Keio collection strains were obtained from the National BioResource Project (NIG, Japan): E. coli. We thank Dirk Linke (Department of Biosciences, University of Oslo) for his continuing support. This work was funded by the Research Council of Norway Young Researcher grant 249793 (to Jack C. Leo).

1. Strains and Plasmids
<ol>
	<li>Bacterial strains
	<ol>
		<li>Use the E. coli strains BW251135, JW4179 (BW25113 tamA::kan)7, BL21(DE3)20, and BL21&#916;ABCF21. See Table of Materials for further information.</li>
	</ol>
	</li>
	<li>Bacteriophages
	<ol>
		<li>Use the phage P1vir for the general transduction. Store the phage as a liquid stock with a few drops of chloroform (see step 2.2). For more information, see Table of Materials.</li>
	</ol>
	</li>
	<li>Plasmids
	<ol>
		<li>Use the following plasmids in this protocol: pCP2022, pIBA2-YadA23, and pEibD1024. As control plasmids, use pASK-IBA2 and pET22b (see Table of Materials).</li>
	</ol>
	</li>
	<li>Growth conditions
	<ol>
		<li>Propagate bacteria in a lysogeny broth (LB) medium25 with vigorous shaking (180&#8211;200 rpm) at 37&#160;&#176;C or 30&#160;&#176;C in the case of BL21&#916;ABCF and strains containing pCP20.</li>
		<li>Perform plasmid curing at 43&#160;&#176;C.</li>
		<li>For a solid medium, supplement LB with 1% agar (w/v).</li>
		<li>For top agar, supplement LB with 0.7% agar and 10 mM CaCl2 and autoclave the medium. Use SOC medium for the recovery after electroporation26.</li>
		<li>Use the following concentrations for antibiotics: 100 &#956;g/mL for Amp and 25 &#956;g/mL for Kan.</li>
	</ol>
	</li>
</ol>
2. Preparing a Phage Lysate
<ol>
	<li>Infection of the donor strain
	<ol>
		<li>Grow the donor strain JW4197 in 5 mL of LB medium supplemented with 10 mM CaCl2 and optionally with Kan (25 &#181;g/mL) to an optical density at 600 nm (OD600) of ~1.0. Measure the OD600 value using a spectrophotometer.</li>
		<li>Make a dilution series of an existing P1 phage stock in the LB medium: recommended dilutions are between 10-3 to 10-7.</li>
		<li>Mix 200 &#956;L of the bacterial suspension and 100 &#956;L of a given phage dilution in a 15 mL centrifuge tube or equivalent. Prepare as many tubes as phage dilutions. Incubate the tubes for 20 min at 37&#160;&#176;C without shaking.</li>
		<li>Add ~3 mL of molten top agar (~50&#160;&#176;C) supplemented with 10 mM CaCl2 to the tubes, mix the contents thoroughly by vortexing the tubes shortly, and pour the mixtures onto prewarmed LB plates to make even layers.</li>
		<li>Incubate the plates overnight at 37&#160;&#176;C.</li>
	</ol>
	</li>
	<li>Lysate preparation
	<ol>
		<li>The following day choose a plate with a semi-confluent growth of phage plaques. On a semi-confluent plate, approximately half the surface area of the plate is clear (Figure 3).</li>
		<li>Scrape the top agar layer from such a plate using an inoculation loop or a similar tool and place the top agar in a centrifuge tube. Add 1&#8211;2 mL of LB and a drop of chloroform and vortex the tube vigorously for ~1 min. Add the chloroform in a fume hood.</li>
		<li>Centrifuge the tube for 15 min at 4,000 x g or faster to pellet the agar and bacterial cells.</li>
		<li>Move the supernatant to a fresh microcentrifuge tube, avoiding carrying over any debris from the pellet. Add 2 drops of chloroform and store the lysate at 4&#8211;10&#160;&#176;C. Add chloroform in a fume hood. Do not freeze the phage lysate as this will result in a significant reduction of the number of infectious particles.</li>
	</ol>
	</li>
	<li>Determining the lysate titer
	<ol>
		<li>Grow BW25113 in LB supplemented with 10 mM CaCl2 at 37&#160;&#176;C till the culture reaches an OD600 of ~1.0.</li>
		<li>Prepare a dilution series of the phage lysate in LB (e.g., 10-6&#8211;10-9). 
		NOTE: Be careful to pipet the sample from the top of the lysate to avoid transferring chloroform to the dilutions.</li>
		<li>Mix 200 &#956;L of the bacterial suspension and 100 &#956;L of a given phage dilution in a 15 mL centrifuge tube or equivalent. Prepare as many tubes as phage dilutions. Incubate the tubes for 20 min at 37&#160;&#176;C without shaking.</li>
		<li>Add ~3 mL of molten top agar (~50&#160;&#176;C) supplemented with 10 mM CaCl2 to the tubes, mix the contents thoroughly by vortexing the tubes shortly, and pour the mixture onto prewarmed LB plates to make even layers.</li>
		<li>Incubate the plates overnight at 37&#160;&#176;C.</li>
		<li>The following day count the number of plaques (clear regions in the bacterial mat) for each plate and calculate the titer of the lysate using the following formula: 
		<img alt="Equation 1" src="/files/ftp_upload/58267/58267eq1.jpg" /> 
		Example: On plates with the dilutions 10-7, 10-8, and 10-9, there are 231, 27, and 2 plaques, respectively. As 100 &#956;L of each dilution was used for the infection, and because the titer is expressed as plaque-forming units (pfu)/mL, the plating factor is 10. These numbers are entered into the formula: 
		<img alt="Equation 2" src="/files/ftp_upload/58267/58267eq2.jpg" /> 
		Thus, the titer is approximately 2 x 1010 infectious phage particles/mL.</li>
	</ol>
	</li>
</ol>
3. P1 Transduction
<ol>
	<li>Preparing the recipient cells
	<ol>
		<li>Grow the recipient strain BL21&#916;ABCF in LB supplemented to an optical density at 600 nm (OD600) of ~1.0. Use a spectrophotometer to measure the OD600 value.</li>
		<li>Calculate the volume of the phage lysate needed to achieve a multiplicity of infection (MOI) value of 0.5. To calculate the MOI, estimate the number of bacteria based on the OD600 of the culture. Assume that an OD600 value of 1.0 corresponds to ~109 cfu/mL. Calculate the volume required based on the known titer of the lysate. 
		Example (based on the titer calculated in the example after step 2.3.6): 
		<img alt="Equation 3" src="/files/ftp_upload/58267/58267eq3.jpg" /></li>
		<li>Add CaCl2 to the recipient strain culture to 10 mM and mix. Take 1 mL of the culture for transduction.</li>
	</ol>
	</li>
	<li>Performing transduction
	<ol>
		<li>Add the appropriate volume of phage lysate to 1 mL of recipient culture (including CaCl2 at 10 mM) as calculated in step 3.1.2 and mix gently. 
		NOTE: Be careful to pipette the sample from the top of the lysate to avoid transferring chloroform to the mix.</li>
		<li>Statically incubate the mix for 20 min at 37&#160;&#176;C.</li>
		<li>Stop the infection by adding sodium citrate, pH 5.5, to 100 mM.</li>
		<li>Centrifuge the bacteria (5,000 x g for 2 min) and remove the supernatant, then resuspend them in 1 mL of fresh LB supplemented with 100 mM sodium citrate, pH 5.5.</li>
		<li>Wash the cells twice more as in step 3.2.4 to ensure the removal of free phages and calcium.</li>
		<li>Resuspend the bacteria in 1 mL of fresh LB supplemented with 100 mM sodium citrate, pH 5.5. Incubate the bacteria at 30&#160;&#176;C for 1 h with shaking (&#62; 100 rpm).</li>
		<li>Collect the bacteria by centrifugation (5,000 x g for 2 min) and resuspend them in ~100 &#956;L of LB with 100 mM sodium citrate, pH 5.5.</li>
		<li>Spread the bacteria on an LB plate supplemented with Kan at 25 &#181;g/mL and 10 mM sodium citrate, pH 5.5, and grow the bacteria at 30&#160;&#176;C until colonies appear (~24 h).</li>
	</ol>
	</li>
	<li>Selecting the transductants
	<ol>
		<li>Once colonies have grown on the selection plate, restreak them on LB + Kan for single colonies and grow at 30&#160;&#176;C till single colonies appear.</li>
	</ol>
	</li>
</ol>
4. Excising the Kan cassette
<ol>
	<li>The transformation with recombination plasmid
	<ol>
		<li>Make the Kan-resistant BL21&#916;ABCF strain electrocompetent.
