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 methods to be used, apart from the final PCR for the verification. Thus, P1 transduction can compete in expended effort and time with &#955; Red recombination and is much faster than traditional marker exchange mutagenesis.
The presented protocol is very robust and allows for modifications of many of the steps. There are, however, a few critical parameters. For P1 infections, it is necessary to add calcium ions to the medium. Calcium is needed for the adsorption of the phage to the bacteria, and failure to add sufficient calcium to the medium will significantly reduce the efficiency of the infection. Some bacterial strains such as BL21&#916;ABCF tend to aggregate in the presence of CaCl221. In such cases, the bacteria can be grown without CaCl2, which can be added to the suspension shortly before the infection. However, for more conventional strains, 10 mM CaCl2 can be included in the growth medium from the beginning.
Conversely, it is important to remove the free calcium from the medium after the transduction. Citrate is a chelator of calcium ions, and because these are needed for the adsorption of P1 to host cells, removing the free calcium from the medium prevents further infections. If the calcium is not removed, the phages will infect further cells throughout the culture, at best reducing the efficiency of the transduction and at worst lysing the whole culture, including the transductants.
Another critical step is culturing bacteria carrying the plasmid pCP20. pCP20 is a conditionally replicating plasmid that does not replicate at temperatures of 37 &#176;C or higher; thus, to establish the plasmid in the cells, incubations with this plasmid must be performed at 30 &#176;C (or lower), a temperature permissive for the replication of pCP20. For curing pCP20, a high temperature (43 &#176;C) is used. Some strains do not grow well at this temperature; in such cases, 37 &#176;C should suffice, although the plasmid curing will be somewhat less efficient at this temperature.
When plating bacteria to test for antibiotic sensitivity, the order of plate streaking is important. The protocol calls for clones to be streaked first on antibiotic-containing plates and finally on non-selective medium. In this way, the investigator can be sure that any lack of growth on the selective media will be due to antibiotic sensitivity and rather than to a potential lack of material transferred on the plates. Following the protocol, no growth on the LB + Kan plate validates the excision of the Kan resistance cassette; no growth on the LB + Amp plate validates the loss of the recombination plasmid pCP20; strains growing on the LB plate (which did not grow in the same streaking experiment on the selection plates) will contain the positive recombinants.
Most of the other steps allow for considerable leeway. In the protocol as presented, BL21&#916;ABCF is cultured at 30 &#176;C, as this strain does not grow well at 37 &#176;C. However, E. coli strains without growth defects may be cultured at 37 &#176;C (except when transformed with pCP20).
The number of bacteria used for infections can be varied to some extent. The relationship between OD600 and a viable count is roughly linear between an OD600 value of 0.1 and 1.0, where the former corresponds to approximately 108 cfu/mL and the latter to ~109 cfu/mL. However, this relationship may vary to some extent depending on the strain in question, the growth medium, and other factors. It is recommended that the relationship between OD600 and the viable count should be established for each laboratory and strain, particularly for calculating MOI values. The number of bacteria used in infection experiments is not particularly critical, and 109 cfu/mL represents the early stationary phase, which is a reasonable compromise between cell density and the proportion of viable cells in the culture. If a lower number of viable bacteria are used for transduction, the number of phages simply needs to be adjusted accordingly. The MOI itself can also be varied to some extent. The protocol calls for an MOI value of 0.5, although anything between 0.1 and 0.5 should result in a good efficiency. At an MOI of 0.5, the ratio of phages to bacteria is 1:2 (i.e., half the number of phages compared to the number of bacteria). In the example in the protocol, the OD600 of the culture is 1.0, and 1 mL of the culture is used for the transduction; thus, the number of phages required is 5 x 108. An MOI value of 0.5 gives a high level of infection but reduces the number of bacteria that are doubly infected, which would reduce the efficiency of the transduction as the wild-type (infectious) phages far outnumber the transducing phages. A double infection with a transducing phage and an infectious phage would lead to cell lysis, thus eliminating this transductant from the pool of survivors. Therefore, an MOI of 0.5 should not be exceeded.
The protocol also allows for some shortcuts. Rather than preparing lysates from plates, some authors advocate preparing phage lysates in liquid medium29. This can save time as an overnight incubation step is not needed. Similarly, titrating the phage lysate might not be necessary. As noted above, the MOI&#160;is not very critical, and reasonable efficiency can be achieved by simply assuming a titer of 1010 pfu/mL for a standard lysate.
