. 20 (4), 619-626 (2013).</li><li>Novick, A., Szilard, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Description+of+the+chemostat.">Description of the chemostat.</a> Science (New York, N.Y.). 112 (2920), 715-716 (1950).</li><li>Monod, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=La+technique+de+culture+continue.+Th&#233;orie+et+applications.">La technique de culture continue. Th&#233;orie et applications.</a> Ann. Inst. Pasteur. 79 (4), 390-410 (1950).</li><li>Shimizu-Sato, S., Huq, E., Tepperman, J. M., Quail, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+light-switchable+gene+promoter+system.">A light-switchable gene promoter system.</a> Nature Biotechnology. 20, 1041-1044 (2002).</li><li>Kennedy, M. J., Hughes, R. M., Peteya, L. A., Schwartz, J. W., Ehlers, M. D., Tucker, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+blue-light-mediated+induction+of+protein+interactions+in+living+cells.">Rapid blue-light-mediated induction of protein interactions in living cells.</a> Nature Methods. 7, 973-975 (2010).</li><li>Olson, E. J., Tabor, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optogenetic+characterization+methods+overcome+key+challenges+in+synthetic+and+systems+biology.">Optogenetic characterization methods overcome key challenges in synthetic and systems biology.</a> Nature chemical biology. 10 (7), 502-511 (2014).</li><li>Botstein, D., Fink, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Yeast:+An+experimental+organism+for+21st+century+biology.">Yeast: An experimental organism for 21st century biology.</a> Genetics. 189 (3), 695-704 (2011).</li><li>Galanie, S., Smolke, C. 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D., Stuurman, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advanced+methods+of+microscope+control+using+&#181;Manager+software.">Advanced methods of microscope control using &#181;Manager software.</a> Journal of biological methods. 1 (2), (2014).</li><li>Schindelin, J., Arganda-Carreras, I., 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=Fiji:+an+open-source+platform+for+biological-image+analysis.">Fiji: an open-source platform for biological-image analysis.</a> Nature methods. 9 (7), 676-682 (2012).</li><li>Bhattacharya, S., Datta, A., Berg, J. M., Gangopadhyay, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+on+surface+wettability+of+poly(dimethyl)+siloxane+(PDMS)+and+glass+under+oxygen-plasma+treatment+and+correlation+with+bond+strength.">Studies on surface wettability of poly(dimethyl) siloxane (PDMS) and glass under oxygen-plasma treatment and correlation with bond strength.</a> Journal of Microelectromechanical Systems. 14 (3), 590-597 (2005).</li><li>Saldanha, A. J., Brauer, M. J., Botstein, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nutritional+homeostasis+in+batch+and+steady-state+culture+of+yeast.">Nutritional homeostasis in batch and steady-state culture of yeast.</a> Molecular Biology of the Cell. 15, 4089-4104 (2004).</li><li>Boer, V. M., Tai, S. 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=Transcriptional+responses+of+Saccharomyces+cerevisaie+to+preferred+and+nonpreferred+nitrogen+sources+in+glucose-limited+chemostat+cultures.">Transcriptional responses of Saccharomyces cerevisaie to preferred and nonpreferred nitrogen sources in glucose-limited chemostat cultures.</a> FEMS Yeast Research. 7, 604-620 (2007).</li><li>Boer, V. M., Amini, S., Botstein, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+genotype+and+nutrition+on+survival+and+metabolism+of+starving+yeast.">Influence of genotype and nutrition on survival and metabolism of starving yeast.</a> Proceedings of the National Academy of Sciences. 105, 6930-6935 (2008).</li><li>Brauer, M. J., Huttenhower, C., 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=Coordination+of+growth+rate,+cell+cycle,+stress+response+and+metabolic+activity+in+yeast.">Coordination of growth rate, cell cycle, stress response and metabolic activity in yeast.</a> Molecular Biology of the Cell. 19, 352-367 (2008).</li><li>Gresham, D., Dunham, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+enduring+utility+of+continuous+culturing+in+experimental+evolution.">The enduring utility of continuous culturing in experimental evolution.</a> Genomics. 104 (6), 399-405 (2014).</li><li>Liu, H., Yu, X., 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=Photoexcited+CRY2+interacts+with+CIB1+to+regulate+transcription+and+floral+initiation+in+Arabidopsis.">Photoexcited CRY2 interacts with CIB1 to regulate transcription and floral initiation in Arabidopsis.</a> Science (New York, N.Y.). 322 (5907), 1535-1539 (2008).</li><li>McIsaac, R. S., Oakes, B. L., Wang, X., Dummit, K. A., Botstein, D., Noyes, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthetic+gene+expression+perturbation+systems+with+rapid,+tunable,+single-gene+specificity+in+yeast.">Synthetic gene expression perturbation systems with rapid, tunable, single-gene specificity in yeast.</a> Nucleic Acids Research. 41 (4), 1-10 (2013).</li><li>McIsaac, R. S., Petti, A. A., Bussemaker, H. J., Botstein, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perturbation-based+analysis+and+modeling+of+combinatorial+regulation+in+the+yeast+sulfur+assimilation+pathway.">Perturbation-based analysis and modeling of combinatorial regulation in the yeast sulfur assimilation pathway.</a> Molecular biology of the cell. 23 (15), 2993-3007 (2012).</li><li>McIsaac, R. S., Silverman, S. J., 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=Fast-acting+and+nearly+gratuitous+induction+of+gene+expression+and+protein+depletion+in+Saccharomyces+cerevisiae.">Fast-acting and nearly gratuitous induction of gene expression and protein depletion in Saccharomyces cerevisiae.</a> Molecular biology of the cell. 22 (22), 4447-4459 (2011).</li><li>Melendez, J., Patel, M., Oakes, B. L., Xu, P., Morton, P., McClean, M. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+optogenetic+control+of+intracellular+protein+concentration+in+microbial+cell+cultures.">Real-time optogenetic control of intracellular protein concentration in microbial cell cultures.</a> Integrative biology quantitative biosciences from nano to macro. 6 (3), 366-372 (2014).</li><li>Brauer, M. J., Huttenhower, C., 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=Coordination+of+growth+rate,+cell+cycle,+stress+response,+and+metabolic+activity+in+yeast.">Coordination of growth rate, cell cycle, stress response, and metabolic activity in yeast.</a> Molecular biology of the cell. 19 (1), 352-367 (2008).</li><li>Boer, V. M., Crutchfield, C. A., Bradley, P. H., Botstein, D., Rabinowitz, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth-limiting+intracellular+metabolites+in+yeast+growing+under+diverse+nutrient+limitations.">Growth-limiting intracellular metabolites in yeast growing under diverse nutrient limitations.</a> Molecular biology of the cell. 21 (1), 198-211 (2010).</li><li>Markham, B. E., Byrne, W. J., Methods, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Uptake,+storage+and+utilization+of+phosphate+by+yeast.">Uptake, storage and utilization of phosphate by yeast.</a> Journal of the Institute of Brewing. 73, 271-273 (1967).</li><li>Kazemi Seresht, A., Palmqvist, E. 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We designed this apparatus with flexibility in mind. All the code used is free and open-source. The default image analysis process to segment cells is simple and runs quickly. Custom analysis could be implemented by recording user input while analyzing a representative image with the FIJI graphic user interface, converting the input to a beanshell script, and then setting the plugin to call the script. When it is called, this script will be sent a String array called &#34;images&#34; containing the file paths to the most...