		<ol>
			<li>Grow the strain from a single colony in fresh LB (5 mL) supplemented with Kan (25 &#181;g/mL) at 30&#160;&#176;C until the OD600 is ~0.5&#8211;0.7.</li>
			<li>From here on, carry out the following steps at 4&#160;&#176;C or on ice. Centrifuge the bacteria (5,000 x g for 10 min) and remove the supernatant.</li>
			<li>Wash the cell pellet with ice-cold distilled water twice by repeating the previous centrifugation step and, finally, resuspend cell pellet in 100 &#181;L of ice-cold 10% glycerol.</li>
		</ol>
		</li>
		<li>Cool down 1 mm electroporation cuvettes on ice.</li>
		<li>Add 1 pg of plasmid DNA (pCP20) to the cell suspension, mix the suspension gently, and transfer it to a cooled electroporation cuvette.</li>
		<li>Set the electroporator to 1.8 kV and electroporate the cells.</li>
		<li>Rescue transformed cells by adding 1 mL of SOC medium and growing them for 1 h at 30&#160;&#176;C.</li>
		<li>Plate 100 &#181;L of the cells on LB + Amp plates and grow the cells at 30&#160;&#176;C overnight.</li>
	</ol>
	</li>
	<li>Induction of the recombination
	<ol>
		<li>Pick single colonies from the LB + Amp plate and inoculate fresh LB omitting all antibiotics.</li>
		<li>Grow the cells at the non-permissive temperature (43&#160;&#176;C) overnight to induce the expression of FLP recombinase.</li>
	</ol>
	</li>
	<li>Selecting recombinants
	<ol>
		<li>Make serial dilutions and plate 50 &#181;L of a 105-106 dilution on non-selective plates and grow it overnight at 30&#160;&#176;C.</li>
	</ol>
	</li>
</ol>
5. Verification of the Gene Deletion
<ol>
	<li>Verification of the plasmid loss and successful recombination
	<ol>
		<li>Streak single colonies from the plates prepared in step 4.3.1 on LB + Kan, LB + Amp, and LB plates without antibiotics, in this order. To aid in the streaking, use a colony grid (see Supplementary File 1). Grow the plates at 30&#160;&#176;C until colonies appear (~24 h).</li>
		<li>Pick 10&#8211;20 clones that have grown on the non-selective plate but failed to grow on the selective media for further verification.</li>
	</ol>
	</li>
	<li>Additional verification by colony PCR 
	<ol>
		<li>Perform a colony PCR with primers flanking the tamA coding sequence (see Table of Materials).</li>
		<li>Prepare a master PCR mix. The amount of mix needed depends on the number of colonies to be tested (see the example in Table 1). Mix the reagents on ice, add the polymerase last.</li>
		<li>Mix the PCR master mix thoroughly, then dispense 20 &#181;L into tubes of a PCR strip. Pick a small amount of each colony to be screened using a sterile pipette tip and add it to a tube. Remember to include the original recipient strain for comparison. Optionally, also include the donor strain.</li>
		<li>Run a PCR reaction (see Table 2 for the program used).</li>
		<li>Prepare a 1% agarose gel.
		<ol>
			<li>For a 50 mL gel, measure 0.5 g of agarose and add 50 mL of TAE buffer (40 mM Tris, 20 mM acetic acid, and 1 mM EDTA, pH 8.0). Heat the mixture in a microwave oven until all agarose has dissolved.</li>
			<li>Once the agarose has dissolved, cool the solution to approximately 50&#160;&#176;C, and then add 5 &#956;L of DNA staining dye. Mix the solution well and pour it into a gel tray in a casting chamber. Insert one or more rows of well combs so that there are sufficient wells for all samples, as well as molecular size markers. Allow the gel to set for 30 min.</li>
		</ol>
		</li>
		<li>Once the PCR run is complete, add 4 &#181;L of 6x DNA loading dye to each sample. Place the gel in the electrophoresis chamber and add TAE buffer till the wells are covered.</li>
		<li>Apply 10&#8211;15 &#181;L from each PCR reaction to a well in the gel. Also add 5 &#181;L of a molecular size marker. Then, run the gel for 30 min at 75 V.</li>
		<li>Once the run is complete, image the gel using a blue-light imager.</li>
	</ol>
	</li>
</ol>
6. Other Techniques
<ol>
	<li>Protein expression 
	NOTE: The expression of test proteins (YadA and EibD) has been described in detail elsewhere23,24. This is a brief summary of the main steps.
	<ol>
		<li>Transform BL21&#916;ABCF &#916;tamA with the necessary plasmids (pIBA2-YadA and pEibD10 and the corresponding control plasmids) and select for transformants on LB + Amp.</li>
		<li>For the protein expression, inoculate 100 mL of LB medium + Amp with 1 mL of an overnight culture of transformed bacteria and grow these at 30&#160;&#176;C until mid-log phase, at which time, induce the protein production with either anhydrotetracycline (at 100 ng/mL) or isopropyl thiogalactoside (at 0.5 mM).</li>
		<li>After 2 h of induction at 30&#160;&#176;C, measure the turbidity of the cultures and collect a number of cells corresponding to 50 mL at OD600 = 1.0 by centrifuging the cultures for 15 min at 4,000 x g. Wash the pellet 1x with 10 mM HEPES, pH 7.4, and then either store it at -20&#160;&#176;C or process it further as in step 6.2.</li>
	</ol>
	</li>
	<li>Outer membrane extraction 
	NOTE: Outer membrane extraction is performed as described in detail by Leo et al.27. The main steps are summarized below.
	<ol>
		<li>Resuspend the cell pellet in 1 mL of 10 mM HEPES, pH 7.4, supplemented with 10 mM MgCl2 and MnCl2, lysozyme (0.1 mg/mL), and a pinch of DNase I.</li>
		<li>Lyse the cells (e.g., using a bead beater).</li>
		<li>Centrifuge the cells shortly (2 min at 15,600 x g) to remove cell debris and move the supernatant to a fresh tube.</li>
		<li>Centrifuge the cells for 30 min at 16,000 x g, after which, resuspend the pellet in 400 &#956;L of 1% N-lauroyl sarcosine in 10 mM HEPES, pH 7.4.</li>
		<li>Incubate the cells with agitation for 30 min at room temperature, after which, centrifuge them for 30 min as above.</li>
		<li>Wash the translucent pellet 1x with 200 &#956;L of 10 mM HEPES, pH 7.4, and then resuspend it in 30 &#956;L of 10 mM HEPES and add 10 &#956;L of 4x SDS-PAGE sample buffer.</li>
	</ol>
	</li>
	<li>Activity assays 
	NOTE: Perform SDS-PAGE and activity assays for YadA and EibD as described28. The main steps are summarized below.</li>
	<li>SDS-PAGE
	<ol>
		<li>Heat the samples at 50&#160;&#176;C for 5 min before loading them onto a polyacrylamide gel, to avoid denaturing the proteins.</li>
		<li>After the separation in SDS-PAGE, transfer the proteins to a polyvinylidene difluoride (PVDF) membrane.</li>
		<li>After the transfer, block the membrane with 2% skimmed-milk powder in PBS.</li>
	</ol>
	</li>
	<li>YadA-collagen far-western blot
	<ol>
		<li>After the blocking, add bovine collagen type I diluted in blocking buffer to a concentration of 10 &#956;g/mL and incubate the membrane for 1 h.</li>
		<li>Wash the membrane 2x with PBS + 0.05% Tween20 (PBS-T).</li>
		<li>Add the primary antibody (monoclonal anti-collagen COL-1) to the membrane, diluted 1:2,000 in blocking buffer.</li>
		<li>After incubating the membrane for 1 h, wash 2x as mentioned in step 6.3.2.2 and then add the secondary antibody [goat anti-mouse IgG-horseradish peroxidase (HRP) conjugate], diluted 1:10,000 in blocking buffer.</li>
		<li>Incubate the membrane for 1 h, then wash it 2x with PBS-T. Add an enhanced chemiluminescent substrate to the membrane according to the manufacturer&#8217;s instructions and detect the band using a CCD camera.</li>
	</ol>
	</li>
	<li>EibD-IgG binding assay
	<ol>
		<li>After the blocking, add a secondary antibody (goat anti-rabbit HRP), diluted 1:2,000 in blocking buffer.</li>
		<li>Incubate the membrane for 1 h, then wash it 2x with PBS. Perform chemiluminescent detection as mentioned in step 6.3.2.5.</li>
	</ol>
	</li>
</ol>

