Name: The Use of Chemostats in Microbial Systems Biology (Video) | JoVE Journal
Uploaded: 2013-10-14T20:45:00.000+00:00
Duration: 13 min 19 s
Description: Learn chemostat operation for microbial systems biology using continuous culture methods. This technique enables precise control of cell growth rates and facilitates studies of adaptive evolution and gene regulation.

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This work was supported by start up funds form New York University. We thank Maitreya Dunham and Matt Brauer who initially developed the use of Sixfors bioreactors as chemostats.

The principle of continuous culturing using a chemostat can be realized in a variety of implementations. In all chemostats it is essential to have 1) methods for maintaining sterility of all components, 2) a well-mixed culture, 3) appropriate aeration of the culture vessel and 4) a reliable means of media addition and culture removal. Here, we describe the use of a Sixfors bioreactor (Infors Inc) as a chemostat using methods that can be readily adapted to alternative setups.
1. Assembling the Chemostat Vessels
<ol>
	<li>Turn on the Sixfors using the main switch.</li>
	<li>Thoroughly rinse the chemostat vessel, stirrer assembly, and attached tubing with deionized (DI) water. Check all the tubing and o-rings and replace any worn out looking pieces.</li>
	<li>Make sure the drive shaft support base is facing upward in the glass vessel and that the top of the drive shaft is snapped into its housing on the stirrer assembly. Set the stirrer assembly into the glass vessel ensuring the bottom of the drive shaft is seated in the drive shaft support. Use the clamp to secure the stirrer assembly to the glass vessel.</li>
	<li>Fill the vessel with around 300 ml of DI water.</li>
	<li>Remove the caps from a dissolved oxygen (dO2) probe and check the electrolyte level by unscrewing the bottom casing and making sure that the bottom casing is filled halfway with electrolyte solution. Rescrew the bottom casing and insert into the port on the chemostat vessel. Screw until finger tight.</li>
	<li>Take a pH probe and remove it from its storage buffer (3 M KCl). Remove the pH probe cap and attach to fermentor 1. Using the Sixfors control screen, navigate to the fermentor parameter menu and select "calibrate pH". Place the pH probe into a standard pH 7 buffer and record reading as High Read. Repeat with pH 4 buffer and record reading as Low Read. Detach the pH probe from fermentor, wash with DI water and insert the probe into the port on the chemostat vessel. Screw until finger tight.</li>
	<li>Place the chemostat vessel in the rack. Note on the vessel which fermentor (1-6) the pH probe was connected to and calibrated. Place the pH probe cap on the pH probe. Using foil tightly cover the top of the dO2 probe.</li>
	<li>Fold foil over the ends of the tubing attached to the chemostat vessel.</li>
	<li>Repeat steps 1.2-1.8 for all vessels.</li>
	<li>Sterilize the vessels by autoclaving them for 15 min on a liquid cycle.</li>
</ol>
2. Preparing the Media
<ol>
	<li>Establish the limiting range of concentrations for a nutrient by growing batch cultures in different nutrient concentrations. The nutrient is limiting in the range where the final cell density is a linear function of the initial nutrient concentration (Figure 4). Select a nutrient concentration well within the limiting range. Examples of standard media composition for studies with Saccharomyces cerevisiae are available in 7-11.</li>
	<li>Autoclave an empty carboy that is capped with a rubber plug containing an air inlet and media outlet after first ensuring that the end of the media tubing is covered in foil.</li>
	<li>In a separate vessel prepare 10 L of media.</li>
	<li>Attach a 500 ml filter cup to a 100 ml media bottle. Remove the cotton filter with forceps sterilized in ethanol and immediately attach to the filtration port on the media carboy.</li>
	<li>Attach the media carboy to a vacuum source and filter sterilize media by adding it to the filter cup.</li>
	<li>When filtration of media is complete, close off the filtration port with a metal clamp.</li>
</ol>
3. Calibrating dO2 Probes and Setting up Chemostat
<ol>
	<li>After the chemostat vessels have been autoclaved and allowed to cool down place the vessel in its corresponding heat jacket. Connect the temperature probe, pH probe, and dO2 probe. Let the vessel sit with the power on for at least 6 hr to allow the dO2 probes to polarize.</li>
	<li>Place the end of each effluent tube into a separate collecting receptacle. Connect the air supply via an autoclaved filter and turn on airflow. The water in each vessel should flow out of the effluent tubes, which indicates that all seals are properly formed.</li>
	<li>Adjust the height of the effluent tube according to the desired working volume (e.g. 300 ml). We use a ruler that is marked with tube placements calibrated to different working volumes.</li>
	<li>Connect the media carboy to the chemostat vessel. Use ethanol in order to keep tube ends as sterile as possible. Thread the pump tubing through the pump and open clamp. Manually press the pump until the media starts to flow into the chemostat vessel. Release tubing from pump, media should flow freely into chemostat vessel. When the media reaches the effluent tube, reattach tubing to pump and clamp.</li>
	<li>Start running the basic program with impeller set to 400 rpm and the temperature set at 30 &deg;C.</li>
	<li>In order to calibrate dO2 probes, turn off air supply and switch to nitrogen gas. Wait at least one hour and record measure value as "low read". Switch back to air supply (i.e. containing ambient oxygen concentration), wait at least one additional hour and record measurement as "high read".</li>
	<li>Initiate data recording using Iris software.</li>
</ol>
4. Inoculation
<ol>
	<li>Use 70% ethanol to sterilize the top of the culture vessel.</li>
	<li>Remove screw on vessel top and pipette 1 ml of an overnight culture into the chemostat vessel. Retighten screw.</li>
	<li>Indicate the time of inoculation in Iris.</li>
	<li>Wait approximately 24 hr for cultures to reach early stationary phase. As the culture grows, dissolved oxygen and pH will decrease. In the case of glucose-limiting media, dissolved oxygen will return to ~100% in stationary phase. For other nutrient limitations dissolved oxygen will remain &lt;100% in stationary phase.</li>
</ol>
5. Initiating Pumps and Attaining Steady State
<ol>
	<li>The dilution rate is calculated by dividing the flow rate (how much media flows into the vessel per hour) by the culture volume. For example, using a volume of 300 ml a dilution rate of 0.1 means that 30 ml of media is added to the culture every hour. Because the system is under positive pressure the same volume is removed from the vessel ensuring that the culture is continuously diluted. A range of dilution rates (D) can be used that do not exceed the maximum growth rate (&mu;max) of the cells, above which cells will be washed out of the chemostat.</li>
