Name: Imaging Mismatch Repair and Cellular Responses to DNA Damage in Bacillus subtilis (Video) | JoVE Journal
Uploaded: 2010-02-08T17:30:00.000+00:00
Duration: 10 min 28 s
Description: Learn to image DNA repair complexes in live Bacillus subtilis cells using GFP-tagged mismatch repair proteins. This protocol enables real-time visualization of cellular responses to DNA damage.

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The authors wish to thank Drs. Philina S. Lee and Alan D. Grossman for initially training L.A.S. in fluorescence microscopy. The authors also thank Drs. Melanie Berkmen and Hajime Kobayashi for help and tips for imaging. This work was supported by start-up funds from the College of Literature, Science &amp; Arts and from the Department of Molecular, Cellular, and Developmental Biology at the University of Michigan.

Growing cultures of cells for microscopy
1. One or two days prior to imaging, prepare the B. subtilis strain containing the translational fusion protein you wish to visualize. Trial rounds of steps 1A and 1B will be necessary to determine which growth condition provides the best images of your strain. In most cases, cells should be imaged during exponential growth phase.
<ol class="ualpha">
<li>For some B. subtilis strains (in particular, strains that grow poorly due to the integration of the translational fusion between the gene of interest and GFP at its endogenous locus), initial growth for two days prior to imaging is necessary for high quality images. On the first day, streak the B. subtilis strain to be imaged on LB agar with selective antibiotic(s) and incubate overnight at 30°C. On the second day, inoculate 100 μl of 0.85% saline with a single colony and perform three 10-fold serial dilutions on LB agar plates (with selective antibiotics), plating 100 μL of each dilution, followed by incubation overnight at 30°C. On the third day, select the dilution plate with light confluent growth of cells. Light confluent growth on a plate will provide the growth medium with a healthy, exponentially growing starter inoculum to potentially yield excellent imaging results (see step 4). This step is particularly useful for the highest quality membrane imaging with the stain FM4-64. </li>
<li>Other B. subtilis strains may simply be applied to selective LB agar medium for single colonies with a sterile stick the day prior to imaging and incubate overnight at 30°C. The following day, cells on this plate will be sufficient to use as an inoculum for imaging (see step 4). NOTE: For B. subtilis, overnight cultures in liquid medium are not appropriate due to a combination of spore formation and quorum responses, which will cause an extended period of lag phase growth 1.</li>
</ol>
2. On the morning of imaging, place 10 mL of S750 1-3 medium into a 125 mL sterile Erlenmeyer flask. 
3. Resuspend the cells in up to 2 mL of S750 medium from the LB agar plate with single colonies or the light confluent cells to obtain an inoculum. 
4. Add enough S750 medium-cell inoculum to the Erlenmeyer flask containing 10 mL S750 medium for a culture with an initial optical density measured at 600 nm (OD600) of ~0.08 to 0.1. At this step it is important to have at least a 10-fold air to medium ratio. Thus, we use 10 to 12.5 mL of inoculated medium in a 125 mL Erlenmeyer flask. 
5. Grow each culture at 30°C until the culture reaches an OD600 ~0.5-0.8. Shaking water baths are preferred with revolutions per minute from 150-200. Cultures challenged with DNA damaging or mismatch inducing agents should be at an early exponential growth phase OD600 such that the cells will reach the target OD600 with the additional period of growth that is required for treatment (see Table 1) 
 For example, 2-aminopurine requires one hour of treatment, and therefore a culture treated with 2-aminopurine should be at OD600~0.3-0.4, such that the one hour of treatment/growth results in an OD600~0.5-0.8, whereby cells are ready for imaging. 
Sample Preparation
1. Pipette 300 μl of cultured cells into a 1.5 mL microcentrifuge tube.
2. If imaging of cellular membranes is desired, add 1:1000 dilution of FM4-64 membrane stain (Invitrogen) to each sample from a 1 mg/mL stock solution. A titration of FM4-64 may be required to achieve the desired fluorescence signal. A typical titration range is 1:100, 1:1000, and 1:10,000.