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P., Wackernagel, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+disruption+in+Escherichia+coli:+TcR+and+KmR+cassettes+with+the+option+of+Flp-catalyzed+excision+of+the+antibiotic-resistance+determinant.">Gene disruption in Escherichia coli: TcR and KmR cassettes with the option of Flp-catalyzed excision of the antibiotic-resistance determinant.</a> Gene. 158 (1), 9-14 (1995).</li><li>Grosskinsky, U., 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=A+conserved+glycine+residue+of+trimeric+autotransporter+domains+plays+a+key+role+in+Yersinia+adhesin+A+autotransport.">A conserved glycine residue of trimeric autotransporter domains plays a key role in Yersinia adhesin A autotransport.</a> Journal of Bacteriology. 189 (24), 9011-9019 (2007).</li><li>Leo, J. C., 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=The+structure+of+E.+coli+IgG-binding+protein+D+suggests+a+general+model+for+bending+and+binding+in+trimeric+autotransporter+adhesins.">The structure of E. coli IgG-binding protein D suggests a general model for bending and binding in trimeric autotransporter adhesins.</a> Structure. 19 (7), 1021-1030 (1993).</li><li>Bertani, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+on+lysogenesis.+I.+The+mode+of+phage+liberation+by+lysogenic+Escherichia+coli.">Studies on lysogenesis. I. The mode of phage liberation by lysogenic Escherichia coli.</a> Journal of Bacteriology. 62 (3), 293-300 (1951).</li><li>Hanahan, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+on+transformation+of+Escherichia+coli+with+plasmids.">Studies on transformation of Escherichia coli with plasmids.</a> Journal of Molecular Biology. 166 (4), 557-580 (1983).</li><li>Leo, J. C., Oberhettinger, P., Linke, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessing+the+outer+membrane+insertion+and+folding+of+multimeric+transmembrane+&#946;-barrel+proteins.">Assessing the outer membrane insertion and folding of multimeric transmembrane &#946;-barrel proteins.</a> Methods in Molecular Biology. , 157-167 (2015).</li><li>Mikula, K. M., Leo, J. C., &#321;yskowski, A., Kedracka-Krok, S., Pirog, A., Goldman, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+translocation+domain+in+trimeric+autotransporter+adhesins+is+necessary+and+sufficient+for+trimerization+and+autotransportation.">The translocation domain in trimeric autotransporter adhesins is necessary and sufficient for trimerization and autotransportation.</a> Journal of Bacteriology. 194 (4), 827-838 (2012).</li><li>Thomason, L. C., Costantino, N., Court, D. L., Ausubel, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=E.+coli+genome+manipulation+by+P1+transduction.">E. coli genome manipulation by P1 transduction.</a> Current Protocols in Molecular Biology. , (2007).</li><li>Franklin, N. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutation+in+gal+U+gene+of+E.+coli+blocks+phage+P1+infection.">Mutation in gal U gene of E. coli blocks phage P1 infection.</a> Virology. 38 (1), 189-191 (1969).</li><li>Jeong, H., 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=Genome+sequences+of+Escherichia+coli+B+strains+REL606+and+BL21(DE3).">Genome sequences of Escherichia coli B strains REL606 and BL21(DE3).</a> Journal of Molecular Biology. 394 (4), 644-652 (2009).</li><li>Greene, E. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+Sequence+Alignment+during+Homologous+Recombination.">DNA Sequence Alignment during Homologous Recombination.</a> Journal of Biological Chemistry. 291 (22), 11572-11580 (2016).</li><li>Watt, V. M., Ingles, C. J., Urdea, M. S., Rutter, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Homology+requirements+for+recombination+in+Escherichia+coli.">Homology requirements for recombination in Escherichia coli.</a> Proceedings of the National Academy of Sciences of the United States of America. 82 (14), 4768-4772 (1985).</li><li>Ho, T. D., Waldor, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enterohemorrhagic+Escherichia+coli+O157:H7+gal+mutants+are+sensitive+to+bacteriophage+P1+and+defective+in+intestinal+colonization.">Enterohemorrhagic Escherichia coli O157:H7 gal mutants are sensitive to bacteriophage P1 and defective in intestinal colonization.</a> Infection and Immunity. 75 (4), 1661-1666 (2006).</li><li>Ikeda, H., Tomizawa, J. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transducing+fragments+in+generalized+transduction+by+phage+P1.+I.+Molecular+origin+of+the+fragments.">Transducing fragments in generalized transduction by phage P1. I. Molecular origin of the fragments.</a> Journal of Molecular Biology. 14 (1), 85-109 (1965).</li><li>Murooka, Y., Harada, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expansion+of+the+host+range+of+coliphage+P1+and+gene+transfer+from+enteric+bacteria+to+other+Gam-negative+bacteria.">Expansion of the host range of coliphage P1 and gene transfer from enteric bacteria to other Gam-negative bacteria.</a> Applied and Environmental Microbiology. 38 (4), 754-757 (1979).</li><li>Goldberg, R. B., Bender, R. A., Streicher, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+selection+for+P1-sensitive+mutants+of+enteric+bacteria.">Direct selection for P1-sensitive mutants of enteric bacteria.</a> Journal of Bacteriology. 118 (3), 810-814 (1974).</li><li>O'Connor, K. A., Zusman, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coliphage+P1-mediated+transduction+of+cloned+DNA+from+Escherichia+coli+to+Myxococcus+xanthus:+use+for+complementation+and+recombinational+analyses.">Coliphage P1-mediated transduction of cloned DNA from Escherichia coli to Myxococcus xanthus: use for complementation and recombinational analyses.</a> Journal of Bacteriology. 155 (1), 317-329 (1983).</li></ol>

