Name: inducing a site specific replication blockage in e. coli using a fluorescent repressor operator system
Uploaded: 2016-08-21T15:00:00
Description: Watch this Scientific Journal Video about Inducing a Site Specific Replication Blockage in E. coli Using a Fluorescent Repressor Operator System at JoVE.com

This work was supported by the Australian Research Council [DP11010246].

1. Blocking Replication with FROS
<ol>
	<li>Inducing the Replication Blockage
	<ol>
		<li>Dilute a fresh overnight culture of an E. coli strain carrying a tetO array and pKM118 to OD600nm = 0.01 in a dilute complex medium (0.1% tryptone, 0.05% yeast extract, 0.1% NaCl, 0.17 M KH2PO4, 0.72 M K2HPO4) with antibiotics as required for selection. 
		Note: Do not add tetracycline for selection as it is not compatible with this system. Ma...

<ol>
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T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replication+fork+blockage+by+transcription+factor-DNA+complexes+in+Escherichia+coli.">Replication fork blockage by transcription factor-DNA complexes in Escherichia coli.</a> Nucleic Acids Res. 34 (18), 5194-5202 (2006).</li><li>Brewer, B. J., Fangman, W. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+localization+of+replication+origins+on+ARS+plasmids+in+S.+cerevisiae.">The localization of replication origins on ARS plasmids in S. cerevisiae.</a> Cell. 51 (3), 463-471 (1987).</li><li>Jeiranian, H. A., Schalow, B. J., Courcelle, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+UV-induced+replication+intermediates+in+E.+coli+using+two-dimensional+agarose-gel+analysis.">Visualization of UV-induced replication intermediates in E. coli using two-dimensional agarose-gel analysis.</a> J Vis Exp. (46), (2010).</li><li>Southern, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+specific+sequences+among+DNA+fragments+separated+by+gel+electrophoresis.">Detection of specific sequences among DNA fragments separated by gel electrophoresis.</a> J Mol Biol. 98 (3), 503-517 (1975).</li><li>Courcelle, J., Donaldson, J. R., Chow, K. H., Courcelle, C. 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J., Bidnenko, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+pathways+process+stalled+replication+forks.">Multiple pathways process stalled replication forks.</a> Proc Natl Acad Sci U S A. 101 (35), 12783-12788 (2004).</li><li>Michel, B., Boubakri, H., Baharoglu, Z., LeMasson, M., Lestini, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recombination+proteins+and+rescue+of+arrested+replication+forks.">Recombination proteins and rescue of arrested replication forks.</a> DNA Repair (Amst). 6 (7), 967-980 (2007).</li><li>Carl, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Escherichia+coli+mutants+with+temperature-sensitive+synthesis+of+DNA.">Escherichia coli mutants with temperature-sensitive synthesis of DNA.</a> Mol Gen Genet. 109 (2), 107-122 (1970).</li><li>Nawotka, K. A., Huberman, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-dimensional+gel+electrophoretic+method+for+mapping+DNA+replicons.">Two-dimensional gel electrophoretic method for mapping DNA replicons.</a> Mol Cell Biol. 8 (4), 1408-1413 (1988).</li><li>Cole, R. S., Levitan, D., Sinden, R. 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During chromosome duplication, the replication machinery will encounter various impediments that prevent its progress. To ensure the entire single-origin chromosome is replicated, bacteria have numerous pathways for repair of the DNA that then enables the replisome to be reloaded20,21. Lesions, single stranded breaks, double stranded breaks and proteins tightly bound to the DNA may each be dealt with using a dedicated pathway, although there is likely to be significant overlap in these pathways. The most commo...