Optogenetic systems use light to control a growing list of cellular processes including gene expression,1,2,3,4,5 protein localization,6 protein activity,6,7,8 protein binding,8,9,10 and protein degradation.11 A method for culturing cells in a controlled environment with programmed optical stimulation and for measuring their response over biologically relevant timescales is necessary to exploit the potential of these tools for research in cell biology and biotechnology. Our method takes advantage of chemostasis to maintain a constant cell growth rate in a well-mixed, aerated, and temperature-controlled glass culturing vessel12,13 that is exposed to programmed illumination. We image individual cells in the culture effluent with an inverted microscope to measure the response of the culture to programmed illumination. The culturing, sampling, imaging, and image analysis are fully automated so that the fluorescence intensity and cellular morphology of the effluent cell culture can be measured over multiple days without user input.
This protocol can be implemented in most labs familiar with growing cell culture and microscopy, and the apparatus used is inexpensive and made of readily available components. A transparent culturing vessel is placed above a matrix of light emitting diodes (LEDs) capable of emitting 1 &#181;W/cm2-10 mW/cm2 of light. Microbes are grown in the culturing vessel continuously; one peristaltic pump is used to add media at the dilution rate, another is used to withdraw culture at a lesser rate to the microscope, and the difference escapes through an overflow outlet. A heating pad maintains the temperature. Air is continually pumped into the culturing vessel to maintain a positive pressure as well as to mix and aerate the culture. Except for the air pump, power to these devices is regulated by a microcontroller that also receives input from a thermometer and a connected desktop computer. The effluent cell culture is pumped to a microfluidic device on the stage of an inverted microscope. Non-fluorescent and fluorescent images are automatically acquired. The cells in the images are characterized by an algorithm that locates each cell as a region of interest (ROI) and measures the properties of each ROI.
To demonstrate an application of this protocol, we measured the response to varying light intensities of Saccharomyces cerevisiae cells engineered with a blue-light responsive optogenetic system which controls the transcription of fluorescent protein. S. cerevisiae, commonly known as baker&#39;s yeast, was selected because multiple optogenetic systems for controlling gene expression in this system already exist14,15,16. Furthermore, this model organism is commonly used for studies in systems biology17 and as a chassis for biotechnological applications18,19,20. Our representative results demonstrate that this protocol can be used to control transcription of a culture over multiple days by varying input light intensities and measuring the production of a fluorescent reporter.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr >
			<td >Extensive lab manual</td>
			<td >GitHub</td>
			<td >NA</td>
			<td >An extensive, regularly updated lab manual is available in the &#8220;Optogenetic Chemostat Files&#8221; GitHub repository (https://github.com/McCleanResearch/Optogenetic-Chemostat-Files). This also includes a description of the microfluidic mold used to generate the representative results.</td>
		</tr>
		<tr >
			<td >Fritzing Design Viewer</td>
			<td >Fritzing</td>
			<td >NA</td>
			<td >The free, open-sourced software to view and edit the .fzz type circuit board designs is available at &#34;http://fritzing.org/download/&#34;</td>
		</tr>
		<tr >
			<td >Arduino Uno R3 (Atmega328 - assembled)</td>
			<td >Adafruit</td>
			<td >50</td>
			<td >Microcontroller. 1 required.</td>
		</tr>
		<tr >
			<td >Arduino Stackable Header Kit</td>
			<td >SparkFun Electronics</td>
			<td >10007</td>
			<td >Female pin headers for connecting PCB to microcontroller. 1 required.</td>
		</tr>
		<tr >
			<td >Adjustable 30W 110V soldering iron - XY-258 110V</td>
			<td >Adafruit</td>
			<td >180</td>
			<td >For making electrical connections to the PCB. 1 required.</td>
		</tr>
		<tr >
			<td >Soldering iron stand</td>
			<td >Adafruit</td>
			<td >150</td>
			<td >For making electrical connections to the PCB. 1 required.</td>
		</tr>
		<tr >
			<td >Mini Solder spool - 60/40 lead rosin-core solder 0.031&#34; diameter - 100g</td>
			<td >Adafruit</td>
			<td >145</td>
			<td >For making electrical connections to the PCB. 1 required.</td>
		</tr>
		<tr >
			<td >0.1 &#956;F capacitor</td>
			<td >SparkFun Electronics</td>
			<td >COM-08375</td>
			<td >Stabilizes voltage in PCB. 1 required.</td>
		</tr>
		<tr >
			<td >10 &#956;F capacitor</td>
			<td >SparkFun Electronics</td>
			<td >COM-00523</td>
			<td >Stabilizes voltage in PCB. 1 required.</td>
		</tr>
		<tr >
			<td >MAX7219CNG LED Matrix/Digit Display Driver - MAX7219</td>
			<td >Maxim</td>
			<td >MAX7219CNG</td>
			<td >LED driver. 1 required.</td>
		</tr>
		<tr >
			<td >8 pin IC Socket</td>
			<td >Mouser Electronics</td>
			<td >575-144308</td>
			<td >16 required. These will be stacked on top of each other to support the culture vessel above the LED matrix.</td>
		</tr>
		<tr >
			<td >24 Pin IC socket</td>
			<td >Mouser Electronics</td>
			<td >535-24-3518-10</td>
			<td >Optional. Use this to reversibly attach the MAXIM 7219CNG driver to the PCB.</td>
		</tr>
		<tr >
			<td >Digital multimeter</td>
			<td >Adafruit</td>