	<li>Choose pump settings, which specify the number of seconds the pump is on and off, to establish the desired flow rate. The pump delivers media at a rate of ~0.11 ml/sec.</li>
	<li>Define a program using the Sixfors interface that specifies the pump timing, temperature and impellor speed. Start the program.</li>
	<li>Empty the collecting receptacles of liquid and record the time.</li>
	<li>After at least two hours, use a graduated cylinder to measure how much media has been removed from the vessel. This will equal the volume that has been added to the chemostat vessel. Calculate the dilution rate (D = Veffluent/Vculture/time). Adjust pump settings if calculated dilution rate does not match desired dilution rate.</li>
	<li>At steady state, cell density in the chemostat does not change over time. This can be measured without perturbing the culture by sampling the outflow. We operationally define attainment of steady state as identical cell density measurements that are separated by at least one population doubling. Reaching steady state will take approximately eight culture generations after initiation of culture dilution. At steady state, cells grow exponentially (i.e. constant growth rate over time) in nutrient-limited conditions. The doubling time of the population (generation time) is ln(2)/D.</li>
	<li>Continue to periodically measure effluent volumes for the duration of the experiment to ensure that a constant dilution rate is maintained.</li>
</ol>
6. Application 1: Studying Cells Growing at Different Rates in Steady-state Conditions
<ol>
	<li>At steady state in the chemostat, the growth rate of the population of cells is equal to the dilution rate. By systematically altering the dilution rate it is possible to grow cells at different rates. This enables the systematic study of physiological parameters that vary with growth rate including cell volume, cell cycle stage and stress resistance. In addition, steady-state profiles of global mRNA, protein and metabolite levels can be assayed in cells growing at different rates. There are two ways to acquire samples:</li>
	<li>Small samples can be passively obtained by placing the end of the effluent tube into an 1.5 ml or 15 ml tube. A sample of 1-5 ml can be obtained in a few minutes (depending on the flow rate). Many physiological parameters can be measured from these samples.</li>
	<li>Gene expression, or metabolite analyses require larger samples that must be obtained as quickly as possible. Place the end of the effluent tube into a 15 ml conical tube. Release the screw holding the metal end of the effluent tube and push down gently. A ~10 ml sample will rapidly fill your tube. Keep in mind that the volume in the chemostat vessel has changed. If many consecutive samples are taken it may be necessary to reduce flow to maintain a constant dilution rate.</li>
</ol>
7. Application 2: Precise Measurement of Differences in Growth Rates Between Genotypes in Controlled Environments Using Flow Cytometry-based Competition Assays
<ol>
	<li>Chemostats can be used to accurately quantify the effect of nutrient concentration on differences in growth rates (i.e. fitness) between different genetic backgrounds. By co-culturing strains labeled with different fluorescent proteins the rate of change in relative abundance during exponential growth is determined. Performing this assay at different dilution rates (D) allows the study of effects of nutrient concentration (s) on fitness differences.</li>
	<li>Establish steady state cultures of the two strains in separate chemostat vessels with identical dilution rates and a volume of 300 ml.</li>
	<li>Passively sample 1 ml from each vessel. Spin down cells, resuspend in phosphate buffered saline (PBS) and place at 4 &deg;C. These samples contain homogenous cell samples, which are controls for the subsequent flow cytometry analysis.</li>
	<li>Place the effluent tube from one vessel into an autoclaved graduated cylinder. Release the screw holding the metal end of the effluent tube and gently push down to expel cells from the chemostat. When the volume reaches 150 ml, return the metal end of the effluent tube to its original position (300 ml). Repeat with the second vessel, using a different graduated cylinder.</li>
	<li>Use 70% ethanol to maintain sterility while removing screw on the top of the chemostat vessel. Place funnel in opening and pour 150 ml from the other culture into the chemostat vessel. Retighten screw. Repeat with the second vessel, using the second funnel. Each chemostat vessel now contains a mixture of the two strains.</li>
	<li>Obtain a 1 ml sample from each vessel using passive sampling every 2-6 hr. Spin down cells, resuspend in PBS and store at 4 &deg;C. Continue taking samples for 2-3 days carefully recording the time each sample was taken.</li>
	<li>At the end of the experiment, sonicate and dilute samples to ~2 x 106 cells/ml. Using flow cytometry, measure the proportion of the two strains in each sample. As both strains are growing exponentially, the relative growth rate difference is determined by linear regression of ln(strain1/strain2) against time (measured in generations). The slope of the regression is the proportional difference in growth rate (i.e. the fitness advantage) of one strain relative to the other.</li>
	<li>As the mixed culture can take some time to attain a new steady-state, early time points may fit poorly to the regression. This issue is best solved by allowing 2-3 generations to elapse before commencing sampling or removing early points that are clear outliers. Conversely, once one strain has outcompeted the other the data are no longer useful and these points should be excluded.</li>
</ol>
8. Application 3: Experimental Evolution
<ol>
	<li>Experimental evolution performed in chemostats selects for mutants that are increased in fitness in the nutrient-limited environment. Selection is generally performed over hundreds of generations.</li>
	<li>Establish a steady-state chemostat culture using a strain of known genotype (and preferably known genome sequence) and a defined nutrient-limitation.</li>
	<li>To maintain a "fossil record" of the adapting population passively sample chemostat every 2-3 days and store the sample in 15% glycerol at -80 &deg;C.</li>
	<li>Monitor the media reservoir and replenish with fresh media as necessary.</li>
	<li>Maintain the exponentially growing culture for several hundred generations.</li>
	<li>After completion of the selection plate a sample of cells on solid agar plates. After cells have grown into colonies, pick an unbiased sample of colonies using toothpicks and inoculate clones into individual wells of a 96-well plate containing 100 &mu;l of media. Allow clonal samples to grow overnight, add 100 &mu;l of 30% glycerol and store in -80 &deg;C.</li>
	<li>Characterize clones by whole genome sequencing and performing phenotypic assays including assessment of fitness, using a common fluorescently labeled reference strain, as described in Application 2.</li>
</ol>