The authors have nothing to disclose.

P1 transduction is a fast, robust, and reliable method for generating gene deletions in E. coli. This is demonstrated here by transducing a tamA deletion mutant from a Keio donor strain to a BL21-derived recipient. The major stages in the transduction process are the production of the transducing lysate, the transduction itself, the excision of the Kan resistance cassette, and the verification of the knock-out by PCR. In total, the process takes approximately 1 week and requires no molecular biology met...

A common first approach to study the function of a gene is to perform knock-out mutagenesis and observe the resulting phenotype. This is also termed reverse genetics. The bacterium E. coli has been the workhorse of molecular biology for the last 70 years or so, due to the ease of its culturing and its amenability to genetic manipulation1. Several methods have been developed to produce gene deletions in E. coli, including marker exchange mutagenesis2,3 and, more recently, recombineering using the &#955; Red or Rac ET systems4,5,6.
In a widely used system, coding sequences of individual genes are replaced by an antibiotic resistance cassette that can later be excised from the chromosome5,7. The coding sequences are replaced, for instance by a kanamycin (Kan) resistance cassette, which is flanked by flippase (FLP) recognition target (FRT) sites on either side. The FRT sites are recognized by the recombinase FLP, which mediates site-specific recombination between the FRT sites leading to the deletion of the Kan cassette. In this way, a full deletion of a given gene&#39;s coding sequence can be achieved, leaving behind only a minimal scar sequence of approximately 100 base pairs (bp) (Figure 1).
Just over a decade ago, the so-called Keio collection was developed. This is a bacterial library based on a standard laboratory E. coli K12 strain, where almost all non-essential genes were individually deleted by &#955; Red recombination7,8. The clones within this collection each have one coding sequence replaced with an excisable Kan resistance cassette. The Keio collection has proven to be a useful tool for many applications9. One such application is the production of deletion mutants in other E. coli strains. The Kan cassette from a given deletion clone can be mobilized by generally transducing bacteriophages, such as P110,11,12,13,14. A phage stock prepared from such a strain can then be used to infect a recipient E. coli strain of interest, where at a low but reliable frequency the Kan cassette-containing region can be incorporated into the recipient genome by homologous recombination (Figure 2). Transductants can be selected for the growth on the Kan-containing medium. Following this, if removal of the antibiotic resistance cassette is desired, the FLP recombinase can be supplied to the transductant strain in trans. After curing the FLP-containing plasmid, which carries an ampicillin (Amp) resistance marker, Kan and Amp-sensitive clones are screened for, and the correct excision of the wild-type coding sequence and the Kan cassette are verified by&#160;colony PCR.
Here, a detailed protocol is presented, describing each of the steps in producing a knock-out E. coli strain based on the strategy outlined above. As an example, a deletion of the tamA gene is demonstrated. tamA encodes an outer membrane &#946;-barrel protein that is a part of the Transport and Assembly Module (TAM), which is involved in the biogenesis of certain autotransporter proteins and pili15,16,17. This knock-out strain was then used to examine the effect of the tamA deletion on the biogenesis of two trimeric autotransporter adhesins (TAAs), the Yersinia adhesin YadA and the E. coli immunoglobulin (Ig)-binding TAA EibD18,19.