During DNA replication, the replisome faces obstacles on the DNA that impair its progression. DNA damage including lesions and gaps as well as aberrant structures can prevent the replisome from proceeding1. Recently, it has been found that proteins bound to the DNA are the most common source of impediment to replication fork progression2. The knowledge of the events following the encounter of the replisome with a nucleoprotein block has previously been limited by the inability to induce such a block in the chromosome of a living cell at a known location. In vitro analysis has enhanced our understanding of the kinetic behavior of an active replisome when it meets a nucleoprotein blockage3, as well as the mechanistic details of the replisome itself4,5. Current understanding of the repair of replication is generally undertaken with UV as the damaging agent and studied using plasmid DNA in vivo6-8. While the proteins that may be involved in repair of DNA after it encounters an in vivo nucleoprotein block are generally understood from these studies, whether there are variations in the molecular events within the repair pathways owing to the distinct cause in the replication block is still yet to be determined.
Here, we describe a system that allows a nucleoprotein block to be established in a specific location of the chromosome using a Fluorescent Repressor Operator System (FROS). We utilize a strain of E. coli that has had an array of 240 tetO sites incorporated into the chromosome9. Each tetO site within the array has a 10 bp random sequence flanking it to increase the stability of the array by preventing RecA-mediated recombination within the array. This array, and variations of it, were originally used to understand E. coli chromosome dynamics10,11 but were then adapted to prevent in vivo replication12. The array has been found to be stably maintained and to block close to 100% of replication forks when bound by TetR10,12. The use of the similar lacO array in vitro has found as few as 22 sites were sufficient to block 90% of replication, although this shorter array was less effective in vivo13. To adapt the array to create a nucleoprotein blockage, the repressor protein must be highly overproduced under optimized conditions where it then binds to the array to create a roadblock. The formation of the blockage, and its subsequent release, can be monitored through the use of fluorescence microscopy if a fluorescently tagged variant of the Tet repressor is used. The status of replication in each cell is indicated by the number of foci seen, where a single focal point means only one copy of the array is present within the cell and multiple foci are indicative of active replication. This active replication is enabled when the nucleoprotein blockage is reversed by the addition of the gratuitous inducer that decreases the binding affinity of TetR for the operator site sufficiently for the replisome to proceed through the array. The repressor protein is still able to bind to the DNA with high enough affinity that the now multiple copies of the array can be visualized.
More intricate details of the events at the nucleoprotein blockage can be discovered using neutral-neutral two-dimensional agarose gel electrophoresis and Southern hybridization14-16. These techniques allow the analysis of DNA structures across the population. The replication intermediates that are formed during the event, and potentially remain unrepaired, can be visualized. By varying the restriction enzyme and probe utilized, the intermediates can be visualized not only in the array region but also upstream of the array when the replication fork regresses17,18. The regression takes place subsequent to the replisome dissociation; the leading and lagging nascent strands separate from the template strands and anneal to each other as the template strands concurrently re-anneal resulting in a four-way DNA structure (a Holliday junction).
Using this system it has been shown that the replication fork is not stable when it encounters this block18. In addition, temperature sensitive derivatives of replisome components can be utilized to prevent reloading of the replication fork once it has collapsed. Once a block is established, the strain can be shifted to a non-permissive temperature to ensure a synchronous deactivation of the replisome and a controlled prevention of reloading. This temperature-induced deactivation ensures all of the forks within the population have collapsed at a given time and allows the assessment of what happens when the replisome collapses, how the DNA is processed, and what is required to restart the process of DNA replication.
An advantage of the system described here is that the nucleoprotein block is fully reversible; therefore, the ability of the cells to recover from the nucleoprotein block is able to be followed. The addition of anhydrotetracycline to the cells will relieve the tight binding of the repressor, allowing a replication fork to proceed through and the cell to regain viability. The relief of the blockage can be visualized by neutral-neutral two-dimensional agarose gel electrophoresis after 5 min, and by microscopy within 10 min. Furthermore, viability analysis can reveal the ability of the strain to recover from the replication blockage and continue to proliferate.
By altering the genetic background of the strains used in the experimental procedure described here, the repair pathways for this type of blockage can be elucidated.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Tryptone</td>
