The activator module and repressor module functions can be independently measured in intact cells. The activator module, when present in cells containing wild-type NRII and wild-type lacI encoding Lac Repressor, is predicted to form an N-IMPLIES logic gate with respect to ammonia and IPTG (). In the presence of wild-type NRII, the nitrogen-rich state brought about by the presence of ammonia causes the formation of the NRII-PII complex and the rapid dephosphorylation of NRI~P (18). Furthermore, in the absence of IPTG, the constitutively-present Lac Represor blocks the expression of the activator module. Expression of a reporter consisting of the glnK promoter to lacZ, or any other Ntr gene, thus requires the presence of IPTG and the absence of ammonia, providing the N-IMPLIES logic function. After construction of the bacterial strain, we observed that the cells indeed only expressed β-galactosidase in the absence of ammonia and the presence of IPTG.
The activator module is an N-IMPLIES gate with positive feedback
The activator module also displayed toggle-switch function in the absence of the repressor module, as predicted by modeling (Atkinson et al, 2003
, ). For these experiments, where only repression of the activator module was examined, the activator module was integrated into a strain deleted for both glnL
, and the transmitter protein function was complemented by the plasmid p3Y15, encoding NRII2302. The strain also contained a wild-type lacI
, but contained a mutation of lacY
. The mutation of lacY
is essential to allow control of the internal IPTG concentration by variation in the external IPTG concentration (Novice and Weiner, 1957
). The assembly of the strain was as follows: strain M7044 (lacY
) was transduced to glnA::Tn5
by selection for kanamycin resistance and checking for glutamine auxotrophy, forming strain M7044A. This was then transduced to glutamine prototrophy using phage grown on strain SN24 (glnLglnG
), producing strain M7044LG. This strain was then transduced with the activator module, by selection for gentamycin resistance of the integrated rbs
landing pad, producing strain TS1. Finally, strain TS1 was made competant by standard methods and transformed with plasmid p3Y15, with selection for ampicillin resistance.
The activator module can function as a toggle switch
Toggle switch experiments are performed by growing the strain overnight in the absence and presence of 0.1 mM IPTG, resulting in naive and induced cultures. The overnight cultures are then diluted one million-fold into media containing various concentrations of IPTG. Growth is continued for about 15–17 generations, until the cultures were in mid-log phase, and β-galactosidase and glutamine synthetase are measured. The glutamine synthetase measurement provides an indication of the level of activator, while the β-galactosidase measurement provides an indication of the level of repressor. It is expected that the naive and induced cultures should show equivalent levels of β-galactosidase expression, but, if there is hysteresis in the activator module control, should shown very different glutamine synthetase levels. As shown in , this behavior was observed. It should be noted that since the glnL2302 mutation was present on p3Y15, a variety of growth media can be used for the experiment, including complex media such as LB or nutrient broth. To compensate for the loss of normal Ntr regulation, glutamine is included in all media at 0.2% (w/v). [Note that glutamine does not survive autoclaving and filter sterilized glutamine must be added to the medium after autoclaving.]
The repressor module, when present in cells containing a deletion of the natural lacI and wild-type lacZYA and Ntr system, is predicted to display OR gate function with regard to ammonia and IPTG control of lacZYA expression (). This is because ammonia blocks the formation of the activator, while IPTG blocks the function of Lac Repressor produced from the repressor module. Consequently, either stimulus provides full expression of lacZYA (). The designed strain (3.300 containing the repressor module) was observed to indeed show the OR gate function.