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The authors declare that they have no competing financial interests.

Chemostats enable the cultivation of microbes in growth-controlled steady-state conditions. The cells grow continuously at a constant rate resulting in an invariant external environment. This is in contrast to batch culture methods in which the external environment is continuously changing and the rate of cell growth is determined by the complex interaction of environment and genotype. Thus, a major advantage of culturing microbes in chemostats over batch cultures is the ability to experimentally control the growth rate ...

The growth of cells is regulated by complex networks of interacting genetic and environmental factors1,2. The multifactorial regulation of cell growth necessitates a system-level approach to its study. However, the rigorous study of regulated cell growth is challenged by the difficulty of experimentally controlling the rate at which cells grow. Moreover, in even the simplest experiments extracellular conditions are frequently dynamic and complex as cells continuously alter their environment as they proliferate. A solution to these problems is provided by the chemostat: a method of culturing cells that enables experimental control of cell growth rates in defined, invariant and controlled environments.
The method of continuous culturing using a chemostat was independently described by Monod3 and Novick &amp; Szilard4 in 1950. As originally conceived, cells are grown in a fixed volume of media that is continually diluted by addition of new media and simultaneous removal of old media and cells (Figure 1). Coupled ordinary differential equations (Figure 2) describe the rate of change in cell density (x) and the concentration of a growth-limiting nutrient (s) in the chemostat vessel. Importantly, this system of equations predicts a single (nonzero) stable steady-state (Figure 3) with the remarkable implication that at steady-state, the specific growth rate of the cells (i.e. the exponential growth rate constant) is equal to the rate at which the culture is diluted (D). By varying the dilution rate it is possible to establish steady-state populations of cells at different growth rates and under different conditions of nutrient limitation.
The experimental control of growth rate using chemostats was critical to the development of an understanding of how cell physiology changes with rates of growth5,6. However, this former mainstay of microbiological methods became increasingly obscure during the explosion in molecular biology research during the late twentieth century. Today, renewed interest in growth control in both microbes and multicellular organisms and the advent of genome-scale methods for systems-level analysis has renewed motivation for the use of chemostats. Here, we describe three applications that capitalize on the precise control of cell growth rates and the external environment that are uniquely possible using chemostats. First, we describe the use of chemostats to investigate how the abundance of thousands of biomolecules - such as transcripts and metabolites - are coordinately regulated with growth rate. Second, we describe how chemostats can be used to obtain precise estimates of growth-rate differences between different genotypes in nutrient-limited environments using competition experiments. Third, we describe how chemostats can be used to study adaptive evolution of cells growing in constant nutrient-poor environments. These examples exemplify the ways in which chemostats are enabling systems-level investigations of cell growth regulation, gene by environment interactions and adaptive evolution.