			<td >2034</td>
			<td >For troubleshooting electronics. 1 required.</td>
		</tr>
		<tr >
			<td >Break Away Headers - 40-pin Male (Long Centered, PTH, 0.1&#34;)</td>
			<td >SparkFun Electronics</td>
			<td >PRT-12693</td>
			<td >Male pin headers for connected LED matrix to printed circuit board. Ends can be trimmed with wire cutters. 1 set required.&#160;</td>
		</tr>
		<tr >
			<td >Flush diagonal wire cutters</td>
			<td >Adafruit</td>
			<td >152</td>
			<td >For trimming long pin headers and cutting power cables. 1 required.</td>
		</tr>
		<tr >
			<td >Premium Female/Female Jumper Wires - 40 x 12&#34; (300mm)</td>
			<td >Adafruit</td>
			<td >793</td>
			<td >Wire ribbon for connecting breadboard to LED matrix. Can be connected end-to-end with male pin-headers to be longer. 1 required.</td>
		</tr>
		<tr >
			<td >Half-size breadboard</td>
			<td >Adafruit</td>
			<td >64</td>
			<td >The LED matrix will connect to this and the culturing vessel will rest above it.</td>
		</tr>
		<tr >
			<td >Miniature 8x8 Blue LED Matrix</td>
			<td >Adafruit</td>
			<td >956</td>
			<td >Light source. Dominant wavelength is 470nm (blue). 1 required. Alternative miniature LED matrices from the same vendor are available with dominant wavelengths: 624 nm (red), 588 nm (yellow), 525 nm (green), 572 nm (yellow-green), and white.</td>
		</tr>
		<tr >
			<td >Stackable header-3 pin</td>
			<td >SparkFun Electronics</td>
			<td >13875</td>
			<td >8 required.</td>
		</tr>
		<tr >
			<td >Resistor Kit - 1/4W (500 total)</td>
			<td >SparkFun Electronics</td>
			<td >10969</td>
			<td >For electronics. 1 required.</td>
		</tr>
		<tr >
			<td >&#160;IRL520N MOSFET</td>
			<td >International Rectifier</td>
			<td >IRL520N</td>
			<td >Voltage regulating switch for controlling DC current. 4 required.</td>
		</tr>
		<tr >
			<td >Hook-Up Wire - Assortment (Solid Core, 22 AWG)</td>
			<td >SparkFun Electronics</td>
			<td >PRT 11367</td>
			<td >Wire for electronics. 1 required.</td>
		</tr>
		<tr >
			<td >5V 2A (2000mA) switching power supply - UL Listed</td>
			<td >Adafruit</td>
			<td >276</td>
			<td >Power supply for the heating pad and Arduino. 2 required.</td>
		</tr>
		<tr >
			<td >12 VDC 1000mA regulated switching power adapter - UL listed</td>
			<td >Adafruit</td>
			<td >798</td>
			<td >For peristaltic pumps. 2 required.</td>
		</tr>
		<tr >
			<td >Electric Heating Pad - 10cm x 5cm</td>
			<td >Adafruit</td>
			<td >1481</td>
			<td >For heating the bioreactor. 1 required.</td>
		</tr>
		<tr >
			<td >Low flow variable flow peristaltic pump</td>
			<td >Fisher Scientific</td>
			<td >13-876-1</td>
			<td >For pumping media. 1 required</td>
		</tr>
		<tr >
			<td >Medium flow variable flow peristaltic pump</td>
			<td >Fisher Scientific</td>
			<td >13-876-2</td>
			<td >For pumping culture. 1 required.</td>
		</tr>
		<tr >
			<td >9 VDC 1000mA regulated switching power adapter - UL listed</td>
			<td >Adafruit</td>
			<td >63</td>
			<td >For microcontroller power supply. Order 1.</td>
		</tr>
		<tr >
			<td >High Temp Waterproof DS18B20 Digital temperature sensor + extras</td>
			<td >Adafruit</td>
			<td >642</td>
			<td >Thermometer for the bioreactor. 1 required.</td>
		</tr>
		<tr >
			<td >Micromanager</td>
			<td >Micromanager</td>
			<td >NA</td>
			<td >The free, open-sourced microscope control software is available at &#34;https://micro-manager.org/wiki/Download_Micro-Manager_Latest_Release&#34;</td>
		</tr>
		<tr >
			<td >FIJI</td>
			<td >ImageJ</td>
			<td >NA</td>
			<td >The free, open-sourced image analysis software is available at &#34;http://fiji.sc/&#34;</td>
		</tr>
		<tr >
			<td >Arduino Integrated Development Environment</td>
			<td >Arduino</td>
			<td >NA</td>
			<td >The free, open-sourced IDE is available at &#34;https://www.arduino.cc/en/Main/Software&#34;</td>
		</tr>
		<tr >
			<td >Custom code</td>
			<td >GitHub</td>
			<td >NA</td>
			<td >The custom microcontroller code and &#34;Bioreactor Controller&#34; plugin are available in the &#8220;Optogenetic Chemostat Files&#8221; GitHub repository (https://github.com/McCleanResearch/Optogenetic-Chemostat-Files).</td>
		</tr>
		<tr >
			<td >USB Cable A to B - 6 Foot</td>
			<td >SparkFun Electronics</td>
			<td >CAB-00512</td>
			<td >Used to download data to microcontroller. 1 required.</td>
		</tr>
		<tr >
			<td >bioreactorTimecourse_example.csv</td>
			<td >GitHub</td>
			<td >NA</td>
			<td >The advantage of loading LED matrix values from a CSV file is that a program can be called by the plugin to update those values based on image analysis results, and those values can be reloaded to the microcontroller, enabling feed-back control. It is available from the &#8220;Optogenetic Chemostat Files&#8221; GitHub repository (https://github.com/McCleanResearch/Optogenetic-Chemostat-Files).</td>
		</tr>
		<tr >
			<td >Tota-frost gels (diffusion paper)</td>
			<td >B&#38;H</td>
			<td >B&#38;H # LOFSFTL 
			MFR # T1-72</td>
			<td >For LED matrix. 1 required.</td>
		</tr>
		<tr >
			<td >Kitting Sheet Crosslink 1/4x12x24in</td>
			<td >Grainger, inc</td>
			<td >20JL37</td>
			<td >Black foam for culturing vessel enclosure. 4 required.</td>
		</tr>
		<tr >
			<td >Standard Photodiode Power Sensor, Si, 200 - 1100 nm, 50 mW&#160;</td>
			<td >Thorlabs</td>
			<td >S120VC</td>
			<td >For measuring light intensity. 1 required.</td>
		</tr>
		<tr >
			<td >Labelling Tape</td>
			<td >Fisher Scientific</td>
			<td >159015N</td>
			<td >For labelling and securing loose components. 1 required.</td>
		</tr>
		<tr >