<table>
	<tbody>
		<tr>
			<td>Strains</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>E. coli BW25113</td>
			<td>NIG</td>
			<td>ME6092</td>
			<td>Wild-type strain of Keio collection</td>
		</tr>
		<tr>
			<td>E. coli BL21(DE3)</td>
			<td>Merck</td>
			<td>69450-3</td>
			<td>Expression strain</td>
		</tr>
		<tr>
			<td>E. coli BL21DABCF</td>
			<td>Addgene</td>
			<td>102270</td>
			<td>Derived from BL21(DE3)</td>
		</tr>
		<tr>
			<td>E. coli JW4179</td>
			<td>NIG</td>
			<td>JW4179-KC</td>
			<td>tamA deletion mutant</td>
		</tr>
		<tr>
			<td>P1 vir</td>
			<td>NIG</td>
			<td>HR16</td>
			<td>Generally transducing bacteriophage</td>
		</tr>
		<tr>
			<td>Plasmids</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>pCP20</td>
			<td>CGSC</td>
			<td>14177</td>
			<td>conditionally replicating plasmid with FLP</td>
		</tr>
		<tr>
			<td>pASK-IBA2</td>
			<td>IBA GmbH</td>
			<td>2-1301-000</td>
			<td>expression vector</td>
		</tr>
		<tr>
			<td>pEibD10</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>for production of EibD; plasmid available on request</td>
		</tr>
		<tr>
			<td>pET22b+</td>
			<td>Merck</td>
			<td>69744-3</td>
			<td>expression vector</td>
		</tr>
		<tr>
			<td>pIBA2-YadA</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>for production of YadA; plasmid available on request</td>
		</tr>
		<tr>
			<td>Chemicals</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Acetic acid</td>
			<td>ThermoFisher</td>
			<td>33209</td>
			<td></td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>BD Bacto</td>
			<td>214010</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Lonza</td>
			<td>50004</td>
			<td></td>
		</tr>
		<tr>
			<td>Ampicillin</td>
			<td>Applichem</td>
			<td>A0839</td>
			<td></td>
		</tr>
		<tr>
			<td>Anhydrotetracycline</td>
			<td>Abcam</td>
			<td>ab145350</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-collagen type I antibody COL-1</td>
			<td>Sigma</td>
			<td>C2456</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine collagen type I</td>
			<td>Sigma</td>
			<td>C9791</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium chloride</td>
			<td>Merck</td>
			<td>102382</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Merck</td>
			<td>102445</td>
			<td></td>
		</tr>
		<tr>
			<td>Di-sodium hydrogen phosphate</td>
			<td>VWR</td>
			<td>28029</td>
			<td></td>
		</tr>
		<tr>
			<td>DNA dye</td>
			<td>Thermo</td>
			<td>S33102</td>
			<td></td>
		</tr>
		<tr>
			<td>DNA molecular size marker</td>
			<td>New England BioLabs</td>
			<td>N3232S</td>
			<td></td>
		</tr>
		<tr>
			<td>DNase I</td>
			<td>Sigma</td>
			<td>DN25</td>
			<td></td>
		</tr>
		<tr>
			<td>dNTP mix</td>
			<td>New England Biolabs</td>
			<td>N0447</td>
			<td></td>
		</tr>
		<tr>
			<td>ECL HRP substrate</td>
			<td>Advansta</td>
			<td>K-12045</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Applichem</td>
			<td>A2937</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>VWR</td>
			<td>24388</td>
			<td></td>
		</tr>
		<tr>
			<td>goat anti-mouse IgG-HRP</td>
			<td>Santa Cruz</td>
			<td>sc-2005</td>
			<td></td>
		</tr>
		<tr>
			<td>goat anti-rabbit IgG-HRP</td>
			<td>Agrisera</td>
			<td>AS10668</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>VWR</td>
			<td>30487</td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropyl thiogalactoside</td>
			<td>VWR</td>
			<td>43714</td>
			<td></td>
		</tr>
		<tr>
			<td>Kanamycin</td>
			<td>Applichem</td>
			<td>A1493</td>
			<td></td>
		</tr>
		<tr>
			<td>Lysozyme</td>
			<td>Applichem</td>
			<td>A4972</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium chloride</td>
			<td>VWR</td>
			<td>25108</td>
			<td></td>
		</tr>
		<tr>
			<td>Manganese chloride</td>
			<td>Sigma</td>
			<td>221279</td>
			<td></td>
		</tr>
		<tr>
			<td>N-lauroyl sarcosine</td>
			<td>Sigma</td>
			<td>L9150</td>
			<td></td>
		</tr>
		<tr>
			<td>Skim milk powder</td>
			<td>Sigma</td>
			<td>70166</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>VWR</td>
			<td>27808</td>
			<td></td>
		</tr>
		<tr>
			<td>tamA forward primer</td>
			<td>Invitrogen</td>
			<td>N/A</td>
			<td>Sequence 5&#39;-GAAAAAAGGATATTCAGGAGAAAATGTG-3&#39;</td>
		</tr>
		<tr>
			<td>tamA reverse primer</td>
			<td>Invitrogen</td>
			<td>N/A</td>
			<td>Sequence 5&#39;-TCATAATTCTGGCCCCAGACC-3&#39;</td>
		</tr>
		<tr>
			<td>Taq DNA polymerase</td>
			<td>New England Biolabs</td>
			<td>M0267</td>
			<td></td>
		</tr>
		<tr>
			<td>Tri-sodium citrate</td>
			<td>Merck</td>
			<td>106448</td>
			<td></td>
		</tr>
		<tr>
			<td>Tryptone</td>
			<td>VWR</td>
			<td>84610</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween20</td>
			<td>Sigma</td>
			<td>P1379</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast extract</td>
			<td>Merck</td>
			<td>103753</td>
			<td></td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose gel electrophoresis chamber</td>
			<td>Hoefer</td>
			<td>SUB13</td>
			<td></td>
		</tr>
		<tr>
			<td>Bead beater</td>
			<td>Thermo</td>
			<td>FP120A-115</td>
			<td></td>
		</tr>
		<tr>
			<td>CCD camera</td>
			<td>Kodak</td>
			<td>4000R</td>
			<td></td>
		</tr>
		<tr>
			<td>Electroporation cuvettes</td>
			<td>Bio-Rad</td>
			<td>165-2089</td>
			<td></td>
		</tr>
		<tr>
			<td>Electroporation unit</td>
			<td>Bio-Rad</td>
			<td>1652100</td>
			<td></td>
		</tr>
		<tr>
			<td>Gel imager</td>
			<td>Nippon Genetics</td>
			<td>GP-03LED</td>
			<td></td>
		</tr>
		<tr>
			<td>Incubating shaker</td>
			<td>Infors HT</td>
			<td>Minitron</td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>VWR</td>
			<td>390-0482</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge</td>
			<td>Eppendorf</td>
			<td>5415D</td>
			<td></td>
		</tr>
		<tr>
			<td>Microwave oven</td>
			<td>Samsung</td>
			<td>CM1099A</td>
			<td></td>
		</tr>
		<tr>
			<td>PCR machine</td>
			<td>Biometra</td>
			<td>Tpersonal</td>
			<td></td>
		</tr>
		<tr>
			<td>PCR strips</td>
			<td>Axygen</td>
			<td>PCR-0208-CP-C</td>
			<td></td>
		</tr>
		<tr>
			<td>pH meter</td>
			<td>Hanna Instruments</td>
			<td>HI2211-01</td>
			<td></td>
		</tr>
		<tr>
			<td>PVDF membrane</td>
			<td>ThermoFisher</td>
			<td>88518</td>
			<td></td>
		</tr>
		<tr>
			<td>SDS-PAG electrophoresis chamber</td>
			<td>ThermoFisher</td>
			<td>A25977</td>
			<td></td>
		</tr>
		<tr>
			<td>Tabletop centrifuge</td>
			<td>Beckman Coulter</td>
			<td>B06322</td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex mixer</td>
			<td>Scientific Industries</td>
			<td>SI-0236</td>
			<td></td>
		</tr>
		<tr>
			<td>Water bath</td>
			<td>GFL</td>
			<td>D3006</td>
			<td></td>
		</tr>
		<tr>
			<td>Wet transfer unit</td>
			<td>Hoefer</td>
			<td>TE22</td>
			<td></td>
		</tr>
	</tbody>
</table>

