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

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...

Watch this Scientific Journal Video about The Use of Chemostats in Microbial Systems Biology at JoVE.com

The Use of Chemostats in Microbial Systems Biology

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 ...

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Research

JoVE Journal

Biology

30.8K Views.  New York University.  Celgroei tarief is een gereguleerd proces en een primaire determinant van cel fysiologie. Continue kweken met chemostaten maakt extrinsieke controle cellen groei met nutriënten limitatie de studie van moleculaire netwerken die celgroei en hoe deze netwerken ontwikkelen om optimale celgroei regelen vergemakkelijken. 

Video: The Use of Chemostats in Microbial Systems Biology

Design and Use of a Low Cost, Automated Morbidostat for Adaptive Evolution of Bacteria Under Antibiotic Drug Selection

We describe a low cost, configurable morbidostat for characterizing the evolutionary pathway of antibiotic resistance. The morbidostat is a bacterial culture device that continuously monitors bacterial growth and dynamically adjusts the drug concentration to constantly challenge the bacteria as they evolve to acquire drug resistance. The device features a working volume of ~10 ml and is fully automated and equipped with optical density measurement and micro-pumps for medium and drug delivery. To validate the platform, we measured the stepwise acquisition of trimethoprim resistance in Escherichia coli MG 1655, and integrated the device with a multiplexed microfluidic platform to investigate cell morphology and antibiotic susceptibility. The approach can be up-scaled to laboratory studies of antibiotic drug resistance, and is extendible to adaptive evolution for strain improvements in metabolic engineering and other bacterial culture experiments.

We describe a low cost, configurable morbidostat that enables the characterization of antibiotic drug resistance by dynamically adjusting the drug concentration. The device can be integrated with a multiplexed microfluidic platform. The approach can be scaled up for laboratory antibiotic drug resistance studies.

We describe a low cost, configurable morbidostat for characterizing the evolutionary pathway of antibiotic resistance. The morbidostat is a bacterial ...

Genetics

Procedure for Adaptive Laboratory Evolution of Microorganisms Using a Chemostat

Natural evolution involves genetic diversity such as environmental change and a selection between small populations. Adaptive laboratory evolution (ALE) refers to the experimental situation in which evolution is observed using living organisms under controlled conditions and stressors; organisms are thereby artificially forced to make evolutionary changes. Microorganisms are subject to a variety of stressors in the environment and are capable of regulating certain stress-inducible proteins to increase their chances of survival. Naturally occurring spontaneous mutations bring about changes in a microorganism's genome that affect its chances of survival. Long-term exposure to chemostat culture provokes an accumulation of spontaneous mutations and renders the most adaptable strain dominant. Compared to the colony transfer and serial transfer methods, chemostat culture entails the highest number of cell divisions and, therefore, the highest number of diverse populations. Although chemostat culture for ALE requires more complicated culture devices, it is less labor intensive once the operation begins. Comparative genomic and transcriptome analyses of the adapted strain provide evolutionary clues as to how the stressors contribute to mutations that overcome the stress. The goal of the current paper is to bring about accelerated evolution of microorganisms under controlled laboratory conditions.

Here, we present a protocol to obtain adaptive laboratory evolution of microorganisms under conditions using chemostat culture. Also, genomic analysis of the evolved strain is discussed.

Natural evolution involves genetic diversity such as environmental change and a selection between small populations. Adaptive laboratory evolution (ALE) ...

Chemotactic Response of Marine Micro-Organisms to Micro-Scale Nutrient Layers

The degree to which planktonic microbes can exploit microscale resource patches will have considerable implications for oceanic trophodynamics and biogeochemical flux. However, to take advantage of nutrient patches in the ocean, swimming microbes must overcome the influences of physical forces including molecular diffusion and turbulent shear, which will limit the availability of patches and the ability of bacteria to locate them. Until recently, methodological limitations have precluded direct examinations of microbial behaviour within patchy habitats and realistic small-scale flow conditions. Hence, much of our current knowledge regarding microbial behaviour in the ocean has been procured from theoretical predictions. To obtain new information on microbial foraging behaviour in the ocean we have applied soft lithographic fabrication techniques to develop 2 microfluidic devices, which we have used to create (i) microscale nutrient patches with dimensions and diffusive characteristics relevant to oceanic processes and (ii) microscale vortices, with shear rates corresponding to those expected in the ocean. These microfluidic devices have permitted a first direct examination of microbial swimming and chemotactic behaviour within a heterogeneous and dynamic seascape. The combined use of epifluorescence and phase contrast microscopy allow direct examinations of the physical dimensions and diffusive characteristics of nutrient patches, while observing the population-level aggregative response, in addition to the swimming behaviour of individual microbes. These experiments have revealed that some species of phytoplankton, heterotrophic bacteria and phagotrophic protists are adept at locating and exploiting diffusing microscale resource patches within very short time frames. We have also shown that up to moderate shear rates, marine bacteria are able to  fight the flow  and swim through their environment at their own accord. However, beyond a threshold high shear level, bacteria are aligned in the shear flow and are less capable of swimming without disturbance from the flow. Microfluidics represents a novel and inexpensive approach for studying aquatic microbial ecology, and due to its suitability for accurately creating realistic flow fields and substrate gradients at the microscale, is ideally applicable to examinations of microbial behaviour at the smallest scales of interaction.  We therefore suggest that microfluidics represents a valuable tool for obtaining a better understanding of the ecology of microorganisms in the ocean.