			<td >Compact Power and Energy Meter Console, Digital 4&#34; LCD</td>
			<td >Thorlabs</td>
			<td >PM100D</td>
			<td >For measuring light intensity. 1 required.</td>
		</tr>
		<tr >
			<td >100mL GL45 hybridization glass bottle</td>
			<td >Bellco Glass, Inc.</td>
			<td >(7910-40150)</td>
			<td >Bioreactor vessel. 1 required.</td>
		</tr>
		<tr >
			<td >Six port assembly</td>
			<td >Bellco Glass, Inc.</td>
			<td >Custom&#160;</td>
			<td >For the bioreactor vessel. Tubing Specs: .125&#34; OD x .055&#34;ID. Port A: 1.0&#34; long above cap slug and to bottom of tube. Ports B,C,E,F: 1.0&#34; long above cap slug, 33 mm long below. Port D: 1.0&#34; long above cap slug, 65 mm&#160; long below. 1 required. Includes 45 mm diameter polypropylene open top screw cap and a white silicone gasket to ensure a tight seal between the cap and the vessel.&#160;</td>
		</tr>
		<tr >
			<td >Scotch Magic Tape 3105, 3/4 x 300 Inches, Pack of 3</td>
			<td >Amazon</td>
			<td >B0009F3P3U</td>
			<td >Clear scotch tape. This is available from many other vendors. It is used to cover markings on the culturing vessel and to secure the coverglass with the PDMS channel to the aluminum support frame.</td>
		</tr>
		<tr >
			<td >1/16&#34; ID x 3/16&#34; OD x 1/16&#34; Wall Tygon Sanitary Silicone Tubing</td>
			<td >United States Plastic Corp.</td>
			<td >57288</td>
			<td >Tubing. ~25&#39; required.</td>
		</tr>
		<tr >
			<td >Cole-Parmer Twistit white rubber stopper, size 10</td>
			<td >Cole-Parmer</td>
			<td >EW-62992-32</td>
			<td >Media flask stopper and effluent flask stopper. 2 required.</td>
		</tr>
		<tr >
			<td >2L Laboratory Flask</td>
			<td >Pyrex</td>
			<td >4980</td>
			<td >Media flask and effluent flask. 2 required.</td>
		</tr>
		<tr >
			<td >Day pinchcock</td>
			<td >Fisher Scientific</td>
			<td >5867</td>
			<td >For pinching tubes shut. 3 required.</td>
		</tr>
		<tr >
			<td >Replacement tubing assembly 1/16&#34; ID</td>
			<td >Traceable Products</td>
			<td >3372</td>
			<td >The peristaltic pumps come with a set of tubes, but they wear out after weeks of use.</td>
		</tr>
		<tr >
			<td >Replacement tubing assembly 1/50&#34; ID</td>
			<td >Traceable Products</td>
			<td >3371</td>
			<td >The peristaltic pumps come with a set of tubes, but they wear out after weeks of use.</td>
		</tr>
		<tr >
			<td >Male luer with lock ring x 1/16&#34; hose barb, Nylon, 25/pk</td>
			<td >Cole-Parmer</td>
			<td >EW-45505-00</td>
			<td >Connectors. ~10 luers are required.</td>
		</tr>
		<tr >
			<td >Male luer with lock ring x 1/8&#34; hose barb, Nylon, 25/pk</td>
			<td >Cole-Parmer</td>
			<td >EW-45505-04</td>
			<td >Connectors. 5 required, one for each rubber stopper hole to fill with tubing.</td>
		</tr>
		<tr >
			<td >Female luer x 1/16&#34; hose barb adapter, Nylon, 25/pk</td>
			<td >Cole-Parmer</td>
			<td >EW-45502-00</td>
			<td >Connectors. ~10 luers required.</td>
		</tr>
		<tr >
			<td >Female luer x 3/16&#34; hose barb adapter</td>
			<td >Cole-Parmer</td>
			<td >EW-45502-08</td>
			<td >Connectors. ~10 luers required.</td>
		</tr>
		<tr >
			<td >Cole-Parmer Luer Accessory, Female Luer Cap, Nylon, 25/Pk</td>
			<td >Cole-Parmer</td>
			<td >SC-45502-28</td>
			<td ></td>
		</tr>
		<tr >
			<td >Cole-Parmer Luer Accessory, Male Luer Lock Plug, Nylon, 25/Pk</td>
			<td >Cole-Parmer</td>
			<td >EW-45505-56</td>
			<td ></td>
		</tr>
		<tr >
			<td >Microbore PTFE Tubing, 0.022&#34;ID x 0.042&#34;OD, 100 ft/roll</td>
			<td >Cole-Parmer</td>
			<td >EW-06417-21</td>
			<td >Tubing. 1 roll required.</td>
		</tr>
		<tr >
			<td >Masterflex platinum-cured silicone tubing, L/S 13, 25 ft</td>
			<td >Cole-Parmer</td>
			<td >EW-96410-13</td>
			<td >Tubing. ~25&#39; required.</td>
		</tr>
		<tr >
			<td >3/16&#34; ID x 1/4&#34; OD x 1/32&#34; Wall Tygon Sanitary Silicone Tubing</td>
			<td >United States Plastic Corp.</td>
			<td >57293</td>
			<td >Tubing. ~1&#39; required.</td>
		</tr>
		<tr >
			<td >Vacuum filter</td>
			<td >Fisher Scientific</td>
			<td >974107</td>
			<td >Nalgene vacuum filter for sterile filtering media.</td>
		</tr>
		<tr >
			<td >Aquel Oxy-Boost 200</td>
			<td >Rena Aquatic Supply</td>
			<td >AP200</td>
			<td >Dual diaphram adjustable flow air pump for aerating and mixing media. 1 required.&#160;</td>
		</tr>
		<tr >
			<td >0.2 &#956;m pore syringe filter</td>
			<td >Corning International</td>
			<td >431229</td>
			<td >This ensures that air from the aquarium pump does not contaminate the apparatus. 1 required.</td>
		</tr>
		<tr >
			<td >Slygard 184 Silicone Elastomer Kit</td>
			<td >Dow Corning</td>
			<td >Slygard 184</td>
			<td >For microfluidic device. 1 required.</td>
		</tr>
		<tr >
			<td >American Safety Razor GEM Scientific Single-Edge Razor Blades</td>
			<td >Fisher Scientific</td>
			<td >17989000</td>
			<td >For cutting tubes and PDMS. 1 blade required.</td>
		</tr>
		<tr >
			<td >Harris Uni-Core hole puncher 1.2mm ID</td>
			<td >Sigma-Aldrich</td>
			<td >WHAWB100028 ALDRICH</td>
			<td >For punching inlet/outlet in microfluidic device. 1 required.</td>
		</tr>
		<tr >
			<td >Microscope cover glass 22x60-1.5</td>
			<td >Fisher Scientific</td>
			<td >12-544-G</td>
			<td >For microfluidic device. 1 required.</td>
		</tr>
		<tr >
			<td >Rectangular aluminum frame with a square window</td>
			<td >Custom</td>
			<td >Custom</td>
			<td >To support the microfluidic channel. Outer dimensions: 3 inches x 1.25 inches. 
			Inner dimmensions (cut out portion): 7/8 inches x 7/8 inches 
			Thickness: ~1/32 inches</td>
		</tr>
	</tbody>
</table>