producing gene deletions in escherichia coli by p1 transduction with excisable antibiotic resistance cassettes

A first approach to study the function of an unknown gene in bacteria is to create a knock-out of this gene. Here, we describe a robust and fast protocol for transferring gene deletion mutations from one Escherichia coli strain to another by using generalized transduction with the bacteriophage P1. This method requires that the mutation be selectable (e.g., based on gene disruptions using antibiotic cassette insertions). Such antibiotic cassettes can be mobilized from a donor strain and introduced into a recipient strain of interest to quickly and easily generate a gene deletion mutant. The antibiotic cassette can be designed to include flippase recognition sites that allow the excision of the cassette by a site-specific recombinase to produce a clean knock-out with only a ~100-base-pair-long scar sequence in the genome. We demonstrate the protocol by knocking out the tamA gene encoding an assembly factor involved in autotransporter biogenesis and test the effect of this knock-out on the biogenesis and function of two trimeric autotransporter adhesins. Though gene deletion by P1 transduction has its limitations, the ease and speed of its implementation make it an attractive alternative to other methods of gene deletion.

Generation of a tamA Knock-out of BL21&#916;ABCF:
The strategy outlined above has previously been used to produce a derivative strain of BL21(DE3), a standard laboratory strain used for protein production, which is optimized for outer membrane protein production and called BL21&#916;ABCF21. This strain lacks four genes coding for abundant outer membrane proteins and, consequently, is able to produce...

Watch this Scientific Journal Video about Producing Gene Deletions in Escherichia coli by P1 Transduction with Excisable Antibiotic Resistance Cassettes at JoVE.com

Producing Gene Deletions in Escherichia coli by P1 Transduction with Excisable Antibiotic Resistance Cassettes

Here we present a protocol for the use of pre-existing antibiotic resistance-cassette deletion constructs as a basis for making deletion mutants in other E. coli strains. Such deletion mutations can be mobilized and inserted into the corresponding locus of a recipient strain using P1 bacteriophage transduction.

Producing Gene Deletions in Escherichia coli by P1 Transduction with Excisable Antibiotic Resistance Cassettes

A first approach to study the function of an unknown gene in bacteria is to create a knock-out of this gene. Here, we describe a robust and fast protocol ...

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16.9K Views.  University of Oslo. Here we present a protocol for the use of pre-existing antibiotic resistance-cassette deletion constructs as a basis for making deletion mutants in other E. coli strains. Such deletion mutations can be mobilized and inserted into the corresponding locus of a recipient strain using P1 bacteriophage transduction. 

Video: Producing Gene Deletions in Escherichia coli by P1 Transduction with Excisable Antibiotic Resistance Cassettes

Rearing and Double-stranded RNA-mediated Gene Knockdown in the Hide Beetle, Dermestes maculatus

Advances in genomics have raised the possibility of probing biodiversity at an unprecedented scale. However, sequence alone will not be informative without tools to study gene function. The development and sharing of detailed protocols for the establishment of new model systems in laboratories, and for tools to carry out functional studies, is thus crucial for leveraging the power of genomics. Coleoptera (beetles) are the largest clade of insects and occupy virtually all types of habitats on the planet. In addition to providing ideal models for fundamental research, studies of beetles can have impacts on pest control as they are often pests of households, agriculture, and food industries. Detailed protocols for rearing and maintenance of D. maculatus laboratory colonies and for carrying out dsRNA-mediated interference in D. maculatus are presented. Both embryonic and parental RNAi procedures-including apparatus set up, preparation, injection, and post-injection recovery-are described. Methods are also presented for analyzing embryonic phenotypes, including viability, patterning defects in hatched larvae, and cuticle preparations for unhatched larvae. These assays, together with in situ hybridization and immunostaining for molecular markers, make D. maculatus an accessible model system for basic and applied research. They further provide useful information for establishing procedures in other emerging insect model systems.

Rearing and Double-stranded RNA-mediated Gene Knockdown in the Hide Beetle, Dermestes maculatus

Here, we present protocols for rearing an intermediate-germ beetle, Dermestes maculatus (D. maculatus) in the lab. We also share protocols for embryonic and parental RNAi and methods for analyzing embryonic phenotypes to study gene function in this species.

Advances in genomics have raised the possibility of probing biodiversity at an unprecedented scale. However, sequence alone will not be informative ...

QTL Mapping and CRISPR/Cas9 Editing to Identify a Drug Resistance Gene in Toxoplasma gondii

Scientific knowledge is intrinsically linked to available technologies and methods. This article will present two methods that allowed for the identification and verification of a drug resistance gene in the Apicomplexan parasite Toxoplasma gondii, the method of Quantitative Trait Locus (QTL) mapping using a Whole Genome Sequence (WGS) -based genetic map and the method of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 -based gene editing. The approach of QTL mapping allows one to test if there is a correlation between a genomic region(s) and a phenotype. Two datasets are required to run a QTL scan, a genetic map based on the progeny of a recombinant cross and a quantifiable phenotype assessed in each of the progeny of that cross. These datasets are then formatted to be compatible with R/qtl software that generates a QTL scan to identify significant loci correlated with the phenotype. Although this can greatly narrow the search window of possible candidates, QTLs span regions containing a number of genes from which the causal gene needs to be identified. Having WGS of the progeny was critical to identify the causal drug resistance mutation at the gene level. Once identified, the candidate mutation can be verified by genetic manipulation of drug sensitive parasites. The most facile and efficient method to genetically modify T. gondii is the CRISPR/Cas9 system. This system comprised of just 2 components both encoded on a single plasmid, a single guide RNA (gRNA) containing a 20 bp sequence complementary to the genomic target and the Cas9 endonuclease that generates a double-strand DNA break (DSB) at the target, repair of which allows for insertion or deletion of sequences around the break site. This article provides detailed protocols to use CRISPR/Cas9 based genome editing tools to verify the gene responsible for sinefungin resistance and to construct transgenic parasites.