The repressor module functions as an OR logic gate
Procedures for clock experiments
We have used a variety of growth media for clock experiments; a very good medium providing rapid growth of the cells and good oscillatory function is W-salts based glucose-glutamine-caseamino acids, with the following formula (per liter): 10.5 g K2HPO4, 4.5 g KH2PO4, 0.65 mL of 1 M MgSO4, 0.04 g thiamine, 0.04 g tryptophan, 0.5 g glutamine, 10 g glucose 5 g Bacto caseamino acids, 0.1 g ampicillin, and 1 mL of 34 mg/mL chloramphenicol. The medium is filter sterilized, owing to its labile components. A single colony of the clock strain is picked from an LB + ampicillin plate and innoculated into 2 mL of medium, and allowed to grow to saturation. 1.2 mL of the overnight culture is used to innoculate 120 mL of medium containing 0.5 mM IPTG. This culture is incubated until the turbidity reaches the desired turbidity for the clock experiment; typically 10–12 hr incubation provides an OD600 of ~0.6, which gives good results. The cells are harvested by centrifugation, resuspended in 120 mL of fresh medium lacking IPTG, repelleted, again resuspended in 120 mL of fresh medium lacking IPTG, and introduced into the continuous culture device. The volumes stated above can be scaled as appropriate for a variety of working volumes required by different fermentors. The optical density of the culture can be corrected in the initial stages of the run by control of the fermentor nutrient pump. Best oscillatory behavior was observed when the continuous culture was run at OD600 of 0.5–0.6. Cells are grown in the continuous culture device at a constant optical density by controlling the nutrient flow. Samples are removed periodically (or obtained from the fermentor efflux) and assayed for β-galactosidase and glutamine synthetase.
We have used two methods for conducting the continuous culture experiments, one utilizing a standard laboratory fermentor that was not desiged to function as a turbidostat, and the other using a custom-built turbidostat. Both methods will be briefly described here. It is important to note that the clock strain does not grow at a constant rate in continuous culture; rather, the presence of the synthetic genetic clock causes growth to slow down as the activator module and lacZYA
expression are derepressed and to speed up as the activator module and lacZYA
are repressed. Thus, to maintain a constant culture optical density, the nutrient flow must be continuously adjusted. When using a standard laboratory fermentor not designed to function as a turbidostat, this is accomplished by periodically sampling the fermenter efflux, measuring OD600
, and manually adjusting the nutrient flow rate so as to hold the culture at constant turbidity (12). Since clock experiments typically are run for 80 hrs or more, the experiments require lots of coffee and/or several people to take shifts minding the fermenter. Nevertheless, good oscillatory function can be easily observed in such experiments, despite the fairly crude control of the culture turbidity (Atkinson et al, 2003
Results of clock experiments using a standard laboratory continuous culture device
For experiments with a typical laboratory fermentor, the starting culture at approximately the desired working OD600 is introduced directly into the reactor, and the optical density is corrected to the desired optical density by manipulation of the nutrient pump. Filtered air is pumped into the reactor and vigorous stirring is used to provide good aeration. Medium is pumped into the reactor from a 4L reservior, to which fresh sterile medium is added aseptically as needed. Samples are collected from the efflux line directly into a clean disposable cuvette; the OD600 of the culture is measured, and aliquots are used for the β-galactosidase and glutamine synthetase assay (12).
To automate the clock experiments, we developed a turbidostat as depicted in . The reactor is a 500 mL Erlenmeyer flask that contains a 120 mL culture that is kept at constant optical density by varying the media flow appropriately. A close-up view of the reactor is shown in ; it has four connections to the outside: one line to pour media in, one line to suck culture out when the volume slightly exceeds 120 ml (waste line), one line to take samples, and one line to pump in filtered air. The air pump is a large fish tank aerator connected to a standard 0.22 μm filter to provide sterility. The reactor is contained within a standard small laboratory incubator, and good mixing of the culture is provided by placing the flask on a standard stir plate and including a magnetic stir bar in the vessle (not depicted). In addition to the stirring, the pumping in of air contributes to mixing within the reactor.
Schematic diagram for a home-made turbidostat
Close-up view of the reactor for a home-made turbidostat
The media is pumped in from a media reservoir at a variable speed. To maintain sterility and accomodate the large volumes of medium required, we use a home-made medium “tree” that consists of up to 6 one-gallon bottles connected in a daisy chain by sterilizable tygon tubing. This apparatus is sterilized dry under a tin-foil tent, placed into a bacteriological hood while still hot, and the filter sterilized medium is introduced after it cools sufficiently. The waste line sucks culture out and has twice the flow rate of incoming media, and it sucks culture out only when the volume goes higher than 120 mL. The waste line needs to have a stronger flow rate than the media line in order to keep the volume constant. The depicted design was chosen, as opposed to having the same flow rate of media in and culture out, because fluctuations in the flow rate of the lines, even if both lines are connected to the same pump, makes the control of the volume more difficult. Any slight difference in flowing rates may lead to volume accumulation or reduction, and since each experiment lasts several days, this constitutes a problem. The waste flow rate is made double to incoming medium flow rate by using tubing of twice the diameter and the same pump ().