design and implementation of an automated illuminating, culturing, and sampling system for microbial optogenetic applications

Optogenetic systems utilize genetically-encoded proteins that change conformation in response to specific wavelengths of light to alter cellular processes. There is a need for culturing and measuring systems that incorporate programmed illumination and stimulation of optogenetic systems. We present a protocol for building and using a continuous culturing apparatus to illuminate microbial cells with programmed doses of light, and automatically acquire and analyze images of cells in the effluent. The operation of this apparatus as a chemostat allows the growth rate and the cellular environment to be tightly controlled. The effluent of the continuous cell culture is regularly sampled and the cells are imaged by multi-channel microscopy. The culturing, sampling, imaging, and image analysis are fully automated so that dynamic responses in the fluorescence intensity and cellular morphology of cells sampled from the culture effluent are measured over multiple days without user input. We demonstrate the utility of this culturing apparatus by dynamically inducing protein production in a strain of Saccharomyces cerevisiae engineered with an optogenetic system that activates transcription.

This apparatus was used to stimulate a culture of S. cerevisiae expressing yellow fluorescent protein (YFP) in response to blue-light via an inducible optogenetic transcription system based on the CRY2/CIB1 protein pair30. Cells were grown chemostatically in phosphate-limited media with an average dilution rate of 0.2 &#177; 0.008. Phosphate limitation is commonly used in S. cerevisiae chemostat experiments to control growth rate and the effects o...

Watch this Scientific Journal Video about Design and Implementation of an Automated Illuminating, Culturing, and Sampling System for Microbial Optogenetic Applications at JoVE.com

Design and Implementation of an Automated Illuminating, Culturing, and Sampling System for Microbial Optogenetic Applications

We designed a continuous culturing apparatus for use with optogenetic systems to illuminate cultures of microbes and regularly image cells in the effluent with an inverted microscope. The culturing, sampling, imaging, and image analysis are fully automated so that dynamic responses to illumination can be measured over several days.

Optogenetic systems utilize genetically-encoded proteins that change conformation in response to specific wavelengths of light to alter cellular ...

The overall goal of this procedure is to assemble a continuous culturing vessel, in which the response to illumination of optogenetic microbes can be measured with a microscope automatically in real time and over multiple days. This method can help answer key questions in cellular biology and metabolic engineering, such as how dynamic gene expression can elicit responses which differ from static gene expression. The main advantage of this method it that continuous culture enables measurements to be collected for multiple days.To begin, solder the ground line, the data line, and the positive voltage line of the digital thermometer to the printed circuit board. Clip off one pin from a female three pin header and trim the remaining two pins. Solder this in the pair of holes labeled R2, and connect the two soldered pins by inserting a 4.7 kilo ohm resistor in the pin header.Next connect the remaining components to the circuit board. Overlay the circuit board on to the micro controller. Set up the light proof enclosure, and install all software as described in the accompanying text protocol.First, place the long end of the aluminum port through the gasket into the culturing vessel and screw on the cap. Cover one short aluminum port outlet with medium diameter silicone tubing. Plug the distill end by inserting a female luer lock, and then connecting a male luer lock plug.This outlet is supplementary and will not be used. Connect two short segments of medium diameter silicone tubing with male and female luer locks. Then connect one end to a short aluminum port outlet, and plug the distill end.The vessel will be inoculated through this tube. Next, connect two medium tubes to the ends of the medium diameter peristaltic tubing and connectors. Connect one end to a short aluminum port, and plug the other.Later, this will be connected to the media flask. Connect a medium diameter silicone tube to the longest aluminum port. The tube should be long enough to reach the media flask.Connect another short segment a few inches long to the end, and then insert it into the rubber stopper on the media flask. Clamp the tube. When later unclamped, this connection allows the culture to be mixed, aerated, and kept at a positive pressure by the incoming bubbles.Insert a medium diameter tube long enough to reach the bottom of the flask into the stopper through which media will be transferred. Connect another medium diameter tube to this one, and then a wide diameter tube on the end. Plug the distill end.Fill the third hole in the stopper with a short segment of medium diameter tubing, connected to two other segments of medium diameter tubing, and plugged at the distill end. This will be connected to a vacuum pump, and later to an aquarium pump. Insert a male luer lock into a segment of narrow diameter silicone tubing and then firmly insert PTFE tubing.Then attach a female luer lock to a medium diameter tube. Thread the female lock along the PTFE tube to connect the locks and the other to a short aluminum port. Ensure that the PTFE tube inside the culturing vessel dangles below the second longest aluminum tube, and above the bottom.Trim as needed. Media will be sampled from here to the imaging channel, and the narrow tubing minimizes the intermediate volume. Next, connect the exposed narrow silicone tube to the narrow peristaltic tube and connectors.To this connect three segments of narrow diameter tubing and two segments of PTFE tubing, alternating them. Connect this to the effluent flask, via medium diameter tubing. Then connect one end of a medium diameter silicone tube to the second longest tube of the aluminum port in the other end of the effluent flask.This aluminum tube sets the culture volume, and excess culture overflows into the effluent container. Wrap the lids of the flask with aluminum foil, and then autoclave the assembly for 30 minutes at 121 degrees Celsius and 15 PSI. Attach the vacuum filter to a 100 milliliter bottle.Remove the nipple cover, and then remove the white plug from the nipple of the vacuum filter with sterile tweezers. Connect the wide silicone tube to the nipple, the media flask's other free tube to a vacuum pump, and ensure that the third tube connecting the media flask to the culturing vessel is clamped shut. Fill the filter with media, turn on the vacuum pump, and then filter the rest of the media.Clamp the medium silicone tubing connected to the vacuum and turn off the vacuum pump. Remove the luers from the intermediate segment of medium silicone tubing connected to the vacuum pump and insert the blue end of a sterile syringe air filter into this tube. Then clamp the wide silicone tube and disconnect the male luer from it.Disconnect the plug from the female luer of the culturing vessel's media inlet tube. Connect these luers to enable media to be pumped into the culturing vessel. Then, remove the filter from the 100 milliliter bottle, and attach the bottle cap.Use this media to prepare a starter culture. Set the continuous culturing assembly near a microscope with the media flask higher than the culturing vessel, and the effluent flask lower than the culturing vessel to prevent back flow. Securely tape down the rubber stoppers on the media and effluent flasks.Next unplug the ends of the PTFE tube from the narrow silicone tube connecting them and plug these ends into the inlet and outlet of a prepared microfluidic device. Then push media into the culturing vessel, by connecting the white end of the syringe air filter to the aquarium pump. When media reaches the level of the effluent port wrap the medium media inlet tubing and connectors around the slow peristaltic pump, and the narrow sample outlet tubing and connectors around the fast peristaltic pump.Aerate the media by unclamping the air tube between the media flask and culturing vessel. Then, tape the heating pad and thermometer to the culturing vessel so that it's temperature can be controlled. Coil the media tubing around the culturing vessel, so the entering media will be at the same temperature as the vessel.Then insert the culturing vessel into the black foam enclosure over the LED matrix and ensure that the tubes are not pinched. Use the bioreactor controller plugin to set the slow media pump on for one tenth of every 30 second interval. The flow rate will be very low, but greater than the rate of evaporation.Clamp the incoming air tube between the media flask and culturing vessel. Use a pipette to inoculate the culturing vessel with one milliliter from a starter culture. Then clamp the distill end of this tube, and unclamp the air tube.Let the culture grow overnight. Cover the enclosure so that no light enters. The next day, use the bioreactor controller plugin to set the peristaltic pumps to rotate for a greater proportion of time, corresponding to the desired media and sampling flow rates.Let the culture density equilibrate overnight. The next day fill the stage position list and micro manager with a set of non overlapping positions where cells pumped into the microfluidic channel will be in the focal plane. Then open the bio reactor controller plugin.Select the desired LED matrix time course, imaging channels, and other experimental settings from the prompts, and then begin collecting data. This apparatus was used to stimulate a culture of saccharomyces cerevisiae, expressing yellow fluorescent protein in response to blue light via an inducible optogenetic transcription system. Base contrast and fluorescent images of the microfluidic channel were acquired.Cell outlines were identified from the phase contrast images, mapped to the fluorescent images, and the fluorescent images were recorded in real time. The heat map here shows the fluorescence distribution of a culture due to yellow fluorescent protein production upon activation of the optogenetic system. The fluorescence of approximately of 170, 000 cells was analyzed from over 30, 000 images, acquired from 28 stage positions over 70 hours.During this study, the culture was exposed to varying intensities of blue light for six hour intervals, followed by six hours of complete darkness. Cells had been exposed to light prior to the first measurement which is why it's fluorescence intensity is decreasing during the first dark period. Once mastered, this technique can be done in eight hours, over the course of three days, if it is performed properly.While attempting this procedure, it's very important to use sterile technique. Following this procedure, other methods, like real time feedback control, can be used to control protein concentration. This technique will enable organisms with optogenetic constructs to be studied dynamically, over longer periods of time, longer than what is possible using plates or batch culture.After watching this video, you should have a good understanding of how to integrate a continuous culturing apparatus, a light, and a microscope, to stimulate and study optogenetic microbes.