3. Allow the samples to sit at room temperature for up to ten minutes or while the slide is being prepared with 1% agarose in 1X Spizizens medium as described below (see step 1 of "slide preparation").
4. If concentration of cells is required, we prefer filtering cells using a 0.2 μm filter with a vacuum apparatus. Cells can then be gently washed from the surface of the filter with PBS, another appropriate buffer or medium prior to visualization.
Slide Preparation
1. Prepare 1% agarose with 1X Spizizens. Add 5 mL of 10X Spizizens stock to 45 ml ddH2O along with 0.5 g agarose, and microwave to melt. Hot molten agarose is difficult to pipette, and solidified agarose will not enter the pipette tip. The agarose should be equilibrated in a water bath to a temperature of ~55-65°C for it to enter the pipette tip without solidifying within the tip.
2. Draw 20 μl of Spizizens with 1% melted agarose into the pipette tip. Pipette a single drop (approximately 1-2 μL) of the agarose solution into each slide well (we use 15-well slides, see Table 2) to form shallow agarose pads. The agarose will solidify immediately. Slides must also be made immediately prior to application of the cells. If the agarose pads are left at room temperature alone, they can dehydrate within minutes and become unusable. 
Agarose pads should be centered and fill each well but have minimum height to avoid disrupting the coverslip; if droplets are too high (bubble-like) or if the sample spreads beyond the well boundary, simply swipe the well clean with a Kimwipe and apply a new agarose pad. 
3. Apply the entire 300 μl sample (see step 6) to the slide, distributed on the top of each agarose pad. This amount of cells should be sufficient to cover each well on the slide.
4. Allow the slide loaded with cells to sit at room temperature (23°C) for approximately ten minutes. This step will allow for the cells to settle on the agarose pad becoming immobile and ready for imaging. For specialized needs this temperature may need to be changed, for example, when performing experiments that require a temperature shift4.
5. Aspirate away the excess medium from each well without removing the pads themselves.
6. Apply the coverslip 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.
Visualization of cells
1. Many different fluorescence microscopes will work well. It is critical to have a 100X oil immersion lens. The Simmons Lab uses:
<ul>
 <li>Olympus BX61 microscope equipped with 1,45 NA TIRFM 100X oil immersion objective lens</li>
 <li>Hamamatsu ORCAR2 CCD cooled camera</li>
 <li>Lumen 200 arc metal (Prior) light source. </li>
 <li>For detection of GFP (FITC), filter excitation 460-500 and emission 510-560</li>
 <li>For detection of FM4-64 (TRITC), filter excitation 510-560 and emission 572-648. </li>
 <li>Images were captured using SlideBook 4.2 (Figure 1) </li>
</ul>
2. Bring the cells into focus using white light.
3. Capture the image using appropriate filters for each fluorophore. 
Representative Results
Representative images are shown (Figure 1). GFP foci should be well defined, while FM4-64 staining should be bright and clear 4-7. GFP or membrane images can be pseudo-colored with any color, to allow for the highest quality image to be presented without losing any data.
<img src="/files/ftp_upload/1736/1736fig1.jpg" alt="Bacterial cell microscopy image showing GFP-tagged proteins with 2AP/MMC, fluorescence analysis." data-altupdatedat="1788235205000" data-alt="Figure 1"> Figure 1: Representative GFP images of B. subtilis translational fusion proteins. GFP proteins are shown in green, while FM4-64 membrane staining is shown in red. The white scale bar indicates 3 μm. (A) MutS-GFP [relevant genotype: mutS-gfp (spc), amyE::PspacmutL (cat)] in the presence of 600 μg/mL 2-aminopurine and 1 mM IPTG. FM4-64 signal is not presented in order to more clearly show the MutS-GFP foci. (B) MutL-GFP [relevant genotype: mutL::mutL-GFP (spc)] in the presence of 2-aminopurine. (C) DnaX-GFP [relevant genotype: dnaX::dnaX-GFP (spc)]. (D) RecA-GFP [relevant genotype: recA::recA-GFP (spc)] in the presence of 20 ng/mL mitomycin C. (E) TagC-GFP [relevant genotype: tagC::tagC-GFP (spc)] in the presence of 1 μg/mL mitomycin C. 

