This protocol in herbals continuous measurement of genetic circuits in E.coli and the calculation of the signal variability and signal-to-noise ratio in individual bacterial cell to assess circuit performance. The main advantages of this technique are that it is easily adaptable to different strains and labs, requires minimal training and no special equipment, and is relatively inexpensive. Noise is a significant role in determining the functionality of biological systems.
Currently, there is an interest in building large scale gene circuits. These systems can be inhibited by noise Be sure to dry the samples well, to keep that chill pads at four degrees Celsius for as long as possible, and to cut and divide the parts patiently and gently. To prepare the bacteria for the analysis inoculate a single colony from a transfected E.coli culture.
For example, a circuit with a GFP reporter. In a glass tube containing five milliliters of LB broth supplement it with the relevant antibiotics. Grow the cells at 37 degrees Celsius and 250 revolutions per minute in a shaking incubator for two hours until the broth is cloudy.
At the end of the incubation, spin down one milliliter of dilution solution in a two milliliter mini micro centrifuge, and transfer 30 microliters of bacteria and the entire volume of dilution solution into a new two milliliter tube. Then, incubate the culture for one hour with shaking before seeding 40 to 60 microliters of this suspension onto M9 culture plates. To prepare the bacteria for the analysis, inoculate a single colony from a transfected E.Coli culture.
For example, a circuit with a GFP reporter in a glass tube containing five milliliters of LB broth supplemented with the relevant antibiotics. Grow the cells at 37 degrees Celsius and 250 revolutions per minute in a shaking incubator for two hours until the broth is cloudy. At the end of the incubation, spin down one milliliter of dilution solution in a two milliliter mini micro centrifuge tube and transfer 30 microliters of bacteria and the entire volume of dilution solution into a new two milliliter tube.
Then incubate the culture for one hour with shaking. To prepare microscope plates, mix 112.5 milligrams of low melting agar with 40 milligrams of agar, and one milliliter of 2%casamino acid in a 25 milliliter Erlenmeyer flask. Next, add 8.92 milliliters of minimal medium over the inner lips of a 25 milliliter Erlenmeyer flask and microwave the resulting solution in short, two to three second bursts.
Then set the water bath to 55 degrees Celsius. When the solution is clear, place the 25 milliliter Erlenmeyer flask in a 60 degrees Celsius water bath and allow the agar solution to cool until its temperature falls to about 45 to 50 degrees Celsius. While the agar cooling, clean the lab bench with 70%ethanol and smooth a piece of lab tape onto the cleaned bench.
Place one glass cover slip onto the tape and place a second cover slip close by. Add cooled agar solution to the appropriate antibiotics and solution. Pour 1.5 milliliters of freshly prepared to gel solution onto the cover slip placed over the tape and place the second cover slip onto the gel, taking care to avoid bubbles.
Return the Erlenmeyer to the hot water bath and cover the cover slips for 20 minutes. Flip the cover slips for a one hour incubation at four degrees Celsius. At the end of the incubation, remove the cover slips from the dry gel and cut a small pad from the agarose.
During the incubation, add six microliter droplets of the prepared bacterial suspension into a 35 millimeter dish and allow the drops to dry for at least 15 to 30 minutes. Then carefully place the pad at a time onto one droplet of bacteria and allow the samples to set for 20 minutes at room temperature. To place the agar bed without smearing the sample, carefully position the cool pad onto the droplet with gentle tapping.
To image the samples, remove the flask from the water and allow the gel solution to cool to body temperature. Pour three millimeters of the gel onto the perimeter of the sample plate. When the agarose has solidified, seal the plate with tape and use a 25 gauge needle to piece several holes into the tape.
Then place the plate upside down at four degrees Celsius for at least 30 minutes to allow the agarose to fully solidify, while preventing cell mitosis. Within 24 hours, use a fluorescence confocal microscope to locate the sample at the lowest magnification and engage the automatic focus system of the microscope. Apply oil to the appropriate objective and use the platform controller to move the plate to carefully spread the oil.
Then engage the automatic focus again and follow the default suggestion for Z step cross-sections to image the sample. The E.coli should appear as black oblong shapes on an off white background. And the dynamic range of the luminance should show a spike at its center.
Mitosis events can first be detected by fluorescence microscopy after 30 minutes. Although individual cells may be hard to detect at low magnification. Bacterial cell cultures prepared at an incorrect dilution ratio can demonstrate unchanging numbers of cells, and samples prepared without plate sealing can exhibit excessive shrinkage, resulting in a loss of focus.
Cells can also indent the gel in their search for nutrients, resulting in stacking of the bacterial cells on top of each other and compromising the cell monolayer, effectively preventing cell detection and reliable fluorescent signal measurement. As suggested by these data, the cell stage in the division cycle does not affect the noise level, as different ranges of area demonstrate the same trend. Further, no substantial change in signal-to-noise ratio is observed between GFP and superfold GFP, as illustrated by these representative data.
When performing this procedure, take care to determine the correct dilution ratio for the bacterial strain used for the experiment. Comparing the signal-to-noise ratio of each regulated circuits to this base line allows selection of the best performance circuit and can reveal interesting phenomena