design-implementation-an-automated-illuminating-culturing-sampling

Research

JoVE Journal

Bioengineering

GENPLAT: an Automated Platform for Biomass Enzyme Discovery and Cocktail Optimization

The high cost of enzymes for biomass deconstruction is a major impediment to the economic conversion of lignocellulosic feedstocks to liquid transportation fuels such as ethanol. We have developed an integrated high throughput platform, called GENPLAT, for the discovery and development of novel enzymes and enzyme cocktails for the release of sugars from diverse pretreatment/biomass combinations. GENPLAT comprises four elements: individual pure enzymes, statistical design of experiments, robotic pipeting of biomass slurries and enzymes, and automated colorimeteric determination of released Glc and Xyl. Individual enzymes are produced by expression in Pichia pastoris or Trichoderma reesei, or by chromatographic purification from commercial cocktails or from extracts of novel microorganisms. Simplex lattice (fractional factorial) mixture models are designed using commercial Design of Experiment statistical software. Enzyme mixtures of high complexity are constructed using robotic pipeting into a 96-well format. The measurement of released Glc and Xyl is automated using enzyme-linked colorimetric assays. Optimized enzyme mixtures containing as many as 16 components have been tested on a variety of feedstock and pretreatment combinations.
GENPLAT is adaptable to mixtures of pure enzymes, mixtures of commercial products (e.g., Accellerase 1000 and Novozyme 188), extracts of novel microbes, or combinations thereof. To make and test mixtures of &tilde;10 pure enzymes requires less than 100 &mu;g of each protein and fewer than 100 total reactions, when operated at a final total loading of 15 mg protein/g glucan. We use enzymes from several sources. Enzymes can be purified from natural sources such as fungal cultures (e.g., Aspergillus niger, Cochliobolus carbonum, and Galerina marginata), or they can be made by expression of the encoding genes (obtained from the increasing number of microbial genome sequences) in hosts such as E. coli, Pichia pastoris, or a filamentous fungus such as T. reesei. Proteins can also be purified from commercial enzyme cocktails (e.g., Multifect Xylanase, Novozyme 188). An increasing number of pure enzymes, including glycosyl hydrolases, cell wall-active esterases, proteases, and lyases, are available from commercial sources, e.g., Megazyme, Inc. (www.megazyme.com), NZYTech (www.nzytech.com), and PROZOMIX (www.prozomix.com).
Design-Expert software (Stat-Ease, Inc.) is used to create simplex-lattice designs and to analyze responses (in this case, Glc and Xyl release). Mixtures contain 4-20 components, which can vary in proportion between 0 and 100%. Assay points typically include the extreme vertices with a sufficient number of intervening points to generate a valid model. In the terminology of experimental design, most of our studies are "mixture" experiments, meaning that the sum of all components adds to a total fixed protein loading (expressed as mg/g glucan). The number of mixtures in the simplex-lattice depends on both the number of components in the mixture and the degree of polynomial (quadratic or cubic). For example, a 6-component experiment will entail 63 separate reactions with an augmented special cubic model, which can detect three-way interactions, whereas only 23 individual reactions are necessary with an augmented quadratic model. For mixtures containing more than eight components, a quadratic experimental design is more practical, and in our experience such models are usually statistically valid.
All enzyme loadings are expressed as a percentage of the final total loading (which for our experiments is typically 15 mg protein/g glucan). For "core" enzymes, the lower percentage limit is set to 5%. This limit was derived from our experience in which yields of Glc and/or Xyl were very low if any core enzyme was present at 0%. Poor models result from too many samples showing very low Glc or Xyl yields. Setting a lower limit in turn determines an upper limit. That is, for a six-component experiment, if the lower limit for each single component is set to 5%, then the upper limit of each single component will be 75%. The lower limits of all other enzymes considered as "accessory" are set to 0%. "Core" and "accessory" are somewhat arbitrary designations and will differ depending on the substrate, but in our studies the core enzymes for release of Glc from corn stover comprise the following enzymes from T. reesei: CBH1 (also known as Cel7A), CBH2 (Cel6A), EG1(Cel7B), BG (&beta;-glucosidase), EX3 (endo-&beta;1,4-xylanase, GH10), and BX (&beta;-xylosidase).
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	 </div>

GENPLAT (GLBRC Enzyme Platform) is an automated platform for discovery and optimization of enzyme cocktails for biomass degradation. It can be adapted to multiple feedstocks and mixtures of enzymes containing multiple components.