Despite the ease of the technique, P1 transduction is not universally applicable, and several conditions must be met for it to be useful. Firstly, a donor strain with a selectable knock-out allele must be available. This is usually accomplished by using a deletion where an antibiotic resistance cassette has replaced the gene of interest. The Keio collection is particularly useful in this regard, as it offers a ready-to-use library of antibiotic cassette-based gene knock-outs covering almost all non-essential genes in E. coli K12. This collection is particularly useful for knocking out conserved genes found in most E. coli strains (i.e., constituents of the E. coli core genome). For less common genes, such as virulence factors of pathogenic E. coli strains or rare metabolic pathway genes, the mutation may need to be created de novo. In such cases, P1 transduction may well not be the method of choice. In addition to essential genes, genes that are required for P1 infection, such as galU, mutations of which lead to P1 resistance30, are poor targets for a deletion by P1 transduction. Another note when using the Keio collection, specifically, is that a few strains carry a duplication of the targeted gene, where only one copy was disrupted by the Kan resistance cassette8. In such cases, the gene of interest may be essential; investigators are recommended to check updated annotations for such genes8. However, given these restraints, P1 transduction allows the deletion of most genes in laboratory E. coli strains. For example, the differences between BW25113 and BL21(DE3) are small and affect only a handful of protein-coding genes31.
Secondly, it is important to emphasize that the recipient strain must be capable of a homologous recombination for the transduction to work. A strain lacking the recombinase RecA can, therefore, not be modified by this method. This excludes all standard cloning strains, such as DH5&#945;, HB101, TOP10, and XL-1 Blue. RecA can mediate recombinations between identical stretches as short as 8 bp; however, a longer region of high similarity will increase the probability of recombination significantly32,33. Another problem, particularly with clinical E. coli strains, is that long O-antigen chains may mask the receptor for P1, which lies in the core oligosaccharide of lipopolysaccharide34. In addition to these requirements for the recipient strain, the P1 strain used for transduction should be a vir mutant; this mutation is required for a full lytic infection35.
A third caveat of P1 transduction is that the gene to be knocked out should reside in a region with high similarity in the flanking regions between the donor and recipient strains. If there is a lack of synteny between the strains, the replacement of the target gene coding sequence with the Kan cassette may well fail, due to differences in the content of the flanking regions. Therefore, before embarking on P1-mediated gene deletion, investigators should check the sequences of the donor and recipient strains to make sure the flanking sequences are homologous. Of course, this is only possible if the strains have been sequenced. In the case of strains with an unknown sequence, P1 transduction may either fail or cause other problems, such as a gene conversion in the flanking regions. P1 can transduce approximately 90 kilobase pairs of DNA; if there are differences in the gene content in the regions around the target gene (e.g., small deletions or insertions), it is likely that these will revert to the donor sequence. This might have unintended consequences on the phenotype of the recipient strain. Therefore, where possible, the sequences of the donor and recipient around the gene of interest should always be compared prior to P1 transduction.
In conclusion, P1 transduction is a rapid way of transferring a specific gene knock-out to a number of strains once the initial knock-out mutation has been generated. Though the technique has its limitations, the ease and speed of its implementation make it an attractive alternative to other methods of gene deletion. P1 only infects E. coli, which normally restricts its use to this species. However, some variants of P1 have been developed that have a broader host range and can infect other species within the family Enterobacteriaceae, and even some other &#947;-proteobacterial species, albeit at reduced efficiency36,37. Even the transduction of cloned DNA from E. coli to Myxococcus xanthus, a &#948;-proteobacterium, has been reported38. In future experiments, these variants could be augmented with the vir mutation to broaden the range of recipient strains used in antibiotic cassette-based gene deletion by general transduction.