QTL Mapping and CRISPR/Cas9 Editing to Identify a Drug Resistance Gene in Toxoplasma gondii

Details are presented on how QTL mapping with a whole genome sequence based genetic map can be used to identify a drug resistance gene in Toxoplasma gondii and how this can be verified with the CRISPR/Cas9 system that efficiently edits a genomic target, in this case the drug resistance gene.

Scientific knowledge is intrinsically linked to available technologies and methods. This article will present two methods that allowed for the ...

Determination of the Optimal Chromosomal Location(s) for a DNA Element in Escherichia coli Using a Novel Transposon-mediated Approach

The optimal chromosomal position(s) of a given DNA element was/were determined by transposon-mediated random insertion followed by fitness selection. In bacteria, the impact of the genetic context on the function of a genetic element can be difficult to assess. Several mechanisms, including topological effects, transcriptional interference from neighboring genes, and/or replication-associated gene dosage, may affect the function of a given genetic element. Here, we describe a method that permits the random integration of a DNA element into the chromosome of Escherichia coli and select the most favorable locations using a simple growth competition experiment. The method takes advantage of a well-described transposon-based system of random insertion, coupled with a selection of the fittest clone(s) by growth advantage, a procedure that is easily adjustable to experimental needs. The nature of the fittest clone(s) can be determined by whole-genome sequencing on a complex multi-clonal population or by easy gene walking for the rapid identification of selected clones. Here, the non-coding DNA region DARS2, which controls the initiation of chromosome replication in E. coli, was used as an example. The function of DARS2 is known to be affected by replication-associated gene dosage; the closer DARS2 gets to the origin of DNA replication, the more active it becomes. DARS2 was randomly inserted into the chromosome of a DARS2-deleted strain. The resultant clones containing individual insertions were pooled and competed against one another for hundreds of generations. Finally, the fittest clones were characterized and found to contain DARS2 inserted in close proximity to the original DARS2 location.

Determination of the Optimal Chromosomal Locations for a DNA Element in Escherichia coli Using a Novel Transposon-mediated Approach

Here, the power of a transposon-mediated random insertion of a non-coding DNA element was used to resolve its optimal chromosomal position.

The optimal chromosomal position(s) of a given DNA element was/were determined by transposon-mediated random insertion followed by fitness selection. In ...

A Standard Methodology to Examine On-site Mutagenicity As a Function of Point Mutation Repair Catalyzed by CRISPR/Cas9 and SsODN in Human Cells

Combinatorial gene editing using CRISPR/Cas9 and single-stranded oligonucleotides is an effective strategy for the correction of single-base point mutations, which often are responsible for a variety of human inherited disorders. Using a well-established cell-based model system, the point mutation of a single-copy mutant eGFP gene integrated into HCT116 cells has been repaired using this combinatorial approach. The analysis of corrected and uncorrected cells reveals both the precision of gene editing and the development of genetic lesions, when indels are created in uncorrected cells in the DNA sequence surrounding the target site. Here, the specific methodology used to analyze this combinatorial approach to the gene editing of a point mutation, coupled with a detailed experimental strategy to measuring indel formation at the target site, is outlined. This protocol outlines a foundational approach and workflow for investigations aimed at developing CRISPR/Cas9-based gene editing for human therapy. The conclusion of this work is that on-site mutagenesis takes place as a result of CRISPR/Cas9 activity during the process of point mutation repair. This work puts in place a standardized methodology to identify the degree of mutagenesis, which should be an important and critical aspect of any approach destined for clinical implementation.

This protocol outlines the workflow of a CRISPR/Cas9-based gene editing system for the repair of point mutations in mammalian cells. Here, we use a combinatorial approach to gene editing with a detailed follow-on experimental strategy for measuring indel formation at the target site&#8212;in essence, analyzing onsite mutagenesis.

Combinatorial gene editing using CRISPR/Cas9 and single-stranded oligonucleotides is an effective strategy for the correction of single-base point ...

Generation of a Gene-disrupted Streptococcus mutans Strain Without Gene Cloning

Typical methods for the elucidation of the function of a particular gene involve comparative phenotypic analyses of the wild-type strain and a strain in which the gene of interest has been disrupted. A gene-disruption DNA construct containing a suitable antibiotic resistance marker gene is useful for the generation of gene-disrupted strains in bacteria. However, conventional construction methods, which require gene cloning steps, involve complex and time-consuming protocols. Here, a relatively facile, rapid, and cost-effective method for targeted gene disruption in Streptococcus mutans is described. The method utilizes a 2-step fusion polymerase chain reaction (PCR) to generate the disruption construct and electroporation for genetic transformation. This method does not require an enzymatic reaction, other than PCR, and additionally offers greater flexibility in terms of the design of the disruption construct. Employment of electroporation facilitates the preparation of competent cells and improves the transformation efficiency. The present method may be adapted for the generation of gene-disrupted strains of various species.

Generation of a Gene-disrupted Streptococcus mutans Strain Without Gene Cloning

We describe a facile, rapid, and relatively inexpensive method for the generation of gene-disrupted Streptococcus mutans strains; this technique may be adapted for the generation of gene-disrupted strains of various species.

Typical methods for the elucidation of the function of a particular gene involve comparative phenotypic analyses of the wild-type strain and a strain in ...

Measuring Gene Expression in Bombarded Barley Aleurone Layers with Increased Throughput

The aleurone layer of barley grains is an important model system for hormone-regulated gene expression in plants. In aleurone cells, genes required for germination or early seedling development are activated by gibberellin (GA), while genes associated with stress responses are activated by abscisic acid (ABA). The mechanisms of GA and ABA signaling can be interrogated by introducing reporter gene constructs into aleurone cells via particle bombardment, with the resulting transient expression measured using enzyme assays. An improved protocol is reported that partially automates and streamlines the grain homogenization step and the enzyme assays, allowing significantly more throughput than existing methods. Homogenization of the grain samples is carried out using an automated tissue homogenizer, and GUS (&#946;-glucuronidase) assays are carried out using a 96-well plate system. Representative results using the protocol suggest that phospholipase D activity may play an important role in the activation of HVA1 gene expression by ABA, through the transcription factor TaABF1.