			<td>Sigma-Aldrich</td>
			<td>16922</td>
			<td>Growth media component</td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>VWR</td>
			<td>27810.364</td>
			<td>Growth media component</td>
		</tr>
		<tr>
			<td>Yeast extract</td>
			<td>Sigma-Aldrich</td>
			<td>92144</td>
			<td>Growth media component</td>
		</tr>
		<tr>
			<td>Potassium phosphate monobasic</td>
			<td>Sigma-Aldrich</td>
			<td>P9791</td>
			<td>Growth media component; Potassium buffer component</td>
		</tr>
		<tr>
			<td>Potassium phosphate dibasic</td>
			<td>Sigma-Aldrich</td>
			<td>P3786</td>
			<td>Growth media component; Potassium buffer component</td>
		</tr>
		<tr>
			<td>L-Arabinose</td>
			<td>Sigma-Aldrich</td>
			<td>A3256</td>
			<td>For induction of TetR-YFP production</td>
		</tr>
		<tr>
			<td>Anhydrotetracycline hydrochloride</td>
			<td>Sigma-Aldrich</td>
			<td>37919</td>
			<td>Release of replication bloackage</td>
		</tr>
		<tr>
			<td>Axioskop 2 Fluorescence microscope</td>
			<td>Zeiss</td>
			<td>452310</td>
			<td>Visualization of cells</td>
		</tr>
		<tr>
			<td>eYFP filter set</td>
			<td>Chroma Technology</td>
			<td>41028</td>
			<td>Visualization of YFP</td>
		</tr>
		<tr>
			<td>CCD camera</td>
			<td>Hamamatsu</td>
			<td>Orca-AG</td>
			<td>Visualization of cells</td>
		</tr>
		<tr>
			<td>MetaMorph&#160; Software (Molecular Devices)</td>
			<td>SDR Scientific</td>
			<td>31282</td>
			<td>Version 7.8.0.0 used in the preparation of this manuscript</td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Bioline</td>
			<td>BIO-41025</td>
			<td>For agarose plugs and gel electrophoresis</td>
		</tr>
		<tr>
			<td>Original Glass Water Repellent (Rain-X)</td>
			<td>Autobarn</td>
			<td>DIO1470</td>
			<td>For agarose plug manufacture</td>
		</tr>
		<tr>
			<td>TRIS</td>
			<td>VWR</td>
			<td>VWRC103157P</td>
			<td>TE, TBE buffer component</td>
		</tr>
		<tr>
			<td>Ethylene diaminetetraacetic acid</td>
			<td>Ajax Finechem</td>
			<td>AJA180</td>
			<td>0.5 M EDTA disodium salt solution adjusted to pH 8.0 with NaOH.</td>
		</tr>
		<tr>
			<td>Sodium azide</td>
			<td>Sigma-Aldrich</td>
			<td>S2002</td>
			<td>Bacteriostatic agent</td>
		</tr>
		<tr>
			<td>Hydrochloric acid</td>
			<td>Sigma-Aldrich</td>
			<td>258148</td>
			<td>TE buffer component</td>
		</tr>
		<tr>
			<td>Sodium deoxycholate</td>
			<td>Sigma-Aldrich</td>
			<td>D6750</td>
			<td>Cell lysis buffer component</td>
		</tr>
		<tr>
			<td>N-Lauroylsarcosine sodium salt (Sarkosyl)</td>
			<td>Sigma-Aldrich</td>
			<td>L5125</td>
			<td>Cell lysis and ESP buffer component</td>
		</tr>
		<tr>
			<td>Rnase A</td>
			<td>Sigma-Aldrich</td>
			<td>R6513</td>
			<td>Cell lysis buffer component</td>
		</tr>
		<tr>
			<td>Lysozyme</td>
			<td>Amresco</td>
			<td>6300</td>
			<td>Cell lysis buffer component</td>
		</tr>
		<tr>
			<td>Proteinase K</td>
			<td>Amresco</td>
			<td>AM0706</td>
			<td>ESP buffer component</td>
		</tr>
		<tr>
			<td>Sub-Cell Model 192 Cell</td>
			<td>BioRad</td>
			<td>1704507</td>
			<td>Electrophoresis system</td>
		</tr>
		<tr>
			<td>UV transilluminator 2000</td>
			<td>BioRad</td>
			<td>1708110</td>
			<td>Visualization of DNA</td>
		</tr>
		<tr>
			<td>Ethidium Bromide</td>
			<td>BioRad</td>
			<td>1610433</td>
			<td>Visualization of DNA</td>
		</tr>
		<tr>
			<td>Boric acid</td>
			<td>VWR</td>
			<td>PROL20185.360</td>
			<td>TBE component</td>
		</tr>
		<tr>
			<td>Hybond-XL nylon memrbane</td>
			<td>Amersham</td>
			<td>RPN203S</td>
			<td>Zeta-Probe Memrbane (BioRad 1620159) can also be used</td>
		</tr>
		<tr>
			<td>3MM Whatman chromatography paper</td>
			<td>GE Healthcare Life Sciences</td>
			<td>3030690</td>
			<td>Southern blotting</td>
		</tr>
		<tr>
			<td>HL-2000 Hybrilinker</td>
			<td>UVP</td>
			<td>95-0031-01/02</td>
			<td>Crosslinking of DNA and hybridization</td>
		</tr>
		<tr>
			<td>Deoxyribonucleic acid from salmon sperm</td>
			<td>Sigma-Aldrich</td>
			<td>31149</td>
			<td>Hybridization buffer component</td>
		</tr>
		<tr>
			<td>Sodium hydroxide</td>
			<td>Sigma-Aldrich</td>
			<td>S5881</td>
			<td>Denaturation buffer component</td>
		</tr>
		<tr>
			<td>Trisodium citrate dihydrate</td>
			<td>VWR</td>
			<td>PROL27833.363</td>
			<td>Transfer buffer</td>
		</tr>
		<tr>
			<td>Sodium dodecyl sulphate (SDS)</td>
			<td>Amresco</td>
			<td>227</td>
			<td>Wash buffer component</td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Sigma-Aldrich</td>
			<td>A7906</td>
			<td>Hybridization buffer component</td>
		</tr>
		<tr>
			<td>Random Hexamer Primers</td>
			<td>Bioline</td>
			<td>BIO-38028</td>
			<td></td>
		</tr>
		<tr>
			<td>Klenow fragment</td>
			<td>New England BioLabs</td>
			<td>M0212L</td>
			<td></td>
		</tr>
		<tr>
			<td>dNTP Set</td>
			<td>Bioline</td>
			<td>BIO-39025</td>
			<td></td>
		</tr>
		<tr>
			<td>Adenosine 5&#8217;-triphosphate-32P-ATP</td>
			<td>PerkinElmer</td>
			<td>BLU502A</td>
			<td></td>
		</tr>
		<tr>
			<td>Storage Phosphor Screen</td>
			<td>GE Healthcare Life Sciences</td>
			<td>GEHE28-9564-76</td>
			<td>BAS-IP MS 3543 E multipurpose standard 35x43cm screen</td>
		</tr>
		<tr>
			<td>Typhoon FLA 7000</td>
			<td>GE Healthcare Life Sciences</td>
			<td>28-9558-09</td>
			<td>Visualization of blot</td>
		</tr>
		<tr>
			<td>Hybridization bottle</td>
			<td>UVP</td>
			<td>07-0194-02</td>
			<td>35 x 300mm</td>
		</tr>
	</tbody>
</table>