<table><tbody><tr><td>Infors-HT Sixfors Chemostat</td><td>Appropriate Technical Resources, Inc.</td><td></td><td>&amp;nbsp;</td></tr><tr><td>Glass Bottle 9.5 L</td><td>Fisher Scientific</td><td>02-887-1</td><td>For Media Vessel and Hosing</td></tr><tr><td>Pinchcock</td><td>Fisher Scientific</td><td>05-867</td><td>For Media Vessel and Hosing</td></tr><tr><td>Stopper, Size 12, Green Neoprene</td><td>Cole-Palmer</td><td>EW-62991-42</td><td>For Media Vessel and Hosing</td></tr><tr><td>Straight Connector</td><td>Cole-Palmer</td><td>EW-30703-02</td><td>For Media Vessel and Hosing</td></tr><tr><td>General purpose ties 4 in</td><td>Fisher Scientific</td><td>NC9557052</td><td>For Media Vessel and Hosing</td></tr><tr><td>Tubing, Silicone Rubber</td><td>Small Parts</td><td>B000FMWTDE</td><td>For Media Vessel and Hosing</td></tr><tr><td>Tubing, Silicone, 3/8 in OD</td><td>Fisher Scientific</td><td>02-587-1Q</td><td>For Media Vessel and Hosing</td></tr><tr><td>Tubing, Silicone, 7/32 in OD</td><td>Fisher Scientific</td><td>02-587-1E</td><td>For Media Vessel and Hosing</td></tr><tr><td>Tubing, Stainless Steel, 3/16 in OD</td><td>McMaster-Carr</td><td>6100K164</td><td>For Media Vessel and Hosing</td></tr><tr><td>Tubing, Stainless Steel, 3/8 in OD</td><td>McMaster-Carr</td><td>6100K161</td><td>For Media Vessel and Hosing</td></tr><tr><td>Hook Connectors</td><td>Fisher Scientific</td><td>14-66-18Q</td><td>For Media Vessel and Hosing</td></tr><tr><td>Ratchet Clamp</td><td>Cole-Palmer</td><td>EW-06403-11</td><td>For Media Vessel and Hosing</td></tr><tr><td>Luer, Female</td><td>Cole-Palmer</td><td>EW-45512-34</td><td>For Media Vessel and Hosing</td></tr><tr><td>Luer, Male</td><td>Cole-Palmer</td><td>EW-45513-04</td><td>For Media Vessel and Hosing</td></tr><tr><td>Millipore Aervent MTGR05010 62 mm Filter, 0.2 &amp;mu;m</td><td>Fisher Scientific</td><td>MTGR05010</td><td>For Media Vessel and Hosing</td></tr><tr><td>PTFE Acrodisc CR 13 mm filters, 0.2 &amp;mu;m</td><td>Fisher Scientific</td><td>NC9131037</td><td>For Media Vessel and Hosing</td></tr><tr><td>Direct-Reading Flowtube for Air</td><td>Cole-Palmer</td><td>EW-32047-77</td><td>For Nitrogen Gas Setup</td></tr><tr><td>Direct-Reading Flowtube for Nitrogen</td><td>Cole-Palmer</td><td>EW-32048-63</td><td>For Nitrogen Gas Setup</td></tr><tr><td>Gas Proportioner Multitube Frames</td><td>Cole-Palmer</td><td>EW-03218-50</td><td>For Nitrogen Gas Setup</td></tr><tr><td>Regulator, Two-Stage Analytical</td><td>Airgas</td><td>Y12-N145D580</td><td>For Nitrogen Gas Setup</td></tr><tr><td>Hose Adaptor, Stainless Steel</td><td>Airgas</td><td>Y99-26450</td><td>For Nitrogen Gas Setup</td></tr><tr><td>Hose Male Adaptor</td><td>Airgas</td><td>WES544</td><td>For Nitrogen Gas Setup</td></tr><tr><td>Norprene Tubing</td><td>US Plastics</td><td>57280</td><td>For Nitrogen Gas Setup</td></tr><tr><td>Tripod Base</td><td>Cole-Palmer</td><td>EW-03218-58</td><td>For Nitrogen Gas Setup</td></tr><tr><td>Valve Cartridges</td><td>Cole-Palmer</td><td>EW-03217-92</td><td>For Nitrogen Gas Setup</td></tr><tr><td>Carboy 10 L</td><td>Fisher Scientific</td><td>02-963-2A</td><td>For Media