This protocol allows us to quantify excision statistics on a single-cell basis, as opposed to looking at bulk behavior. At this level, we can track the behavior of individual cells over time, including their TnpA and TnpB levels for cells that excise versus cells that don't, which can allow us to, for example, measure the effects of identified mutational events on growth rate. This method is a simple, low-cost way to measure live cell mutational dynamics.
The general approach is applicable to any system that requires live cell fluorescent imaging. Prepare a slide by boiling M63 with 0.5%glucose and 1.5%agarose in the microwave to melt the agarose. Ensure that the medium is completely melted and well-mixed.
Allow the mixture to cool to 55 degrees Celsius before adding antibiotics and inducers. Place a microscope slide on the workbench. Stack one slide perpendicular to the first.
Ensure that there is a gap equal to one slide thickness between the bottom and top slide. Pipette one milliliter of the M63 agarose mixture into the gap between the slide slowly to create a small gel square. Once the gel has solidified, slide the top slide to remove it.
Trim the agarose pad with a razor blade or knife. Then, pipette 2.5 microliters of the bacterial culture and put the cover slip on top. Seal the space between the slide and the cover slip with epoxy.
Allow the epoxy to dry and the cells to settle onto the agarose pad for one hour at 37 degrees Celsius. Place the prepared sample on a fluorescence microscope in an environment heated and maintained at 37 degrees Celsius. For each wavelength, find the field-of-view containing minimal fluorescence.
Set up a protocol to acquire images in a grid at different wavelengths and at regular time intervals. Transposon excision events result in the reconstitution of the promoter for mCerulean3, allowing the identification of cells undergoing transposon activity by bright blue fluorescence. TnpB enhances transposon excision rate.
Cells expressing accessory protein TnpB experience four to five times higher levels of transposon activity compared to TnpB-negative cells. In TnpB-positive cells, cells undergoing transposon excision events have higher expression levels of mCherry TnpB than the general population. Moreover, for cells undergoing excision events, TnpB-positive cells express only marginally higher levels of venous TnpA-transposase than TnpB-negative cells, which is higher than the yellow fluorescence of the general population.
The most likely place to struggle is in the preparation of the cell culture for the experiment. You should make sure cells are grown to exponential phase and that the slide is inoculated with an appropriate number of cells. Remember to watch out for bubbles when sealing the slide so the agarose gel does not dry out.