inducing a site specific replication blockage in e. coli using a fluorescent repressor operator system

Obstacles present on DNA, including tightly-bound proteins and various lesions, can severely inhibit the progression of the cell&#39;s replication machinery. The stalling of a replisome can lead to its dissociation from the chromosome, either in part or its entirety, leading to the collapse of the replication fork. The recovery from this collapse is a necessity for the cell to accurately complete chromosomal duplication and subsequently divide. Therefore, when the collapse occurs, the cell has evolved diverse mechanisms that take place to restore the DNA fork and allow replication to be completed with high fidelity. Previously, these replication repair pathways in bacteria have been studied using UV damage, which has the disadvantage of not being localized to a known site. This manuscript describes a system utilizing a Fluorescence Repressor Operator System (FROS) to create a site-specific protein block that can induce the stalling and collapse of replication forks in Escherichia coli. Protocols detail how the status of replication can be visualized in single living cells using fluorescence microscopy and DNA replication intermediates can be analyzed by 2-dimensional agarose gel electrophoresis. Temperature sensitive mutants of replisome components (e.g. DnaBts) can be incorporated into the system to induce a synchronous collapse of the replication forks. Furthermore, the roles of the recombination proteins and helicases that are involved in these processes can be studied using genetic knockouts within this system.

The FROS is an inducible, site-specific nucleoprotein block that enables replication intermediates to be visualized in living cells12,18. A general experimental design for sampling cells is illustrated in Figure 1. The timing of the sampling and variations in genetic background make this a versatile system for studying the repair of such a block. The schematic illustrates how temperature sensitive mutants, such as dnaBts and dnaCts that have b...

Watch this Scientific Journal Video about Inducing a Site Specific Replication Blockage in E. coli Using a Fluorescent Repressor Operator System at JoVE.com

Inducing a Site Specific Replication Blockage in E. coli Using a Fluorescent Repressor Operator System

We describe here a system utilizing a site-specific, reversible in vivo protein block to stall and collapse replication forks in Escherichia coli. The establishment of the replication block is evaluated by fluorescence microscopy and neutral-neutral 2-dimensional agarose gel electrophoresis is used to visualize replication intermediates.

Inducing a Site Specific Replication Blockage in E. coli Using a Fluorescent Repressor Operator System

Obstacles present on DNA, including tightly-bound proteins and various lesions, can severely inhibit the progression of the cell&#39;s replication ...