<table border="1" cellspacing="0" cellpadding="0">
<tbody>
 <tr>
 <td>Treatment /concentration or amount</td>
 <td>Function</td>
 <td>Time of incubation</td>
 </tr>
 <tr>
 <td>Mitomycin C (MMC) [20 to 150 ng/mL]</td>
 <td>DNA alkylating agent</td>
 <td>1 hour</td>
 </tr>
 <tr>
 <td>2-aminopurine (2-AP) [600 μg/mL]</td>
 <td>mismatch inducing</td>
 <td>1 hour</td>
 </tr>
 <tr>
 <td>Hydroxyurea (HU)</td>
 <td>depletes dNTPs</td>
 <td>3 hours</td>
 </tr>
 <tr>
 <td>ultraviolet light (UV) 20 J/m2</td>
 <td>Thymine-thymine dimers and 6-4 photoproducts</td>
 <td>varies depending on source, usually 20 seconds</td>
 </tr>
 <tr>
 <td>grays (ionizing radiation) 5 to 100 Gy</td>
 <td>Double strand breaks, single strand breaks and base damage sites</td>
 <td>varies depending on source </td>
 </tr>
</tbody>
</table>
Table 1. Summary of DNA damaging and mismatch inducing treatments

<table border="1" cellspacing="0" cellpadding="0">
<tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments (optional)</td>
 </tr>
 <tr>
 <td>Multitest slide, 15-well</td>
 <td>MP Biomedicals</td>
 <td>6041505E</td>
 <td> </td>
 </tr>
 <tr>
 <td>Microscope Cover Glass</td>
 <td>Fisher</td>
 <td>12-544-B</td>
 <td> </td>
 </tr>
 <tr>
 <td>Agarose</td>
 <td>Fisher</td>
 <td>BP160-500</td>
 <td> </td>
 </tr>
 <tr>
 <td>Mitomycin C</td>
 <td>Sigma</td>
 <td>M0503-2MG</td>
 <td>mutagen, wear gloves</td>
 </tr>
 <tr>
 <td>2-Aminopurine</td>
 <td>Sigma</td>
 <td>A3509-250MG</td>
 <td>gloves</td>
 </tr>
 <tr>
 <td>Hydroxyurea</td>
 <td>Sigma</td>
 <td>H8627-25G</td>
 <td>gloves</td>
 </tr>
 <tr>
 <td>FM4-64</td>
 <td>Invitrogen</td>
 <td>T13320</td>
 <td>light sensitive</td>
 </tr>
</tbody>
</table>
Table 2. List of specific reagents and equipment:

<ol><li>Hardwood, C. R., Cutting, S. M. Molecular Biological Methods for Bacillus. , John Wiley and Sons. Chichester. (1990).</li>
<li>Berkmen, M. B., Grossman, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Spatial+and+temporal+organization+of+the+Bacillus+subtilis+replication+cycle%5BTitle%5D)+AND+%22Mol.+Microbiol%22%5BJournal%5D)">Spatial and temporal organization of the Bacillus subtilis replication cycle.</a> Mol. Microbiol. 62, 57-71 (2006).</li>
<li>Simmons, L. A., Grossman, A. D., Walker, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Clp+and+Lon+proteases+occupy+distinct+subcellular+positions+in+Bacillus+subtilis%5BTitle%5D)+AND+%22J.+Bacteriol%22%5BJournal%5D)">Clp and Lon proteases occupy distinct subcellular positions in Bacillus subtilis.</a> J. Bacteriol. 190, 6758-6768 (2008).</li>
<li>Simmons, L. A., Grossman, A. D., Walker, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Replication+is+required+for+the+RecA+localization+response+to+DNA+damage+in+Bacillus+subtilis%5BTitle%5D)+AND+%22Proc.+Natl.+Acad.+Sci.+U+S+A%22%5BJournal%5D)">Replication is required for the RecA localization response to DNA damage in Bacillus subtilis.</a> Proc. Natl. Acad. Sci. U S A. 104, 1360-1365 (2007).</li>
<li>Simmons, L. A., Davies, B. W., Grossman, A. D., Walker, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((b+Clamp+Directs+Localization+of+Mismatch+Repair+in+Bacillus+subtilis%5BTitle%5D)+AND+%22Mol.+Cell%22%5BJournal%5D)">b Clamp Directs Localization of Mismatch Repair in Bacillus subtilis.</a> Mol. Cell. 29, 291-301 (2008).</li>
<li>Simmons, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Comparison+of+responses+to+double-strand+breaks+between+Escherichia+coli+and+Bacillus+subtilis+reveals+different+requirements+for+SOS+induction%5BTitle%5D)+AND+%22J.+Bacteriol%22%5BJournal%5D)">Comparison of responses to double-strand breaks between Escherichia coli and Bacillus subtilis reveals different requirements for SOS induction.</a> J. Bacteriol. 191, 1152-1161 (2009).</li>
<li>Smith, B. T., Grossman, A. D., Walker, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Visualization+of+mismatch+repair+in+bacterial+cells%5BTitle%5D)+AND+%22Mol.+Cell%22%5BJournal%5D)">Visualization of mismatch repair in bacterial cells.</a> Mol. Cell. 8, 1197-1206 (2001).</li>
</ol>

Trial and error are required to find exposure conditions for the highest quality images for each strain; we find that 1 millisecond is appropriate for white light images, while exposures of 100 to 2000 ms are appropriate for GFP (FITC) and FM4-64 (TRITC) images. Exposure time will vary depending on the imaging equipment used. We recommend the use of one strain per 15-well microscope slide for the simplest imaging, as pad quality and diffusion of cells from the pad border could complicate strain differentiation if multipl...