An improved protocol is presented for the measurement of transient gene expression from reporter constructs in barley aleurone cells after particle bombardment. The combination of automated grain grinding with 96-well plate enzyme assays provides high throughput for the procedure.

The aleurone layer of barley grains is an important model system for hormone-regulated gene expression in plants. In aleurone cells, genes required for ...

Testing the Role of Multicopy Plasmids in the Evolution of Antibiotic Resistance

Multicopy plasmids are extremely abundant in prokaryotes but their role in bacterial evolution remains poorly understood. We recently showed that the increase in gene copy number per cell provided by multicopy plasmids could accelerate the evolution of plasmid-encoded genes. In this work, we present an experimental system to test the ability of multicopy plasmids to promote gene evolution. Using simple molecular biology methods, we constructed a model system where an antibiotic resistance gene can be inserted into Escherichia coli MG1655, either in the chromosome or on a multicopy plasmid. We use an experimental evolution approach to propagate the different strains under increasing concentrations of antibiotics and we measure survival of bacterial populations over time. The choice of the antibiotic molecule and the resistance gene is so that the gene can only confer resistance through the acquisition of mutations. This &#34;evolutionary rescue&#34; approach provides a simple method to test the potential of multicopy plasmids to promote the acquisition of antibiotic resistance. In the next step of the experimental system, the molecular bases of antibiotic resistance are characterized. To identify mutations responsible for the acquisition of antibiotic resistance we use deep DNA sequencing of samples obtained from whole populations and clones. Finally, to confirm the role of the mutations in the gene under study, we reconstruct them in the parental background and test the resistance phenotype of the resulting strains.

Here we present an experimental method to test the role of multicopy plasmids in the evolution of antibiotic resistance.

Multicopy plasmids are extremely abundant in prokaryotes but their role in bacterial evolution remains poorly understood. We recently showed that the ...

Candidate Gene Testing in Clinical Cohort Studies with Multiplexed Genotyping and Mass Spectrometry

Complex diseases are often underpinned by multiple common genetic variants that contribute to disease susceptibility. Here, we describe a cost-effective tag single nucleotide polymorphism (SNP) approach using a multiplexed genotyping assay with mass spectrometry, to investigate gene pathway associations in clinical cohorts. We investigate the food allergy candidate locus Interleukin13 (IL13) as an example. This method efficiently maximizes the coverage by taking advantage of shared linkage disequilibrium (LD) within a region. Selected LD SNPs are then designed into a multiplexed assay, enabling up to 40 different SNPs to be analyzed simultaneously, boosting cost-effectiveness. Polymerase chain reaction (PCR) is used to amplify the target loci, followed by single nucleotide extension, and the amplicons are then measured using matrix-assisted laser desorption/ionization-time of flight(MALDI-TOF) mass spectrometry. The raw output is analyzed with the genotype calling software, using stringent quality control definitions and cut-offs, and high probability genotypes are determined and output for data analysis.

Identification of genetic variants contributing to complex human disease allows us to identify novel mechanisms. Here, we demonstrate a multiplex genotyping approach to candidate genes or gene pathway analysis that maximizes the coverage at low cost and is amenable to cohort-based studies.

Complex diseases are often underpinned by multiple common genetic variants that contribute to disease susceptibility. Here, we describe a cost-effective ...

CRISPR/Cas9 Ribonucleoprotein-mediated Precise Gene Editing by Tube Electroporation

Gene editing nucleases, represented by CRISPR-associated protein 9 (Cas9), are becoming mainstream tools in biomedical research. Successful delivery of CRISPR/Cas9 elements into the target cells by transfection is a prerequisite for efficient gene editing. This protocol demonstrates that tube electroporation (TE) machine-mediated delivery of CRISPR/Cas9 ribonucleoprotein (RNP), along with single-stranded oligodeoxynucleotide (ssODN) donor templates to different types of mammalian cells, leads to robust precise gene editing events. First, TE was applied to deliver CRISPR/Cas9 RNP and ssODNs to induce disease-causing mutations in the interleukin 2 receptor subunit gamma (IL2RG) gene and sepiapterin reductase (SPR) gene in rabbit fibroblast cells. Precise mutation rates of 3.57%-20% were achieved as determined by bacterial TA cloning sequencing. The same strategy was then used in human iPSCs on several clinically relevant genes including epidermal growth factor receptor (EGFR), myosin binding protein C, cardiac (Mybpc3), and hemoglobin subunit beta (HBB). Consistently, highly precise mutation rates were achieved (11.65%-37.92%) as determined by deep sequencing (DeepSeq). The present work demonstrates that tube electroporation of CRISPR/Cas9 RNP represents an efficient transfection protocol for gene editing in mammalian cells.

Presented here is a protocol for efficient CRISPR/Cas9 ribonucleoprotein-mediated gene editing in mammalian cells using tube electroporation.

Gene editing nucleases, represented by CRISPR-associated protein 9 (Cas9), are becoming mainstream tools in biomedical research. Successful delivery of ...

Quantification of Plasmid-Mediated Antibiotic Resistance in an Experimental Evolution Approach

Plasmids play a major role in microbial ecology and evolution as vehicles of lateral gene transfer and reservoirs of accessory gene functions in microbial populations. This is especially the case under rapidly changing environments such as fluctuating antibiotics exposure. We recently showed that plasmids maintain antibiotic resistance genes in Escherichia coli without positive selection for the plasmid presence. Here we describe an experimental system that allows following both the plasmid genotype and phenotype in long-term evolution experiments. We use molecular techniques to design a model plasmid that is subsequently introduced to an experimental evolution batch system approach in an E. coli host. We follow the plasmid frequency over time by applying replica plating of the E. coli populations while quantifying the antibiotic resistance persistence. In addition, we monitor the conformation of plasmids in host cells by analyzing the extent of plasmid multimer formation by plasmid nicking and agarose gel electrophoresis. Such an approach allows us to visualize not only the genome size of evolving plasmids but also their topological conformation-a factor highly important for plasmid inheritance. Our system combines molecular strategies with traditional microbiology approaches and provides a set-up to follow plasmids in bacterial populations over a long time. The presented approach can be applied to study a wide range of mobile genetic elements in the future.

Our experimental approach provides a strategy to follow plasmid abundance and antibiotic resistance over time in bacterial populations.

Plasmids play a major role in microbial ecology and evolution as vehicles of lateral gene transfer and reservoirs of accessory gene functions in microbial ...