The overall goal of this experiment is to stall a replication fork at a nucleoprotein block and observe the DNA structures at the site of the block for the purpose of studying DNA repair pathways. This method can help answer key questions about DNA replication and repair, such as how the DNA is processed when the cell's replication apparatus encounters a protein roadblock. The main advantage of this technique is that we can reversibly stall a replication fork at a specific location on the chromosome across an entire population of living cells.Demonstrating this procedure will be the following members of my laboratory, Dr.Karla Mettrick, and graduate student, Georgia Weaver, and Tayla-Ann Corocher. An E.coli strain carrying a tetracycline operator array in the pKM1 plasmid is used for this procedure. pKM1 encodes for TetR-YFP, an inducible yellow fluorescent protein-tagged tetracycline repressor protein.Dilute a fresh overnight culture of this E.coli strain to an optical density at 600 nanometers of 0.01 in a dilute complex medium with antibiotics as required for selection. Grow the culture at 30 degrees celsius with shaking to an optical density at 600 nanometers of 0.05 to 0.1. Remove a 10 milliliter sample to serve as the uninduced control.Add 0.01%arabinose to the remaining culture to induce the production of TetR-YFP from pKM1. Continue to grow both the uninduced and induced cultures at 30 degrees celsius with shaking. After one hour, check for the presence of a single focal point within each cell of the induced culture using fluorescence microscopy.If the induced cells are confirmed blocked, indicated by more than 70%of cells having a single focus, record the optical density and remove 7.5 milliliters of the sample for analysis by two-dimensional gel electrophoresis. Remove an equivalent sample from the uninduced control culture. Prior to fluorescence microscopy, prepare the agarose pads for preventing movement of the cells during visualization.For each agarose pad, pipette 500 microliters of molten agarose onto a microscope slide and overlay the coverslip immediately before the agarose solidifies. Store the slides between wet tissues at four degrees celsius until needed. When it is time to check the cells, remove the coverslip from the agarose pad and pipette 10 microliters of bacterial culture onto the center of the pad.After approximately five minutes, when the agarose pad has dried, replace the coverslip. Place a drop of immersion oil onto the coverslip and place the slide under the optics. Visualize the cells using phase microscopy at 100x magnification.On the Acquire dialog box within the imaging software, set Exposure Time to 100 milliseconds. Select Acquire to capture the image. Do not move the stage.In the Acquired dialog box, select YFP as the illumination for the external shutter linked to the camera. Set the Exposure Time to 1, 000 milliseconds. Turn off the stage light and select Acquire to capture the fluorescence image.To begin this procedure, add sodium azide to previously-collected culture samples to a final concentration of 0.1%and incubate on ice for at least five minutes. Centrifuge the cells at 5, 000 times g for ten minutes. Discard the supernatant.Resuspend each cell pellet in 200 microliters of PIV buffer and transfer the cells to a microfuge tube. Microcentrifuge to pellet the cells and discard the supernatant. Treat a microscope slide with 40 microliters of an appropriate glass water repellent or silicone solution.Rub the slide with a tissue until it is dry. Resuspend the cells with the lowest cell density in 50 microliters of PIV and place at 50 degrees celsius in a heat block. For all other samples, adjust the volumes of PIV so the final cell density is the same in each tube.Add an equal volume of freshly-prepared 0.8%Agarose solution in PIV to the samples. Keep the samples in the heat block to ensure they are above 50 degrees celsius to prevent solidification. Place 20 microliter drops of the cell-agarose suspension on the treated slide to produce plugs that are hemispherical.When the plugs have solidified, gently slide all of the plugs from one sample into a single microfuge tube and add one milliliter of cell lysis buffer. Incubate at 37 degrees celsius for two hours. After two hours, remove the cell lysis buffer and add 1 milliliter of EDTA-Sarkosyl-Proteinase K or ESP solution.Incubate at 50 degrees celsius overnight or until the plugs are transparent. Once the plugs are transparent, remove the ESP buffer and transfer the plugs to a 15-milliliter tube. Add 12 milliliters of TE buffer and let stand for 30 minutes.Wash the plugs a total of five times with TE buffer. Store the plugs at four degrees celsius in one milliliter of TE buffer. To analyze the replication intermediates, the DNA in the agarose plugs is digested with an appropriate restriction enzyme for the region of interest.In this example, EcoRV cuts the DNA immediately before the array, within the array, and after the array, to give 5.5 kb and 6.7 kb fragments in the region of interest. To begin this procedure, transfer an agarose plug to a fresh microfuge tube and add 150 microliters of restriction enzyme buffer. Add 25 to 100 units of restriction enzyme and digest at 37 degrees celsius for six to eight hours.Prepare a 0.4%agarose gel at four degrees celsius. Pour 300 milliliters of molten agarose into a gel tray of approximately 25 by 25 centimeters. Insert a comb to make the wells approximately the same width as the diameter of the plug.When the gel is set, remove the comb and move the gel to room temperature. Using an inoculation loop, slide one of the digested agarose plugs into a well, positioning the flat side of the plug against the side of the well where the DNA will enter the gel. Insert a single plug in the same manner into every second well.Pipette molten 0.4%agarose into the wells to seal the plugs in position. Load a one kb DNA ladder in an empty well, leaving a gap between the ladder and samples. Run the gel overnight in 1xTBE at room temperature.On the following day, stain the gel in a water bath containing 0.3 micrograms per milliliter of ethidium bromide for 20 minutes. Visualize the DNA with a long-wave UV transilluminator and cut out each lane fragment with a straight even cut minimizing the inclusion of excess agarose on either side of the lane. Place the excised gel lane in a gel tray at 90 degrees to the direction of DNA migration.The next step is to prepare 300 milliliters of agarose for the second dimension gel. When the agarose is cooled to 50 degrees celsius, pipette the molten agarose around the gel slices to solidify their position. Pour the remaining agarose into the tray to a depth that is at least level with the gel slices.Once the gel is solidified, electrophorese it in 1xTBE containing 0.3 micrograms per milliliter of ethidium bromide at four degrees celsius until the DNA has migrated approximately 10 centimeters. Excise the blocks where the genomic DNA is present including the gel above it, to include the DNA that is not visible. The DNA within the gel is subsequently visualized by Southern hybridization as described in the text protocol.In this system, a replication block was confirmed by a majority of cells containing one focus corresponding to one copy of the array within the cell. The addition of anhydrotetracycline reversed the block and subsequent duplication of the array was visualized as the accumulation of cells with multiple foci. Cells with blocked replication also showed growth inhibition that was reversed by subsequent growth in the presence of anhydrotetracycline.To analyze the replication intermediates, the DNA was electrophoresed in the first dimension. The DNA of interest was then excised and a second dimension electrophoresis resulted in a diagonal of linear DNA. Southern hybridization of the array DNA revealed two spots in the unblocked sample corresponding to the expected 5.5kb and 6.7kb fragments of the array.The blocked sample showed a decrease in the 5.5kb spot and the addition of an elliptical signal indicating an accumulation of Y-shaped DNA. Addition of anhydrotetracycline eliminated the Y-shaped DNA signal. Another common intermediate is the Holliday junction visualized as a cone signal at the top of the Y-arc and a spike from the linear DNA at the end of the Y-arc.Once mastered, this technique can be done in eight days if it is preformed properly. While a 2D procedure, it is important to remember that the quality of the DNA plugs is critical for the optimal visualization of the DNA structures.

inducing-site-specific-replication-blockage-e-coli-using-fluorescent

Research

JoVE Journal

Biology

Transformation of Plasmid DNA into E. coli Using the Heat Shock Method

Transformation of plasmid DNA into E. coli using the heat shock method is a basic technique of molecular biology. It consists of inserting a foreign plasmid or ligation product into bacteria. This video protocol describes the traditional method of transformation using commercially available chemically competent bacteria from Genlantis. After a short incubation in ice, a mixture of chemically competent bacteria and DNA is placed at 42&deg;C for 45 seconds (heat shock) and then placed back in ice. SOC media is added and the transformed cells are incubated at 37&deg;C for 30 min with agitation. To be assured of isolating colonies irrespective of transformation efficiency, two quantities of transformed bacteria are plated. This traditional protocol can be used successfully to transform most commercially available competent bacteria. The turbocells from Genlantis can also be used in a novel 3-minute transformation protocol, described in the instruction manual.

Transformation of plasmid DNA into E. coli using the heat shock method is a basic technique of molecular biology. It consists of inserting a foreign ...