<table><tbody><tr><td colspan="4">Specific solution recipes&lt;sup&gt;1&lt;/sup&gt;:</td></tr><tr><td colspan="4">&lt;strong&gt;10x S7&lt;sub&gt;50&lt;/sub&gt; salts&lt;/strong&gt;&lt;br/&gt; 0.5 M MOPS&lt;br/&gt; 100 mM Ammonium Sulfate&lt;br/&gt; 50 mM Potassium Phosphate Monobasic&lt;br/&gt; Filter sterilize, and wrap S7&lt;sub&gt;50&lt;/sub&gt; media in foil prior to storage</td></tr><tr><td colspan="4">&lt;strong&gt;100x Metals&lt;/strong&gt;&lt;br/&gt; 0.2 M MgCl&lt;sub&gt;2&lt;/sub&gt;&lt;br/&gt; 70 mM CaCl&lt;sub&gt;2&lt;/sub&gt;&lt;br/&gt; 5 mM MnCl&lt;sub&gt;2&lt;/sub&gt;&lt;br/&gt; 0.1 mM ZnCl&lt;sub&gt;2&lt;/sub&gt;&lt;br/&gt; 100 &amp;#x03BC;g/mL Thiamine HCl&lt;br/&gt; 2 mM HCl&lt;br/&gt; 0.5 mM FeCl&lt;sub&gt;3&lt;/sub&gt;*&lt;br/&gt; dH&lt;sub&gt;2&lt;/sub&gt;O to final volume&lt;br/&gt; *FeCl&lt;sub&gt;3&lt;/sub&gt; should be added last, to prevent precipitation.&lt;br/&gt; After filter sterilization, wrap 100x Metal solution in foil.</td></tr><tr><td colspan="4">&lt;strong&gt;S7&lt;sub&gt;50&lt;/sub&gt; media&lt;/strong&gt;&lt;br/&gt; 1x S7&lt;sub&gt;50&lt;/sub&gt; salts&lt;br/&gt; 1x Metal&lt;br/&gt; 1% Glucose&lt;br/&gt; 0.1% Glutamate&lt;br/&gt; 40 &amp;#x03BC;g/mL Tryptophan&lt;br/&gt; 40 &amp;#x03BC;g/mL Phenylalanine&lt;br/&gt; distilled H&lt;sub&gt;2&lt;/sub&gt;O to final volume</td></tr><tr><td colspan="4">&lt;strong&gt;10x Spizizens (grams/L)&lt;/strong&gt;&lt;br/&gt; 151.4 mM Ammonium Sulfate (20g/L)&lt;br/&gt; 803.8 mM Potassium Phosphate Monobasic (140g/L)&lt;br/&gt; 440.9 mM Potassium Phosphate Dibasic (60g/L)&lt;br/&gt; 34.0 mM Sodium Citrate (10g/L)&lt;br/&gt; 16.6 mM MgSO&lt;sub&gt;4&lt;/sub&gt; (2g/L)&lt;br/&gt; dH&lt;sub&gt;2&lt;/sub&gt;O to final volume&lt;br/&gt; Filter sterilize</td></tr></tbody></table>

imaging mismatch repair and cellular responses to dna damage in bacillus subtilis

Imaging Mismatch Repair and Cellular Responses to DNA Damage in Bacillus subtilis

Both prokaryotes and eukaryotes respond to DNA damage through a complex set of physiological changes. Alterations in gene expression, the redistribution of existing proteins, and the assembly of new protein complexes can be stimulated by a variety of DNA lesions and mismatched DNA base pairs. Fluorescence microscopy has been used as a powerful experimental tool for visualizing and quantifying these and other responses to DNA lesions and to monitor DNA replication status within the complex subcellular architecture of a living cell. Translational fusions between fluorescent reporter proteins and components of the DNA replication and repair machinery have been used to determine the cues that target DNA repair proteins to their cognate lesions in vivo and to understand how these proteins are organized within bacterial cells. In addition, transcriptional and translational fusions linked to DNA damage inducible promoters have revealed which cells within a population have activated genotoxic stress responses. In this review, we provide a detailed protocol for using fluorescence microscopy to image the assembly of DNA repair and DNA replication complexes in single bacterial cells. In particular, this work focuses on imaging mismatch repair proteins, homologous recombination, DNA replication and an SOS-inducible protein in Bacillus subtilis. All of the procedures described here are easily amenable for imaging protein complexes in a variety of bacterial species.

A detailed protocol is described for imaging the real time formation of DNA repair complexes in Bacillus subtilis cells.

Both prokaryotes and eukaryotes respond to DNA damage through a complex set of physiological changes. Alterations in gene expression, the redistribution ...

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.

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Learn to image DNA repair complexes in live Bacillus subtilis cells using GFP-tagged mismatch repair proteins. This protocol enables real-time visualization of cellular responses to DNA damage.

Imaging Mismatch Repair and Cellular Responses to DNA Damage in Bacillus subtilis

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