The high cost of enzymes for biomass deconstruction is a major impediment to the economic conversion of lignocellulosic feedstocks to liquid ...

Silk Film Culture System for in vitro Analysis and Biomaterial Design

Silk films are promising protein-based biomaterials that can be fabricated with high fidelity and economically within a research laboratory environment 1,2 . These materials are desirable because they possess highly controllable dimensional and material characteristics, are biocompatible and promote cell adhesion, can be modified through topographic patterning or by chemically altering the surface, and can be used as a depot for biologically active molecules for drug delivery related applications 3-8 . In addition, silk films are relatively straightforward to custom design, can be designed to dissolve within minutes or degrade over years in vitro or in vivo, and are produce with the added benefit of being transparent in nature and therefore highly suitable for imaging applications 9-13. The culture system methodology presented here represents a scalable approach for rapid assessments of cell-silk film surface interactions. Of particular interest is the use of surface patterned silk films to study differences in cell proliferation and responses of cells for alignment 12,14 . The seeded cultures were cultured on both micro-patterned and flat silk film substrates, and then assessed through time-lapse phase-contrast imaging, scanning electron microscopy, and biochemical assessment of metabolic activity and nucleic acid content. In summary, the silk film in vitro culture system offers a customizable experimental setup suitable to the study of cell-surface interactions on a biomaterial substrate, which can then be optimized and then translated to in vivo models. Observations using the culture system presented here are currently being used to aid in applications ranging from basic cell interactions to medical device design, and thus are relevant to a broad range of biomedical fields.

Silk Film Culture System for in vitro Analysis and Biomaterial Design

Silk films are a novel class of biomaterials readily customizable for an array of biomedical applications. The presented silk film culture system is highly adaptable to a variety of in vitro analyses. This system represents a biomaterial design platform offering in vitro optimization before direct translation to in vivo models.

Silk films are promising protein-based biomaterials that can be fabricated with high fidelity and economically within a research laboratory environment ...

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

In this protocol the fabrication, experimental setup and basic operation of the recently introduced microfluidic picoliter bioreactor (PLBR) is described in detail. The PLBR can be utilized for the analysis of single bacteria and microcolonies to investigate biotechnological and microbiological related questions concerning, e.g.&#160;cell growth, morphology, stress response, and metabolite or protein production on single-cell level. The device features continuous media flow enabling constant environmental conditions for perturbation studies, but in addition allows fast medium changes as well as oscillating conditions to mimic any desired environmental situation. To fabricate the single use devices, a silicon wafer containing sub micrometer sized SU-8 structures served as the replication mold for rapid polydimethylsiloxane casting. Chips were cut, assembled, connected, and set up onto a high resolution and fully automated microscope suited for time-lapse imaging, a powerful tool for spatio-temporal cell analysis. Here, the biotechnological platform organism Corynebacterium glutamicum&#160;was seeded into the PLBR and cell growth and intracellular fluorescence were followed over several hours unraveling time dependent population heterogeneity on single-cell level, not possible with conventional analysis methods such as flow cytometry. Besides insights into device fabrication, furthermore, the preparation of the preculture, loading, trapping of bacteria, and the PLBR cultivation of single cells and colonies is demonstrated. These devices will add a new dimension in microbiological research to analyze time dependent phenomena of single bacteria under tight environmental control. Due to the simple and relatively short fabrication process the technology can be easily adapted at any microfluidics lab and simply tailored towards specific needs.

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.

In this protocol the fabrication, experimental setup and basic operation of the recently introduced microfluidic picoliter bioreactor (PLBR) is described ...

Automated Robotic Liquid Handling Assembly of Modular DNA Devices

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

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

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

Live Cell Analysis of Shear Stress on Pseudomonas aeruginosa Using an Automated Higher-Throughput Microfluidic System

A higher-throughput microfluidic in vitro bioreactor coupled with fluorescence microscopy has been used to study bacterial biofilm growth and morphology, including Pseudomonas aeruginosa (P. aeruginosa). Here, we will describe how the system can be used to study the growth kinetics and the morphological properties such as the surface roughness and textural entropy of P. aeruginosa strain PA01 that expresses an enhanced green fluorescent protein (PA01-EGFP). A detailed protocol will describe how to grow and seed PA01-EGFP cultures, how to set up the microscope and autorun, and conduct the image analysis to determine growth rate and morphological properties using a variety of shear forces that are controlled by the microfluidic device. This article will provide a detailed description of a technique to improve the study of PA01-EGFP biofilms which eventually can be applied towards other strains of bacteria, fungi, or algae biofilms using the microfluidic platform.

Live Cell Analysis of Shear Stress on Pseudomonas aeruginosa Using an Automated Higher-Throughput Microfluidic System

Here, we describe the use of a higher-throughput microfluidic bioreactor coupled with a fluorescent microscope for the analysis of shear stress effects on Pseudomonas aeruginosa biofilms expressing green fluorescent proteins, including instrument set up, the determination of biofilm coverage, growth rate, and morphological properties.

A higher-throughput microfluidic in vitro bioreactor coupled with fluorescence microscopy has been used to study bacterial biofilm growth and morphology, ...

Purification and Analytics of a Monoclonal Antibody from Chinese Hamster Ovary Cells Using an Automated Microbioreactor System

Monoclonal antibodies (mAbs) are one of the most popular and well-characterized biological products manufactured today. Most commonly produced using Chinese hamster ovary (CHO) cells, culture and process conditions must be optimized to maximize antibody titers and achieve target quality profiles. Typically, this optimization uses automated microscale bioreactors (15 mL) to screen multiple process conditions in parallel. Optimization criteria include culture performance and the critical quality attributes (CQAs) of the monoclonal antibody (mAb) product, which may impact its efficacy and safety. Culture performance metrics include cell growth and nutrient consumption, while the CQAs include the mAb's N-glycosylation and aggregation profiles, charge variants, and molecular weight. This detailed protocol describes how to purify and subsequently analyze HCCF samples produced by an automated microbioreactor system to gain valuable performance metrics and outputs. First, an automated protein A fast protein liquid chromatography (FPLC) method is used to purify the mAb from harvested cell culture samples. Once concentrated, the glycan profiles are analyzed by mass spectrometry using a specific platform (refer to the Table of Materials). Antibody molecular weights and aggregation profiles are determined using size exclusion chromatography-multiple angle light scattering (SEC-MALS), while charge variants are analyzed using microchip capillary zone electrophoresis (mCZE). In addition to the culture performance metrics captured during the bioreactor process (i.e., culture viability, cell counts, and common metabolites including glutamine, glucose, lactate, and ammonia), spent media is analyzed to identify limiting nutrients to improve the feeding strategies and overall process design. Therefore, a detailed protocol for the absolute quantification of amino acids by liquid chromatography-mass spectrometry (LC-MS) of spent media is also described. The methods used in this protocol take advantage of high-throughput platforms that are compatible for large numbers of small-volume samples.