Principles of Site-Specific Recombinase (SSR) Technology

Site-specific recombinase (SSR) technology allows the manipulation of gene structure to explore gene function and has become an integral tool of molecular biology. Site-specific recombinases are proteins that bind to distinct DNA target sequences. The Cre/lox system was first described in bacteriophages during the 1980's. Cre recombinase is a Type I topoisomerase that catalyzes site-specific recombination of DNA between two loxP (locus of X-over P1) sites. The Cre/lox system does not require any cofactors. LoxP sequences contain distinct binding sites for Cre recombinases that surround a directional core sequence where recombination and rearrangement takes place. When cells contain loxP sites and express the Cre recombinase, a recombination event occurs. Double-stranded DNA is cut at both loxP sites by the Cre recombinase, rearranged, and ligated ("scissors and glue"). Products of the recombination event depend on the relative orientation of the asymmetric sequences.
SSR technology is frequently used as a tool to explore gene function. Here the gene of interest is flanked with Cre target sites loxP ("floxed"). Animals are then crossed with animals expressing the Cre recombinase under the control of a tissue-specific promoter. In tissues that express the Cre recombinase it binds to target sequences and excises the floxed gene. Controlled gene deletion allows the investigation of gene function in specific tissues and at distinct time points. Analysis of gene function employing SSR technology --- conditional mutagenesis -- has significant advantages over traditional knock-outs where gene deletion is frequently lethal.

Principles of Site-Specific Recombinase SSR Technology

The advent of site-specific recombinase (SSR) technology and the Cre/lox system has led to numerous advances in molecular biology, and has proven itself as a valuable tool for assessing gene function in transgenic animals. This interview discusses the mechanism of site specific recombination by Cyclization recombinase (Cre) and how the use of this enzyme has led to the development of conditional mutagenesis, which has significant advantages over traditional knock out strategies.

Site-specific recombinase (SSR) technology allows the manipulation of gene structure to explore gene function and has become an integral tool of molecular ...

Visualization of UV-induced Replication Intermediates in E. coli using Two-dimensional Agarose-gel Analysis

Inaccurate replication in the presence of DNA damage is responsible for the majority of cellular rearrangements and mutagenesis observed in all cell types and is widely believed to be directly associated with the development of cancer in humans. DNA damage, such as that induced by UV irradiation, severely impairs the ability of replication to duplicate the genomic template accurately. A number of gene products have been identified that are required when replication encounters DNA lesions in the template. However, a remaining challenge has been to determine how these proteins process lesions during replication in vivo. Using Escherichia coli as a model system, we describe a procedure in which two-dimensional agarose-gel analysis can be used to identify the structural intermediates that arise on replicating plasmids in vivo following UV-induced DNA damage. This procedure has been used to demonstrate that replication forks blocked by UV-induced damage undergo a transient reversal that is stabilized by RecA and several gene products associated with the RecF pathway. The technique demonstrates that these replication intermediates are maintained until a time that correlates with the removal of the lesions by nucleotide excision repair and replication resumes.

Visualization of UV-induced Replication Intermediates in E. coli using Two-dimensional Agarose-gel Analysis

We present a procedure by which two-dimensional agarose-gel analysis can be used to identify the structure of replication intermediates that occur following UV irradiation.

Inaccurate replication in the presence of DNA damage is responsible for the majority of cellular rearrangements and mutagenesis observed in all cell types ...

Mutagenesis and Functional Selection Protocols for Directed Evolution of Proteins in E. coli

The efficient generation of genetic diversity represents an invaluable molecular tool that can be used to label DNA synthesis, to create unique molecular signatures, or to evolve proteins in the laboratory. Here, we present a protocol that allows the generation of large (>1011) mutant libraries for a given target sequence. This method is based on replication of a ColE1 plasmid encoding the desired sequence by a low-fidelity variant of DNA polymerase I (LF-Pol I). The target plasmid is transformed into a mutator strain of E. coli and plated on solid media, yielding between 0.2 and 1 mutations/kb, depending on the location of the target gene. Higher mutation frequencies are achieved by iterating this process of mutagenesis. Compared to alternative methods of mutagenesis, our protocol stands out for its simplicity, as no cloning or PCR are involved. Thus, our method is ideal for mutational labeling of plasmids or other Pol I templates or to explore large sections of sequence space for the evolution of activities not present in the original target. The tight spatial control that PCR or randomized oligonucleotide-based methods offer can also be achieved through subsequent cloning of specific sections of the library. Here we provide protocols showing how to create a random mutant library and how to establish drug-based selections in E. coli to identify mutants exhibiting new biochemical activities.

Mutagenesis and Functional Selection Protocols for Directed Evolution of Proteins in E. coli

Here we demonstrate a simple protocol to create a random mutant library for a given target sequence. We show how this method, which is performed in vivo in Escherichia coli, can be coupled with functional selections to evolve new enzymatic activities.

The efficient generation of genetic diversity represents an invaluable molecular tool that can be used to label DNA synthesis, to create unique molecular ...