The fabrication of microfluidic channels and their implementation in experiments for studying the chemotactic foraging behaviour of marine microbes within a patchy nutrient seascape and the swimming behaviour of bacteria within shear flow are described.

The degree to which planktonic microbes can exploit microscale resource patches will have considerable implications for oceanic trophodynamics and ...

A Tandem Liquid Chromatography&#8211;Mass Spectrometry-based Approach for Metabolite Analysis of Staphylococcus aureus

In an effort to thwart bacterial pathogens, hosts often limit the availability of nutrients at the site of infection. This limitation can alter the abundances of key metabolites to which regulatory factors respond, adjusting cellular metabolism. In recent years, a number of proteins and RNA have emerged as important regulators of virulence gene expression. For example, the CodY protein responds to levels of branched-chain amino acids and GTP and is widely conserved in low G+C Gram-positive bacteria. As a global regulator in Staphylococcus aureus, CodY controls the expression of dozens of virulence and metabolic genes. We hypothesize that S. aureus uses CodY, in part, to alter its metabolic state in an effort to adapt to nutrient-limiting conditions potentially encountered in the host environment. This manuscript describes a method for extracting and analyzing metabolites from S. aureus using liquid chromatography coupled with mass spectrometry, a protocol that was developed to test this hypothesis. The method also highlights best practices that will ensure rigor and reproducibility, such as maintaining biological steady state and constant aeration without the use of continuous chemostat cultures. Relative to the USA200 methicillin-susceptible S. aureus isolate UAMS-1 parental strain, the isogenic codY mutant exhibited significant increases in amino acids derived from aspartate (e.g., threonine and isoleucine) and decreases in their precursors (e.g., aspartate and O-acetylhomoserine). These findings correlate well with transcriptional data obtained with RNA-seq analysis: genes in these pathways were up-regulated between 10- and 800-fold in the codY null mutant. Coupling global analyses of the transcriptome and the metabolome can reveal how bacteria alter their metabolism when faced with environmental or nutritional stress, providing potential insight into the physiological changes associated with nutrient depletion experienced during infection. Such discoveries may pave the way for the development of novel anti-infectives and therapeutics.

Here we describe a protocol for the extraction of metabolites from Staphylococcus aureus and their subsequent analysis via liquid chromatography and mass spectrometry.

In an effort to thwart bacterial pathogens, hosts often limit the availability of nutrients at the site of infection. This limitation can alter the ...

Biochemistry

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 ...

Bioengineering

Designing Automated, High-throughput, Continuous Cell Growth Experiments Using eVOLVER

Continuous culture methods enable cells to be grown under quantitatively controlled environmental conditions, and are thus broadly useful for measuring fitness phenotypes and improving our understanding of how genotypes are shaped by selection. Extensive recent efforts to develop and apply niche continuous culture devices have revealed the benefits of conducting new forms of cell culture control. This includes defining custom selection pressures and increasing throughput for studies ranging from long-term experimental evolution to genome-wide library selections and synthetic gene circuit characterization. The eVOLVER platform was recently developed to meet this growing demand: a continuous culture platform with a high degree of scalability, flexibility, and automation. eVOLVER provides a single standardizing platform that can be (re)-configured and scaled with minimal effort to perform many different types of high-throughput or multi-dimensional growth selection experiments. Here, a protocol is presented to provide users of the eVOLVER framework a description for configuring the system to conduct a custom, large-scale continuous growth experiment. Specifically, the protocol guides users on how to program the system to multiplex two selection pressures &#8212; temperature and osmolarity &#8212; across many eVOLVER vials in order to quantify fitness landscapes of Saccharomyces cerevisiae mutants at fine resolution. We show how the device can be configured both programmatically, through its open-source web-based software, and physically, by arranging fluidic and hardware layouts. The process of physically setting up the device, programming the culture routine, monitoring and interacting with the experiment in real-time over the internet, sampling vials for subsequent offline analysis, and post experiment data analysis are detailed. This should serve as a starting point for researchers across diverse disciplines to apply eVOLVER in the design of their own complex and high-throughput cell growth experiments to study and manipulate biological systems.

