Single-molecule Imaging of Gene Regulation In vivo Using Cotranslational Activation by Cleavage (CoTrAC)

The aim of this procedure is to acquire time-lapse fluorescence images of growing e coli colonies, expressing co track constructs to identify the number of newly produced protein molecules. This is accomplished by first preparing a sample of cells, coex expressing a protein of interest fused to a fluorescent Cora reporter, and a protease that specifically cleaves between the Cora reporter and the protein of interest. The second step is to prepare a gel of low melting temperature Aris made with M nine minimal media.

Next, an AROS gel pad is prepared for use with a temperature controlled sample chamber. The e coli cells are placed on the gel and the chamber is assembled. The final step is to place the chamber on a fluorescence microscope and image several growing cell colonies at regular intervals over multiple generations.

Photobleaching newly produced TRE report molecules at each interval of all ultimately re is used to acquire time traces of protein production in life cells with single molecule and single cell resolution. The advantage of this technique over ex single cell measures using fluorescence protein fusion is that the production of any cancer of protein molecules can be followed with the single molecule procedure and the result affecting that molecules functions. To engineer the strain insert sequences, encoding a membrane localization sequence, a fast maturing fluorescent protein, and a protease recognition sequence and terminal to and in frame with a protein of interest in this specific case, a transcription factor or tf.

In this procedure, the membrane targeted TSR venous reporter was used. The reporter was fused to the protease recognition sequence ubiquitin to count the number of expressed bacteria phage Lambda Repressor, C one protein molecules. After incorporating the construct into the e coli chromosome, the plasmid encoding of protease specific to the recognition sequence is transformed into a strain expressing the fluorescent reporter and protein of interest in translational fusion.

In this protocol, the protease ubiquitin carboxy Hydro one, which cleaves immediately after the C terminal ubiquitin residue was utilized. To begin this protocol, pick one e coli colony from a freshly streak agar plate into one milliliter M nine, minimal medium supplemented with one x minimum essential medium amino acids and appropriate antibiotics. Incubating a shaker at the desired temperature long enough to reach an optical density at 600 nanometers or OD 600 greater than 1.0.

Then dilute the culture into one milliliter of fresh M nine medium with appropriate antibiotics to an OD 600 of 0.02. Incubating a shaker at the appropriate temperature, initial cellular density can be increased if needed for low temperature growth. When the OD 600 equals 0.2 to 0.3, start preparing the ARO gel pad by weighing out 10 to 20 milligrams of low melting temperature aros into 1.5 milliliter micro tube.

Add the appropriate volume M nine, minimal medium without antibiotics to make a 3%arose solution. Then heat the arose solution for 30 minutes at 70 degrees Celsius to melt. Invert the tube to ensure that the solution is completely melted and homogenous.

For gel pad assembly, rinse one pre-cleaned cover glass and one micro aqueduct Slide with sterile water. Dry the cover glass and slide by blowing with compressed air. Place the rubber gasket on the micro aqueduct slide so that it covers the inlets and outlets.

On the glass side of the slide, apply 50 microliters of ARO solution to the center of the micro aqueduct. Slide top the ARO solution with the cleaned dry cover glass. Let the agro solution stand at room temperature for 30 minutes.

When the cells reach an OD 600 of 0.3 to 0.4, they're ready for imaging. Transfer one milliliter of incubated culture into 1.5 milliliter micro centrifuge tube centrifuge at 10, 000 times G for one minute in a benchtop micro centrifuge. After discarding the SUP agent, add one milliliter of fresh M nine medium and resus.

Suspend the pellet gently centrifuge the suspension again at 10, 000 times G for one minute. Then repeat the resus centrifugation step once more, gently resuspend the pellet in one milliliter of M nine medium. At this point, cells can be used for microscope imaging or diluted 10 to 100 fold to ensure low cell densities for time-lapse imaging.

To prepare a sample for imaging, carefully peel off the cover glass from the top of the gel pad. Add one microliter of washed cell culture to the top of the gel pad. Wait for approximately two minutes for the culture to be absorbed by the gel pad.

It is important not to wait so long that the gel pad over dries. The ideal time will vary with temperature and humidity while waiting dry. Another pre-cleaned cover glass.

Once the sample is dried, cover the sample with the new cover glass ensuring that the cover glass and micro aqueduct slide are well aligned. Assemble the temperature controlled growth chamber following the manufacturer's instructions. It can now be used for imaging on the microscope.

To begin imaging, turn on the laser and microscope following the manufacturer's instructions. Lock the assembled chamber to the stage insert on the microscope. Set the temperature of the growth chamber and the objective heater at 37 degrees Celsius or other desired growth temperature.

If imaging above room temperature, the focus or gel padd will usually drift significantly for approximately 15 minutes after the initial temperature shift. In practice. Use this time to find regions of interest and modify imaging scripts as necessary.

For an experiment. Adjust laser excitation intensity so that almost all fluorescent molecules photo bleach within six exposures. Find cells on the microscope and center them within a predefined and aligned imaging region.

Then store the position of each cell more than one cell can be imaged in each image. Acquisition time window for time-lapse imaging over many generations ensure that image cells are initially separated from other cells, but at least a few hundred microns so that other colonies will not enter the imaging region during growth. Using an automated imaging script acquire time-lapse data using the algorithm described in this experimental workflow, which can be found in the text protocol accompanying this video.

These images show typical time lapse data from a code track experiment. Images from a single colony are shown at 25 minute intervals. In this experiment, data was acquired every five minutes from 12 different colonies.

These images represent brightfield images and Venus YFP fluorescence images with the average background subtracted to account for change in background of entire imaging field. Over time when the images are overlaid, they reveal the pole localization of TSR venous UB reporter molecules and heterogeneity in the number of molecules produced in five minute time windows in different cells. Shown here a typical data from a single time point in a code track experiment venous fluorescence images of six 100 millisecond exposures that were acquired at the beginning of each five minute time interval are shown.

Each of the six exposures was separated by 900 milliseconds to allow time for transiently dark venous molecules that have blinked off to become fluorescent for analysis. Image six is subtracted from image one to correct for unbleached molecules and autofluorescence. Background spots in this image are localized to specific cell lineages and quantified by their integrated fluorescence intensity.

The average integrated fluorescence of the colony is then plotted over six images fitting this line to an exponential decay. Plus a constant offset gives a decay halftime of approximately one second. In this experiment, venous molecules photobleach much more quickly than cellular autofluorescence.

Therefore, approximately half of the total number of venous molecules are photo bleached in each frame. For this procedure, conductivity imaging analysis can be performed in other to create trics of the number of protein molecules produced in single cells.