Visualizing Bacteria in Nematodes using Fluorescent Microscopy

Symbioses, the living together of two or more organisms, are widespread throughout all kingdoms of life. As two of the most ubiquitous organisms on earth, nematodes and bacteria form a wide array of symbiotic associations that range from beneficial to pathogenic 1-3. One such association is the mutually beneficial relationship between Xenorhabdus bacteria and Steinernema nematodes, which has emerged as a model system of symbiosis 4. Steinernema nematodes are entomopathogenic, using their bacterial symbiont to kill insects 5. For transmission between insect hosts, the bacteria colonize the intestine of the nematode's infective juvenile stage 6-8. Recently, several other nematode species have been shown to utilize bacteria to kill insects 9-13, and investigations have begun examining the interactions between the nematodes and bacteria in these systems 9.
We describe a method for visualization of a bacterial symbiont within or on a nematode host, taking advantage of the optical transparency of nematodes when viewed by microscopy. The bacteria are engineered to express a fluorescent protein, allowing their visualization by fluorescence microscopy. Many plasmids are available that carry genes encoding proteins that fluoresce at different wavelengths (i.e. green or red), and conjugation of plasmids from a donor Escherichia coli strain into a recipient bacterial symbiont is successful for a broad range of bacteria. The methods described were developed to investigate the association between Steinernema carpocapsae and Xenorhabdus nematophila 14. Similar methods have been used to investigate other nematode-bacterium associations 9,15-18and the approach therefore is generally applicable.
The method allows characterization of bacterial presence and localization within nematodes at different stages of development, providing insights into the nature of the association and the process of colonization 14,16,19. Microscopic analysis reveals both colonization frequency within a population and localization of bacteria to host tissues 14,16,19-21. This is an advantage over other methods of monitoring bacteria within nematode populations, such as sonication 22or grinding 23, which can provide average levels of colonization, but may not, for example, discriminate populations with a high frequency of low symbiont loads from populations with a low frequency of high symbiont loads. Discriminating the frequency and load of colonizing bacteria can be especially important when screening or characterizing bacterial mutants for colonization phenotypes 21,24. Indeed, fluorescence microscopy has been used in high throughput screening of bacterial mutants for defects in colonization 17,18, and is less laborious than other methods, including sonication 22,25-27and individual nematode dissection 28,29.

To study the mutualism between Xenorhabdus bacteria and Steinernema nematodes, methods were developed to monitor bacterial presence and location within nematodes. The experimental approach, which can be applied to other systems, entails engineering bacteria to express the green fluorescent protein and visualizing, using fluorescence microscopy bacteria within the transparent nematode.

 
Symbioses, the living together of two or more organisms, are widespread throughout all kingdoms of life. As two of the most ubiquitous organisms on ...

The Logic, Experimental Steps, and Potential of Heterologous Natural Product Biosynthesis Featuring the Complex Antibiotic Erythromycin A Produced Through E. coli

The heterologous production of complex natural products is an approach designed to address current limitations and future possibilities. It is particularly useful for those compounds which possess therapeutic value but cannot be sufficiently produced or would benefit from an improved form of production. The experimental procedures involved can be subdivided into three components: 1) genetic transfer; 2) heterologous reconstitution; and 3) product analysis. Each experimental component is under continual optimization to meet the challenges and anticipate the opportunities associated with this emerging approach.
Heterologous biosynthesis begins with the identification of a genetic sequence responsible for a valuable natural product. Transferring this sequence to a heterologous host is complicated by the biosynthetic pathway complexity responsible for product formation. The antibiotic erythromycin A is a good example. Twenty genes (totaling &gt;50 kb) are required for eventual biosynthesis. In addition, three of these genes encode megasynthases, multi-domain enzymes each ~300 kDa in size. This genetic material must be designed and transferred to E. coli for reconstituted biosynthesis. The use of PCR isolation, operon construction, multi-cystronic plasmids, and electro-transformation will be described in transferring the erythromycin A genetic cluster to E. coli.
Once transferred, the E. coli cell must support eventual biosynthesis. This process is also challenging given the substantial differences between E. coli and most original hosts responsible for complex natural product formation. The cell must provide necessary substrates to support biosynthesis and coordinately express the transferred genetic cluster to produce active enzymes. In the case of erythromycin A, the E. coli cell had to be engineered to provide the two precursors (propionyl-CoA and (2S)-methylmalonyl-CoA) required for biosynthesis. In addition, gene sequence modifications, plasmid copy number, chaperonin co-expression, post-translational enzymatic modification, and process temperature were also required to allow final erythromycin A formation.
Finally, successful production must be assessed. For the erythromycin A case, we will present two methods. The first is liquid chromatography-mass spectrometry (LC-MS) to confirm and quantify production. The bioactivity of erythromycin A will also be confirmed through use of a bioassay in which the antibiotic activity is tested against Bacillus subtilis. The assessment assays establish erythromycin A biosynthesis from E. coli and set the stage for future engineering efforts to improve or diversify production and for the production of new complex natural compounds using this approach.

The Logic, Experimental Steps, and Potential of Heterologous Natural Product Biosynthesis Featuring the Complex Antibiotic Erythromycin A Produced Through E. coli

The heterologous biosynthesis of erythromycin A through E. coli includes the following experimental steps: 1) genetic transfer; 2) heterologous reconstitution; and 3) product analysis. Each step will be explained in the context of the motivation, potential, and challenges in producing therapeutic natural products using E. coli as a surrogate host.

The heterologous production of complex natural products is an approach designed to address current limitations and future possibilities.  It is ...

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

A streamlined approach to screening for the expression of recombinant membrane proteins in Escherichia coli based on fusion to green fluorescent protein is presented.

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and ...

