Metodo per l'etichettatura di trascrizioni in cellule individuali Escherichia coli per singola molecola di fluorescenza In Situ gli esperimenti di ibridazione

The overall goal of this protocol is to label individual transcripts in E.Coli for fluorescently labeled DNA probes for visualization in single-molecule FiSH experiments. This method can help answer key questions in the systems biology field, such as characterizing transcripts cell to cell ability of field of interest. As well as the transcripts of cell wall localization.

The main advantage of this technique is that it provides quantity of information about the statistics of transcriptional processes. To begin the protocol, grow E.Coli MG1655 in LB medium overnight in an orbital shaker, at 260 RPM and 37 degrees Celsius. The next day, dilute the overnight culture at one to 100 in fresh medium and measure its optical density in a spectrophotometer, up to a value of 0.2 to 0.4.

Next, pellet the cells by centrifugation for five minutes at 4, 500 G at four degrees Celsius. Remove the supernatant from all tubes. Add one milliliter of 1XPBS to each tube and re-suspend the pellet to minimize cell to cell aggregation.

Then add four milliliters of fixation buffer and gently vortex the tube. Incubate the samples in an orbital shaker. Re-suspend the pellet in order to minimize cell to cell aggregation, which may interfere with cell imaging.

After incubation, pellet the cells, remove the supernatant, and add one milliliter of 1XPBS. Transfer suspensions to 1.8 milliliter conical bottom microcentrifuge tubes. Pellet the cells by centrifugation.

Begin bacteria permeabilization by carefully removing the supernatant. Use small volume pipettes to remove all of the liquid outside the pellet. Next, re-suspend the pellet in 300 microliters DEPC treated water.

Add 350 microliters of high grade absolute ethanol and mix the solution gently by inverting the tube. Add another 350 microliters of ethanol and mix again. Then rotate the sample with the tube rotator at 20 RPM for one hour at room temperature.

Pellet the cells from the permeabilization. After centrifugation, remove the supernatant. Add one milliliter of 20%wash buffer to the samples.

Leave them standing for several minutes. Next, pellet cells by centrifugation for seven minutes at 750 G and four degrees Celsius. Remove the supernatant very gently and use small volume pipettes to remove all the liquid outside the pellet.

Then add the oligonucleotide probe set stock solution to the hybridization buffer aliquot so that the final oligonucleotide concentration is 250 nanomolar and vortex the tube vigorously. Suspend the samples in 50 microliters of hybridization buffer that contains the oligonucleotide probes, while avoiding the generation of bubbles. Leave the samples overnight, in the dark, on a heat block at 30 degrees Celsius.

To prepare washing, obtain one new micro-centrifuge tube for each sample to image. Add one milliliter wash buffer to each tube. Add 10 to 15 microliters from the hybridized samples to the new tube that contains the wash buffer.

Pellet the cells by centrifugation. Then, remove the supernatant. Wash the samples two more times.

Re-suspend the pellet in 25 microliters wash buffer. Prepare an agarose gel by adding 150 milligrams of agarose to 10 milliliters of 2X SSC buffer. Microwave the solution for 10 to 15 seconds each time, until agarose fully dissolves.

Then set the tube aside to cool for a few minutes. Next, place a separating, silicone frame on the slide so that the slide’s center remains exposed. Place a 10 millimeter wide, one to two millimeter thick rigid ring in the center of the slide.

Deposit a small amount of gel into the center for sealing and wait a few minutes for the gel to harden. Then add more gel, until a high dome shape is formed. Wait about 10 minutes for the gel to harden and remove the ring.

Deposit 10 microliters of wash bacterial cells onto the gel and seal it with a number zero cover slip slide. Keep the sample in the dark to prevent photo-bleaching. smFiSH measurements of E.coli cells show the level of galK induction in response to variable extra-cellular D-fucose concentrations.

The background signal is shown by a galK deleted strain, or delta galK JW0740 as a negative control. Numerous spots are seen in most cells. Corresponding to single or few galK transcripts when two millimolar D-fucose is added to the bacterial culture.

Whereas, a few spots are seen in the absence of extra-cellular D-fucose. Another example for the application of this protocol is furnished by the sodB gene, with fluorescence and phase contrast images of smFiSH in a wild type or a JW1648 sodB deleted strain. Samples prepared with this protocol may also be imaged using STORM which allows accurate precisioning of transcripts in the cell.

After its development, this technique paved the way for researchers in the field of systems biology to explore the statistics of transcription events in individual cells.