The eVOLVER framework enables high-throughput continuous microbial culture with high resolution and dynamic control over experimental parameters. This protocol demonstrates how to apply the system to conduct a complex fitness experiment, guiding users on programming automated control over many individual cultures, measuring, collecting, and interacting with experimental data in real-time.

Continuous culture methods enable cells to be grown under quantitatively controlled environmental conditions, and are thus broadly useful for measuring ...

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.

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 ...

Generic Protocol for Optimization of Heterologous Protein Production Using Automated Microbioreactor Technology

A core business in industrial biotechnology using microbial production cell factories is the iterative process of strain engineering and optimization of bioprocess conditions. One important aspect is the improvement of cultivation medium to provide an optimal environment for microbial formation of the product of interest. It is well accepted that the media composition can dramatically influence overall bioprocess performance. Nutrition medium optimization is known to improve recombinant protein production with microbial systems and thus, this is a rewarding step in bioprocess development. However, very often standard media recipes are taken from literature, since tailor-made design of the cultivation medium is a tedious task that demands microbioreactor technology for sufficient cultivation throughput, fast product analytics, as well as support by lab robotics to enable reliability in liquid handling steps. Furthermore, advanced mathematical methods are required for rationally analyzing measurement data and efficiently designing parallel experiments such as to achieve optimal information content.
The generic nature of the presented protocol allows for easy adaption to different lab equipment, other expression hosts, and target proteins of interest, as well as further bioprocess parameters. Moreover, other optimization objectives like protein production rate, specific yield, or product quality can be chosen to fit the scope of other optimization studies. The applied Kriging Toolbox (KriKit) is a general tool for Design of Experiments (DOE) that contributes to improved holistic bioprocess optimization. It also supports multi-objective optimization which can be important in optimizing both upstream and downstream processes.

This manuscript describes a generic approach for tailor-made design of microbial cultivation media. This is enabled by an iterative workflow combining Kriging-based experimental design and microbioreactor technology for sufficient cultivation throughput, which is supported by lab robotics to increase reliability and speed in liquid handling media preparation.

A core business in industrial biotechnology using microbial production cell factories is the iterative process of strain engineering and optimization of ...

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.

Conventional microbial cultivation methods usually have cumbersome operations, low throughput, low efficiency, and large consumption of labor and ...

Design and Use of Multiplexed Chemostat Arrays

Chemostats are continuous culture systems in which cells are grown in a tightly controlled, chemically constant environment where culture density is constrained by limiting specific nutrients.1,2 Data from chemostats are highly reproducible for the measurement of quantitative phenotypes as they provide a constant growth rate and environment at steady state. For these reasons, chemostats have become useful tools for fine-scale characterization of physiology through analysis of gene expression3-6 and other characteristics of cultures at steady-state equilibrium.7 Long-term experiments in chemostats can highlight specific trajectories that microbial populations adopt during adaptive evolution in a controlled environment. In fact, chemostats have been used for experimental evolution since their invention.8 A common result in evolution experiments is for each biological replicate to acquire a unique repertoire of mutations.9-13 This diversity suggests that there is much left to be discovered by performing evolution experiments with far greater throughput. 
We present here the design and operation of a relatively simple, low cost array of miniature chemostats&mdash;or ministats&mdash;and validate their use in determination of physiology and in evolution experiments with yeast. This approach entails growth of tens of chemostats run off a single multiplexed peristaltic pump. The cultures are maintained at a 20 ml working volume, which is practical for a variety of applications. It is our hope that increasing throughput, decreasing expense, and providing detailed building and operation instructions may also motivate research and industrial application of this design as a general platform for functionally characterizing large numbers of strains, species, and growth parameters, as well as genetic or drug libraries.

We developed and validated a small-footprint array of miniature chemostats built from readily available parts for low cost. Physiological and experimental evolution results were similar to larger volume chemostats. The ministat array provides a compact, inexpensive, and accessible platform for traditional chemostat experiments, functional genomics, and chemical screening applications.

Chemostats are continuous culture systems in which cells are grown in a tightly controlled, chemically constant environment where culture density is ...

The Use of Chemostats in Microbial Systems Biology

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