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>&lt;strong&gt;Strains&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;em&gt;E. coli&lt;/em&gt; BW25113</td><td>NIG</td><td>ME6092</td><td>Wild-type strain of Keio collection</td></tr><tr><td>&lt;em&gt;E. coli&lt;/em&gt; BL21(DE3)</td><td>Merck</td><td>69450-3</td><td>Expression strain</td></tr><tr><td>&lt;em&gt;E. coli&lt;/em&gt; BL21DABCF</td><td>Addgene</td><td>102270</td><td>Derived from BL21(DE3)</td></tr><tr><td>&lt;em&gt;E. coli&lt;/em&gt; JW4179</td><td>NIG</td><td>JW4179-KC</td><td>&lt;em&gt;tamA&lt;/em&gt; deletion mutant</td></tr><tr><td>P1 &lt;em&gt;vir&lt;/em&gt;</td><td>NIG</td><td>HR16</td><td>Generally transducing bacteriophage</td></tr><tr><td>&lt;strong&gt;Plasmids&lt;/strong&gt;</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>&lt;strong&gt;Chemicals&lt;/strong&gt;</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>&lt;em&gt;N&lt;/em&gt;-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>&lt;em&gt;tamA&lt;/em&gt; forward primer</td><td>Invitrogen</td><td>N/A</td><td>Sequence 5&#039;-GAAAAAAGG&lt;br /&gt;ATATTCAGGAGAAAATGTG-3&#039;</td></tr><tr><td>&lt;em&gt;tamA&lt;/em&gt; reverse primer</td><td>Invitrogen</td><td>N/A</td><td>Sequence 5&#039;-TCATAATT&lt;br /&gt;CTGGCCCCAGACC-3&#039;</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>&lt;strong&gt;Equipment&lt;/strong&gt;</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 more heterologously expressed outer membrane proteins than the wild-type strain. To test whether the TAM is involved in TAA biogenesis, the tamA gene was deleted in this background.
A P1 lysate was prepared from the Keio collection strain JW4179, where the tamA gene (previously called yftM) coding sequence is replaced by a Kan resistance cassette. Then, a transduction experiment was performed with BL21&#916;ABCF as the recipient strain. Several Kan-resistant colonies were obtained, after which two were chosen for the excision of the Kan cassette. The plasmid pCP20, encoding the FLP recombinase, was introduced into these clones and, subsequently, cured by growing at 43 &#176;C in the absence of an antibiotic selection. A number of clones were screened for sensitivity to both Amp (which is a marker of pCP20) and Kan, and several clones sensitive to both were obtained. These clones were verified by colony PCR using primers flanking the tamA coding sequence and found that the tamA gene had successfully been deleted (Figure 4).
TamA&#39;s Role in TAA Biogenesis:
TamA has been shown to be involved in the biogenesis of some classical autotransporters16 and the inverse autotransporter intimin15. To test whether TamA is important for the biogenesis of TAAs, BL21&#916;ABCF &#916;tamA was transformed with plasmids encoding two test proteins, the Yersinia adhesin YadA and the E. coli Ig-binding protein EibD. These proteins are known to express well in E. coli and have been used as models for TAA biogenesis in earlier studies23,28.
After inducing protein production, the outer membrane fractions of the expression cultures were isolated and analyzed by SDS-PAGE. The samples were not boiled to demonstrate trimerization of the proteins. The trimers run at sizes above 100 kDa, whereas the monomers have expected sizes of 45 kDa (YadA) and 51 kDa (EibD). No major differences were observed between the expression levels in BL21&#916;ABCF and BL21&#916;ABCF &#916;tamA, although YadA seems to be produced at somewhat lower levels in the &#916;tamA strain (Figure 5A). However, the opposite appears to be the case for EibD.
To examine whether the lack of TamA might influence the correct folding or transport of the proteins, their ability to bind to ligands was tested. For YadA, this was accomplished by a collagen far-western blot (Figure 5B). YadA, in both strains, bound collagen at a similar level, demonstrating that the protein is correctly folded and functional. Similarly, IgG-binding activities of EibD in the two strains correlated with the expression level (Figure 4C). These results demonstrate that the deletion of tamA does not have a significant effect on TAA biogenesis, at least not for these two model TAAs.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58267/58267fig1.jpg" /> 
Figure 1: Generation of knock-outs with excisable antibiotic cassettes. For producing the gene knock-outs, a gene of interest (Gene B in this example) is replaced by a Kan resistance cassette flanked by FRT sites. The FRT-Kan cassette, in turn, is flanked by short (~50 bp) stretches of sequence homologous to the upstream and downstream regions of the Gene B. The coding sequence of Gene B is exchanged for the FRT-Kan cassette by &#955; Red recombination. Once this is accomplished, the Kan cassette itself can be removed by introducing the FLP recombinase, which will mediate a site-specific recombination between the FRT sites. This excises the Kan cassette, leaving a minimal (~100 bp) scar sequence in the B locus. For full details, see Baba et al.7. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58267/58267fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58267/58267fig2.jpg" /> 
