Baus TLU strains bearing translational fusions of mismatch repair. Proteins fused to GFP are streaked out on selective media. The street cells are used for fresh inoculum into liquid culture.
Cells are grown until OD 600 is 0.3 to 0.4, where upon mismatch inducing treatments are applied of desired and cells grown further until reaching OD 600 of 0.5 to 0.8 cells are removed. The membrane stain FM 4 64 is added if desired, and cells are applied to slides with prepared aros pads and allowed to settle after removal of excess media. By aspiration.
A cover slip is applied and cells are visualized by microscopy. Hi, I'm Dr.Andrew Kow from the lab of Dr.Lyle Simmons in the Department of Molecular, cellular and Developmental Biology at the University of Michigan Ann Arbor. Today we'll show you a procedure used to visualize translational fusions of the DNA mismatch repair proteins as well as other repair and ribosomal proteins in bacillus solus.
We use this procedure to study the interaction between the DNA mismatch repair proteins and the ribosomal proteins in bacillus subtles live cells. So let's get started. To begin this procedure, prepare the B sublist strains containing the translational fusion protein you wish to visualize for some strains.
Starting one day prior to imaging is sufficient. Examples of these include strains that grow well when the translational fusion between GFP and the gene of interest is incorporated into the gene's endogenous locus such as MU LGFP. When starting one day prior to imaging, use a sterile stick under sterile conditions to apply the bacteria to selective LB agro medium on plates.
In order to grow single colonies incubate overnight at 30 degrees Celsius the next day, the colonies will be used for a starter inoculum. Some B sublist strains require two days of growth prior to microscopy for high quality images. Specific strains that require two days growth are those that grow poorly due to the translational fusion such as rec A GFP on the first day streak the bacteria on selective LB AED and incubate overnight.
On the second day, perform a serial dilution. First pick a single colony to inoculate 100 microliters of 0.85%saline, then perform four tenfold serial dilution plate 100 microliters of each saline dilution as well as the starting colony and saline on LB aerates containing selective antibiotics incubate the plates overnight, usually at 30 degrees Celsius after the second overnight incubation. Select the dilution plate which has light confluent growth of cells.
These provide a healthy, exponentially growing starter inoculum, potentially yielding excellent imaging results. This is particularly useful for the highest quality imaging of membrane staining. With FM 4 64 on the morning of imaging place 10 milliliters of S seven 50 medium into a 125 milliliter sterile flas.
Thus maintaining at least a tenfold air to medium ratio. Resus the cells in up to two milliliters of S seven 50 medium from the lppl with single colonies or the like confluence cells to obtain an inoculum. Add enough S seven 50 medium cell inoculum to the Erlin Meyer flask containing 10 milliliters of S seven 50 medium so that the culture has an initial OD 600 of about 0.08 to 0.1.
Next, grow each culture at the appropriate temperature for the B sublist train to be imaged. Shaking water baths are preferred with revolutions per minute from 150 to 200. Continue growing the cultures until they reach an OD 600 of about 0.5 to 0.8.
For cultures challenged with DNA damaging or mismatch inducing agents plan the treatment such that the end of the incubation period with the DNA altering agent coincides with the time of reaching the target OD 600. For example, two amino purine, A DNA based analog, which mimics a mismatched base when incorporated into the genome, requires one hour incubation and leads to focus formation of mismatch repair proteins such as mut LGFP. However, REC A GFP focus formation requires a one hour incubation with mitomycin C or some other DNA damage reagent for induction of doublet strand DNA breaks both two amino purine and mitomycin C are added.
When the cultures reach OD 600 of about 0.3 to 0.4 and after the required one hour of growth with either treatment, the cultures will most likely have reached an OD 600 of 0.5 to 0.8 and are ready for imaging. Start by pipetting 300 microliters of cultured cells into a 1.5 milliliter micro centrifuge tube. If imaging of cellular membranes is desired at a one to 1000 dilution of one milligram per milliliter FM 4 64 membrane stain to each sample, a titration of FM 4 64 may be required to achieve the desired fluorescence signal.
A typical titration range is one to 100, one to 1001 to 10, 000. Allow the samples to sit for up to 10 minutes or while the slide is being prepared. If concentration of the cells is required, filter the cells using a 0.2 micrometer filter with a vacuum apparatus.
Then gently wash the cells off the filter with appropriate buffer or medium while the cell samples are incubating. With FM 4 64. Prepare 1%agros with one x zens by adding five milliliters of 10 x zen stock to 45 milliliters of distilled water along with 0.5 grams of agros and microwaving to melt.
Let the temperature of the aros equilibrate in a water bath to approximately 65 degrees Celsius and draw 20 microliters of species. Ends with 1%melted aros into a pipette tip. Aros that is too hot, is difficult to pipette and cold agros will solidify and not enter the pipette tip.
Next, use a multitest 15. Well slide and pipette a single drop of approximately one to two microliters of the aros solution into each slide. Well to form shallow aros pads, the aros will solidify immediately.
The slides are prepared immediately prior to application of the cells to avoid dehydration, which would make the slides unusable. The aros pads should be centered and fill each well but have minimum height to avoid disrupting the cover slip. If droplets are too high or bubble like or if the sample spreads beyond the well boundary, simply swipe the well clean with a kim wipe and apply a new aros pad.
Now that the slide is ready, apply the entire 300 microliter sample which was previously set aside in the micro centrifuge tube to the slide with the sample distributed on the top of each aeros pad. This amount of cells should be sufficient to cover each well on the slide. Allow the slide loaded with cells to sit at room temperature for approximately 10 minutes.
This step will allow for the cells to settle on the agros pad and become immobile and therefore ready for imaging at the end of the 10 minute incubation. Aspirate away the excess medium from each well without removing the pads themselves. Apply the cover slip to the slide pressing firmly and evenly, but gently across the area of the slide check to make sure that the cover slip is against the slide and that the cover slip is not raised above the slide.
Proceed to imaging many different fluorescence microscopes will work well. It is critical to have a 100 x oil immersion lens. The Simmons lab uses an Olympus BX 61 microscope equipped with 1.45 numerical aperture 100 x oil immersion objective lens suitable for total internal reflection, fluorescence microscopy or turf m.
The microscope is also equipped with a CCD cooled camera and LU 200 arch metal light source. Start by bringing the cells into focus using white light. Use the appropriate filter for each flora.
Four for detection of GFP. Use filter excitation four 60 to 500 and emission five 10 to five 60 for detection of FM 4 64. Use filter excitation five 10 to five 60 and emission 5 72 to 6 48.
Capture the image using the appropriate filters for each floor. Four with the slide book 4.2 software. Here are some representative results of imaging the DNA mismatch repair protein MU L, which has been translationally fused to GFP.
The untreated control culture of MU LGFP shows minimal foci with most cells containing only cytoplasmic levels of the mut LGFP fusion protein. However, the majority of cells grown in the presence of two amino purine, which mimics a mismatched base when incorporated into the genome. Have GFP foci indicating the mutal GFP protein is involved in recognition and repair of the mismatched DNA bases.
Similar results are obtained with the REC A GFP strain grown in the presence of the DNA damage inducing drug mitomycin C.Here the majority of cells contain a focus of rec A GFP upon treatment of mitomycin C indicating the rec A GFP has responded to the DNA damage initiating the repair process without mitomycin C treatment pho IR seen. We've just shown you how to visualize repair proteins in live facius subtlest cells. When doing this procedure.
It's important to remember to take your time and bring the cells in a proper focus and doing so may actually take some practice. So that's it. Thanks for watching and good luck with your experiments.
