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