The sampling pump (Pump 2 in ) intermittently removes culture from the fermentor at a rate that is considerably less than the nutrient flow. Every five minutes a small aliquot of culture is pumped out through the sampling line to a flow cell (). The flow rate of media is changed as a function of this OD600, in order to keep the optical density constant. After each OD600 reading, the sampling pump is shut off, and the flow cell is washed with sterile water, using water that is pumped into the sampling line from a water reservoir by the wash pump (). This wash routine was found to be important, as continuous pumping of the culture through the flow cell and autosampler leads to the formation of a biofilm on the surfaces, fouling the reading of absorbance and contaminating the samples and instruments. Every 30 min, or six OD600 readings, a 2 mL sample of the culture is directed to waste to clear the lines and a 1 ml sample is collected in a well of a deep well 96-well microtitre plate. The automatic sampler allows the sampling line to point to any of the 96 wells in the microtitre plate, or to waste.
The system depicted in is fully computer-controlled. The media, sampling and wash are each pumped by a computer controlled peristaltic pump (media and sampling lines are pumped by VS-series Alitea peristaltic pumps, water is pumped by a S-series Alitea peristaltic pump), while the spectrometer (Ocean Optics SD2000) and autosampler (AIM 3200) also have an interface to the computer. The flow cell to measure absorbance, as well as the pumps, autosampler and spectrometer were purchased from FIAlab Instruments (Bellevue WA).
The software used to control the system was implemented in Labview (National Instruments, Austin TX). Labview is a graphical programming language that eases the interface of the computer with instruments and data acquisition devices. Some instruments, like the autosampler and valves, have an interface to the computer via the serial port and an ascii language, while others like the spectrometer or a data acquisition card, have some other way of interfacing with the computer, but they offer a basic library of functions in Labview to operate them. Essentially any instrument can be controlled from Labview. Besides its ease to interface with instruments, as a programming language Labview offers the capabilities of any other programming language (like C/C++ or Java). Using Labview, we designed the following algorithm to run the system (collect samples and keep the OD constant)
- Set the target OD and the media flow rate (measured as the percentage of the top speed in the pump, initially 50%)
- Every five minutes:
- Pump culture from the flask to the flow cell, and make an OD measurement
- Wash the flow cells with sterile water
- Adjust the media flow rate according to the formula
- Every half an hour (every 6 readings), pump 2 mL of culture to waste and collect 1 ml of culture in a deep well 96-well plate.
Conducting automated experiments requires that the samples be maintained at low temperature (4 °C) to prevent growth of the cultures and changes in the level of β-galactosidase and glutamine synthetase. We observed that samples held at 4 °C displayed stable levels of these two enzymes for several days. To provide uniformity, we routinely maintain all samples at 4 °C for at least 4 hrs before conducting the assays. The autosampler is contained within a standard small refrigerator, which we adapted to our purpose by drilling a small entry port into one side and a small “overflow” port into its base. The entire apparatus, including refrigerator, incubator, computer, pumps, and flow cell easily fits onto one standard laboratory bench.
The automated system utilized miniaturized assays, and uses samples that have been stored at 4 °C. We observe that the miniaturized assays are somewhat noiser than the standard assays, and that some of this may be due to inconsistent resuspension of the settled cells from the stored samples. Thus, we recommend special attention to resuspending the settled cells, by both extensively vortexing the collection plates and pipetting the samples into and out of the well several times before taking the aliquots for measurement. This noisiness notwithstanding, oscillatory behavior can be easily observed using an automated system with samples stored at 4 °C and miniaturized assays ().
Results of a clock experiment employing an automated system and miniaturized β-galactosidase assay.