Preperation</td></tr><tr><td>Steritop Sterile Vacuum Bottle-Top Filters, 1,000 ml, PES membrane; for 45 mm neck size</td><td>Fisher Scientific</td><td>SCGP-T10-RE</td><td>For Media Preperation</td></tr><tr><td>Media Bottle 100 ml, 45 mm neck size</td><td>Fisher Scientific</td><td>FB-800-100</td><td>For Media Preperation</td></tr><tr><td>calcium chloride&amp;middot;2H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Fisher Scientific</td><td>C79-500</td><td>Media Reagents</td></tr><tr><td>sodium chloride</td><td>Fisher Scientific</td><td>BP358-1</td><td>Media Reagents</td></tr><tr><td>magnesium sulfate&amp;middot;7H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma Aldrich</td><td>230391</td><td>Media Reagents</td></tr><tr><td>potassium phosphate monobasic</td><td>Fisher Scientific</td><td>AC424205000</td><td>Media Reagents</td></tr><tr><td>ammonium sulfate</td><td>Fisher Scientific</td><td>AC423400010</td><td>Media Reagents</td></tr><tr><td>potassium chloride</td><td>Sigma Aldrich</td><td>P9541</td><td>Media Reagents</td></tr><tr><td>boric acid</td><td>Sigma Aldrich</td><td>B6768</td><td>Media Reagents</td></tr><tr><td>copper sulfate&amp;middot;5H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma Aldrich</td><td>209198</td><td>Media Reagents</td></tr><tr><td>potassium iodide</td><td>Sigma Aldrich</td><td>60400</td><td>Media Reagents</td></tr><tr><td>ferric chloride&amp;middot;6H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Fisher Scientific</td><td>I88-100</td><td>Media Reagents</td></tr><tr><td>manganese sulfate&amp;middot;H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma Aldrich</td><td>230391</td><td>Media Reagents</td></tr><tr><td>sodium molybdate&amp;middot;2H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma Aldrich</td><td>M7634</td><td>Media Reagents</td></tr><tr><td>zinc sulfate&amp;middot;7H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Fisher Scientific</td><td>Z68-500</td><td>Media Reagents</td></tr><tr><td>biotin</td><td>Fisher Scientific</td><td>BP232-1</td><td>Media Reagents</td></tr><tr><td>calcium pantothenate</td><td>Fisher Scientific</td><td>AC24330-1000</td><td>Media Reagents</td></tr><tr><td>folic acid</td><td>Sigma Aldrich</td><td>F7876</td><td>Media Reagents</td></tr><tr><td>inositol (aka myo-inositol)</td><td>Fisher Scientific</td><td>AC12226-1000</td><td>Media Reagents</td></tr><tr><td>niacin (aka nicotinic acid)</td><td>Sigma Aldrich</td><td>N4126</td><td>Media Reagents</td></tr><tr><td>p-aminobenzoic acid</td><td>Fisher Scientific</td><td>AC14621-2500</td><td>Media Reagents</td></tr><tr><td>pyridoxine HCl</td><td>Sigma Aldrich</td><td>P9755</td><td>Media Reagents</td></tr><tr><td>riboflavin</td><td>Sigma Aldrich</td><td>R4500-25G</td><td>Media Reagents</td></tr><tr><td>thiamine HCl</td><td>Fisher Scientific</td><td>BP892-100</td><td>Media Reagents</td></tr><tr><td>Leucine</td><td>Sigma Aldrich</td><td>L8000-100G</td><td>Media Reagents</td></tr><tr><td>Uracil</td><td>Sigma Aldrich</td><td>U0750</td><td>Media Reagents</td></tr><tr><td>Dextrose</td><td>Fisher Scientific</td><td>DF0155-08-5</td><td>Media Reagents</td></tr></tbody></table>