Detection of Intracellular Gene Expression in Live Cells of Murine, Human and Porcine Origin Using Fluorescence-labeled Nanoparticles

The reprogramming of somatic cells to induced pluripotent stem cells (iPS) has successfully been performed in different mammalian species including mouse, rat, human, pig and others. The verification of iPS clones mainly relies on the detection of the endogenous expression of different pluripotency genes. These genes mostly represent transcription factors which are located in the cell nucleus. Traditionally, the proof of their endogenous expression is supplied by immunohistochemical staining after fixation of the cells. This approach requires replicate cultures of each clone at this early stage to preserve validated clones for further experiments. The present protocol describes an approach with gene-specific nanoparticles which allows the evaluation of intracellular gene expression directly in live cells by fluorescence. The nanoparticles consist of a central gold particle coupled to a capture strand carrying a sequence complementary to the target mRNA as well as a quenched reporter strand. These nanoparticles are actively endocytosed and the target mRNA displaces the reporter strand which then start to fluoresce. Therefore, specific target gene expression can be detected directly under the microscope. In addition, the emitted fluorescence allows the identification, isolation and enrichment of cells expressing a specific gene by flow cytometry. This method can be applied directly to live cells in culture without any manipulation of the target cells.

This manuscript describes a novel technique which allows the detection of intracellular gene expression after endocytosis of fluorescence-labeled nanoparticles directly in live cells. The method does not require manipulation of the cells and is not restricted with respect to the target gene or species.

The reprogramming of somatic cells to induced pluripotent stem cells (iPS) has successfully been performed in different mammalian species including mouse, ...

Validation of a Mouse Model to Disrupt LINC Complexes in a Cell-specific Manner

Nuclear migration and anchorage within developing and adult tissues relies heavily upon large macromolecular protein assemblies called LInkers of the Nucleoskeleton and Cytoskeleton (LINC complexes). These protein scaffolds span the nuclear envelope and connect the interior of the nucleus to components of the surrounding cytoplasmic cytoskeleton. LINC complexes consist of two evolutionary-conserved protein families, Sun proteins and Nesprins that harbor C-terminal molecular signature motifs called the SUN and KASH domains, respectively. Sun proteins are transmembrane proteins of the inner nuclear membrane whose N-terminal nucleoplasmic domain interacts with the nuclear lamina while their C-terminal SUN domains protrudes into the perinuclear space and interacts with the KASH domain of Nesprins. Canonical Nesprin isoforms have a variable sized N-terminus that projects into the cytoplasm and interacts with components of the cytoskeleton. This protocol describes the validation of a dominant-negative transgenic mouse strategy that disrupts endogenous SUN/KASH interactions in a cell-type specific manner. Our approach is based on the Cre/Lox system that bypasses many drawbacks such as perinatal lethality and cell nonautonomous phenotypes that are associated with germline models of LINC complex inactivation. For this reason, this model provides a useful tool to understand the role of LINC complexes during development and homeostasis in a wide array of tissues.

Nuclear envelope proteins play a central role in many basic biological processes and have been implicated in a variety of human diseases. This protocol describes a new Cre/Lox-based mouse model that allows for the spatiotemporal control of LINC complexes disruption.

Nuclear migration and anchorage within developing and adult tissues relies heavily upon large macromolecular protein assemblies called LInkers of the ...

Residue-specific Incorporation of Noncanonical Amino Acids into Model Proteins Using an Escherichia coli Cell-free Transcription-translation System

The canonical set of amino acids leads to an exceptionally wide range of protein functionality. Nevertheless, the set of residues still imposes limitations on potential protein applications. The incorporation of noncanonical amino acids can enlarge this scope. There are two complementary approaches for the incorporation of noncanonical amino acids. For site-specific incorporation, in addition to the endogenous canonical translational machineries, an orthogonal aminoacyl-tRNA-synthetase-tRNA pair must be provided that does not interact with the canonical ones. Consequently, a codon that is not assigned to a canonical amino acid, usually a stop codon, is also required. This genetic code expansion enables the incorporation of a noncanonical amino acid at a single, given site within the protein. The here presented work describes residue-specific incorporation where the genetic code is reassigned within the endogenous translational system. The translation machinery accepts the noncanonical amino acid as a surrogate to incorporate it at canonically prescribed locations, i.e., all occurrences of a canonical amino acid in the protein are replaced by the noncanonical one. The incorporation of noncanonical amino acids can change the protein structure, causing considerably modified physical and chemical properties. Noncanonical amino acid analogs often act as cell growth inhibitors for expression hosts since they modify endogenous proteins, limiting in vivo protein production. In vivo incorporation of toxic noncanonical amino acids into proteins remains particularly challenging. Here, a cell-free approach for a complete replacement of L-arginine by the noncanonical amino acid L-canavanine is presented. It circumvents the inherent difficulties of in vivo expression. Additionally, a protocol to prepare target proteins for mass spectral analysis is included. It is shown that L-lysine can be replaced by L-hydroxy-lysine, albeit with lower efficiency. In principle, any noncanonical amino acid analog can be incorporated using the presented method as long as the endogenous in vitro translation system recognizes it.

Residue-specific Incorporation of Noncanonical Amino Acids into Model Proteins Using an Escherichia coli Cell-free Transcription-translation System

An easy-to-use, cell-free expression protocol for the residue-specific incorporation of noncanonical amino acid analogs into proteins, including downstream analysis, is presented for medical, pharmaceutic, structural and functional studies.