Procedures for the measurement of glutamine synthetase and β-galactosidase for clock and toggle-switch experiments
Glutamine synthetase microassay
- A reaction mix is assembled as follows:
|0.45 M Imidazole pH 7.33||35|
|0.3 M NH2OH-HCl||7|
|0.01 M MnCl2||3.5|
|0.06 M KAs2O4 pH 7.2||35|
|6 mM ADP, pH 7.0||7|
|1.5 mg/ml CTAB||7|
|2.9 % glutamine||12|
|Correct the pH to 7.27 with KOH.|
- Transfer 400 μL of each sample to be assayed to a well in a deep well 96-well plate. Centrifuge to pellet the cells and carefully remove the supernatant and discard. Resuspend the cells in 40 μL of CTAB cell-wash buffer, and transfer to a standard flat-bottom 96 well microtitre plate. The CTAB cell-wash buffer is: 5 mM Imidazole pH 7.15, 0.1 mg/mL CTAB, 0.27 mM MnCl2.
- 100 μL aliquots of the reaction mix are pippetted into the wells; this starts the reaction. ypically, the mix aliquots are added at 20 sec intervals using an 8-channel micropipettor, and later the reactions will be stopped in the same sequence at 20 sec intervals. When all samples have been added, the plate is incubated at 37 °C in a standard microbiological incubator. In a separate plate, a sample from an early time point in the clock experiment (where the glutamine synthetase activity is expected to be high) are set up in triplicate in three separate wells of a microtitre plate. This plate will be used to determine the appropriate length of time to incubate the reactions. At 30 min intervals, stop solution is added to one of the three wells in the test plate, and the reactions are allowed to continue until a dark brown color is obtained upon stopping the test reaction. Typically, a one-hour incubation is sufficient for measurement of the activity. The stop solution is (per L): FeCl3·6H2O 55 g; TCA 20 g, HCl 21 mL.
- To stop the reactions, 100 μL of stop solution is added to each well, in the same sequence and time interval that the reactions were started. Upon addition of the stop solution, the reaction mixtures that contain high glutamine synthetase activity will turn dark brown. Since the cell samples should all have similar OD600, the oscillations in GS level should become immediately apparent as the reactions are stopped.
- The OD540 of the stopped reactions are directly measured in a plate reader. In a separate plate, the OD600 of the original cell samples are directly read. The glutamine synthetase activity, in arbitrary units, is simply the OD540 value divided by the OD600 value.
- For toggle-switch experiments, the cells from 1 mL of the cultures are pelleted in microcentrifuge tubes, and resuspended in 0.1 mL of CTAB-cell wash buffer. Aliquots of 20 μL and 50 μL are then added to the reaction mix in microtitre plates, and the procedure described above is followed.
- The wells of a deep-well 96-well plate are loaded with 400 μL of Z-buffer, 10 μL of 0.1% SDS, and 10 μL of chloroform (add last). The formula for Z-buffer is (per liter): 16.1 g Na2HPO4.7H2O, 5.5 g NaH2PO4.H2O, 0.75 g KCl, 0.246 g MgSO4.7H2O. Immediately prior to the assay, add 0.27 mL of β-mercaptoethanol/100 mL of Z-buffer.
- A 100 μL of the culture sample is added progressively to each well and mixed thoroughly by pipetting (the chloroform must be well mixed into the suspension for uniform cell permeabilization). We typically use an 8-sample multichannel micropipettor to minimize the difference in exposure of samples to the chloroform. The reaction mixtures are then incubated 10 min at room temperature.
- To start the reactions, 100 μL of ONPG (4 mg/mL in 0.1 mM phosphate buffer) is added to the wells in sequence and at fixed intervals, and mixed thoroughly by pipetting.
- Allow the reaction to proceed until the appearance of yellow color in some of the wells. Note that oscillations in the level of β-galactosidase becomes readily apparent at this stage, and some of the wells should never turn bright yellow since they contain very low levels of β-galactosidase. Typically, a 5–10 min incubation is required.
- Reactions are stopped in the sequence they were started and at the same intervals, by adding 200 μL of 1M Na2CO3.
- The stopped reactions are allowed to sit for 10 min for full color development and settling of the cells, then 150μL aliquots (from the top) from each well is transfered to a 96 well flat bottom plate and the OD410 and OD560 are recorded. In a separate series of wells, the OD600 of the original culture samples is measured.
- The β-galactosidase activity is [OD410 - (1.75 · OD560)]/(OD600 · time).