the use of chemostats in microbial systems biology

The Use of Chemostats in Microbial Systems Biology

Cells regulate their rate of growth in response to signals from the external world. As the cell grows, diverse cellular processes must be coordinated including macromolecular synthesis, metabolism and ultimately, commitment to the cell division cycle. The chemostat, a method of experimentally controlling cell growth rate, provides a powerful means of systematically studying how growth rate impacts cellular processes - including gene expression and metabolism - and the regulatory networks that control the rate of cell growth. When maintained for hundreds of generations chemostats can be used to study adaptive evolution of microbes in environmental conditions that limit cell growth. We describe the principle of chemostat cultures, demonstrate their operation and provide examples of their various applications. Following a period of disuse after their introduction in the middle of the twentieth century, the convergence of genome-scale methodologies with a renewed interest in the regulation of cell growth and the molecular basis of adaptive evolution is stimulating a renaissance in the use of chemostats in biological research.

A major advantage of chemostats is the ability to control the growth rate of cells experimentally by varying the dilution rate. In the budding yeast, Saccharomyces cerevisiae, the morphology of a cell is informative of its phase in the cell division cycle. Populations with higher growth rates contain a higher proportion of actively dividing cells as determined by measuring the fraction of unbudded cells (Figure 5A). Analyses of global mRNA expression in chemostat cultures has shown that the expression of...

Cell growth rate is a regulated process and a primary determinant of cell physiology. Continuous culturing using chemostats enables extrinsic control of cell growth rate by nutrient limitation facilitating the study of molecular networks that control cell growth and how those networks evolve to optimize cell growth.

Cells regulate their rate of growth in response to signals from the external world. As the cell grows, diverse cellular processes must be coordinated ...

The overall goals of the following presentation are to describe the principle of the chemos stat, demonstrate their operation, and to provide examples of applications that use chemos stats. This is achieved by first assembling the hemostat vessel inoculating, and then continuously adding new media while removing old media and cells at a constant rate. Mathematical modeling describes the change in cell density and the change in concentration of a growth limiting nutrient within each hemostat.The use of these equations allows for the prediction of a single non-zero stable steady state at which cell and nutrient concentrations are constant, and the specific growth rate of cells is equal to the rate at which the cells are removed. By combining the precise control of cell growth and the environment within the chemos stats with other technologies results that enable systems level investigations into cell growth regulation, genetic changes and adaptive evolution are obtainable. The main advantage of this method of cell culturing over other techniques is that the growth rate is controlled and the cell's external environment remains constant.This method can help answer key questions in the

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JoVE Journal

Biology

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Design and Use of Multiplexed Chemostat Arrays

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

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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.

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Light-Controlled Fermentations for Microbial Chemical and Protein Production

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A Continuous Culture Millifluidic Device for the Study of Escherichia coli under Low and Fluctuating Nutrient Conditions

Learn chemostat operation for microbial systems biology using continuous culture methods. This technique enables precise control of cell growth rates and facilitates studies of adaptive evolution and gene regulation.

The Use of Chemostats in Microbial Systems Biology

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