A detailed protocol for the purification and subsequent analysis of a monoclonal antibody from harvested cell culture fluid (HCCF) of automated microbioreactors has been described. Use of analytics to determine critical quality attributes (CQAs) and maximizing limited sample volume to extract vital information is also presented.

Monoclonal antibodies (mAbs) are one of the most popular and well-characterized biological products manufactured today. Most commonly produced using ...

Design and Analysis for Fall Detection System Simplification

This paper presents a methodology based on multimodal sensors to configure a simple, comfortable and fast fall detection and human activity recognition system that can be easily implemented and adopted. The methodology is based on the configuration of specific types of sensors, machine-learning methods and procedures. The protocol is divided into four phases: (1) database creation (2) data analysis (3) system simplification and (4) evaluation. Using this methodology, we created a multimodal database for fall detection and human activity recognition, namely UP-Fall Detection. It comprises data samples from 17 subjects that perform 5 types of falls and 6 different simple activities, during 3 trials. All information was gathered using 5 wearable sensors (tri-axis accelerometer, gyroscope and light intensity), 1 electroencephalograph helmet, 6 infrared sensors as ambient sensors, and 2 cameras in lateral and front viewpoints. The proposed novel methodology adds some important stages to perform a deep analysis of the following design issues in order to simplify a fall detection system: a) select which sensors or combination of sensors are to be used in a simple fall detection system, b) determine the best placement of the sources of information, and c) select the most suitable machine learning classification method for fall and human activity detection and recognition. Even though some multimodal approaches reported in literature only focus on one or two of the above-mentioned issues, our methodology allows simultaneously solving these three design problems related to a human fall and activity detection and recognition system.

We present a methodology based on multimodal sensors to configure a simple, comfortable and fast fall detection and human activity recognition system. The goal is to build a system for accurate fall detection that can be easily implemented and adopted.

This paper presents a methodology based on multimodal sensors to configure a simple, comfortable and fast fall detection and human activity recognition ...

Automated Counterflow Centrifugal System for Small-Scale Cell Processing

Successful commercialization of gene and cell-based therapies requires manufacturing processes that are cost-effective and scalable. Buffer exchange and product concentration are essential components for most manufacturing processes. However, at the early stages of product development, these steps are often performed manually. Manual dead-end centrifugation for buffer exchange is labor-intensive, costly, and not scalable. A closed automated system can effectively eliminate this laborious step, but implementation can be challenging. Here, we describe a newly developed cell processing device that is suitable for small- to medium-scale cell processing and aims to bridge the gap between manual processing and large-scale automation. This protocol can be easily applied to various cell types and processes by modifying the flow rate and centrifugation speed. Our protocol demonstrated high cell recovery with shorter processing times in comparison to the manual process. Cells recovered from the automated process also maintained their proliferation rates. The device can be applied as a modular component in a closed manufacturing process to accommodate steps such as buffer exchange, cell formulation, and cryopreservation.

Automation is key to upscaling and cost management in cell manufacturing. This manuscript describes the use of a counterflow centrifugal cell processing device for automating the buffer exchange and cell concentration steps for small-scale bioprocessing.

Successful commercialization of gene and cell-based therapies requires manufacturing processes that are cost-effective and scalable. Buffer exchange and ...

Building a Simple and Versatile Illumination System for Optogenetic Experiments

Controlling biological processes using light has increased the accuracy and speed with which researchers can manipulate many biological processes. Optical control allows for an unprecedented ability to dissect function and holds the potential for enabling novel genetic therapies. However, optogenetic experiments require adequate light sources with spatial, temporal, or intensity control, often a bottleneck for researchers. Here we detail how to build a low-cost and versatile LED illumination system that is easily customizable for different available optogenetic tools. This system is configurable for manual or computer control with adjustable LED intensity. We provide an illustrated step-by-step guide for building the circuit, making it computer-controlled, and constructing the LEDs. To facilitate the assembly of this device, we also discuss some basic soldering techniques and explain the circuitry used to control the LEDs. Using our open-source user interface, users can automate precise timing and pulsing of light on a personal computer (PC) or an inexpensive tablet. This automation makes the system useful for experiments that use LEDs to control genes, signaling pathways, and other cellular activities that span large time scales. For this protocol, no prior expertise in electronics is required to build all the parts needed or to use the illumination system to perform optogenetic experiments.

This protocol describes how to perform optogenetic experiments for controlling gene expression with red and far-red light using PhyB and PIF3. Included are step-by-step instructions for building a simple and flexible illumination system, which enables the control of gene expression or other optogenetics with a computer.

Controlling biological processes using light has increased the accuracy and speed with which researchers can manipulate many biological processes. Optical ...

Automated Microbial Cultivation and Adaptive Evolution using Microbial Microdroplet Culture System (MMC)

Conventional microbial cultivation methods usually have cumbersome operations, low throughput, low efficiency, and large consumption of labor and reagents. Moreover, microplate-based high-throughput cultivation methods developed in recent years have poor microbial growth status and experiment parallelization because of their low dissolved oxygen, poor mixture, and severe evaporation and thermal effect. Due to many advantages of micro-droplets, such as small volume, high throughput, and strong controllability, the droplet-based microfluidic technology can overcome these problems, which has been used in many kinds of research of high-throughput microbial cultivation, screening, and evolution. However, most prior studies remain&#160;at the stage of laboratory construction and application. Some key issues, such as high operational requirements, high construction difficulty, and lack of automated integration technology, restrict the wide application of droplet microfluidic technology in microbial research. Here, an automated Microbial Microdroplet Culture system (MMC) was successfully developed based on droplet microfluidic technology, achieving the integration of functions such as inoculation, cultivation, online monitoring, sub-cultivation, sorting, and sampling required by the process of microbial droplet cultivation. In this protocol, wild-type Escherichia coli (E. coli) MG1655 and a methanol-essential E. coli strain (MeSV2.2) were taken as examples to introduce how to use the MMC to conduct automated and relatively high-throughput microbial cultivation and adaptive evolution in detail. This method is easy to operate, consumes less labor and reagents, and has high experimental throughput and good data parallelity, which has great advantages compared with conventional cultivation methods. It provides a low-cost, operation-friendly, and result-reliable experimental platform for scientific researchers to conduct related microbial research.

Automated Microbial Cultivation and Adaptive Evolution using Microbial Microdroplet Culture System MMC

This protocol describes how to use the Microbial Microdroplet Culture system (MMC) to conduct automated microbial cultivation and adaptive evolution. MMC can cultivate and sub-cultivate microorganisms automatically and continuously and monitor online their growth with relatively high throughput and good parallelization, reducing labor and reagent consumption.