Figure 2: Gene deletion by P1 transduction. The donor strain (beige) carries a Kan cassette that has replaced the gene of interest (yellow) on the chromosome (blue). The donor is infected with P1 bacteriophage. The phage multiplies in the donor strain, producing a large number of progenies. Most are wild-type (red genome), but a fraction are transducing phages that have incorporated a portion of the donor strain chromosome rather than phage DNA (blue genome). A proportion of these will contain the Kan cassette (yellow genome). Eventually, the infected host cell lyses and releases the phages into the medium. These are used to prepare a lysate. In the transduction experiment, the recipient strain (light blue) is infected with the lysate prepared from the donor strain. In a minority of cases, the recipient is infected by a transducing phage carrying the Kan cassette (shown here). If the regions adjacent to the Kan cassette undergo homologous recombination, the Kan cassette is incorporated into the recipient chromosome replacing the endogenous allele, resulting in Kan-resistant clones that can be selected for. The addition of FLP recombinase on a curable plasmid excises the Kan cassette, leaving only a short scar sequence (shown here in yellow), which reverts the clone to the Kan-sensitive phenotype. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58267/58267fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58267/58267fig3.jpg" /> 
Figure 3: Examples of plates after a P1 infection. (A) This panel shows a plate with individual plaques. (B) This panel shows a semi-confluent plate. (C) This panel shows an over-infected plate, where almost all the bacteria have been lysed by phage. Some resistant colonies have grown out of the top agar layer. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58267/58267fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/58267/58267fig4.jpg" /> 
Figure 4: Deletion of the tamA coding sequence. The tamA::kan deletion allele was introduced into the strain BL21&#916;ABCF by P1 transduction. After the excision of the Kan cassette, a scar sequence (~100 bp) is all that remains at the tamA locus. This was verified by PCR, using primers flanking the deletion site. In BL21&#916;ABCF and its parent strain, BL21(DE3), the PCR gives a product the length of the tamA coding sequence (the expected size is 1.7 kilobase pairs). In BL21&#916;ABCF, where the Kan cassette has been excised, the product corresponds to the expected scar sequence (145 bp). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58267/58267fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/58267/58267fig5.jpg" /> 
Figure 5: Expression of TAAs by a tamA deletion strain. (A) This panel shows the SDS-PAGE of outer membranes prepared from cells expressing TAAs (YadA or EibD) and vector controls (pIBA2 and pET22). The strains are BL21&#916;ABCF and its derivative strain lacking TamA (&#916;tamA). (B) This panel shows a collagen far-western blot of YadA samples. The YadA samples and pIBA2 controls from panel A were transferred to a PVDF membrane and incubated with collagen type I. They were then probed with an anti-collagen antibody and detected by ECL. (C) This panel shows an antibody binding assay for EibD samples. The EibD samples and pET22 controls from panel A were transferred to a PVDF membrane and incubated with an HRP-conjugated secondary antibody and then detected by ECL. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58267/58267fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Reagent</td>
			<td>1x mix</td>
			<td>7x mix</td>
		</tr>
		<tr>
			<td>PCR-grade water</td>
			<td>17 &#181;L</td>
			<td>119 &#181;L</td>
		</tr>
		<tr>
			<td>10 x polymerase buffer</td>
			<td>2 &#181;L</td>
			<td>14 &#181;L</td>
		</tr>
		<tr>
			<td>10 mM deoxyribonucleotide mix</td>
			<td>0.4 &#181;L</td>
			<td>2.8 &#181;L</td>
		</tr>
		<tr>
			<td>100 &#181;M forward primer</td>
			<td>0.2 &#181;L</td>
			<td>1.4 &#181;L</td>
		</tr>
		<tr>
			<td>100 &#181;M reverse primer</td>
			<td>0.2 &#181;L</td>
			<td>1.4 &#181;L</td>
		</tr>
		<tr>
			<td>Taq DNA polymerase</td>
			<td>0.2 &#181;L</td>
			<td>1.4 &#181;L</td>
		</tr>
		<tr>
			<td>Total</td>
			<td>20 &#181;L</td>
			<td>140 &#181;L</td>
		</tr>
	</tbody>
</table>
Table 1: Colony PCR master mix. The amount of mix depends on the number of colonies to be screened. In addition, prepare a control reaction (original recipient strain). For instance, if screening five clones, six reactions are needed, including the control. It is worth preparing an extra reaction to make sure there is enough PCR mix for all samples (repeated pipetting amplifies small pipetting errors). In this example, prepare a 7x mix (5 colonies, 1 control, and 1 extra reaction).
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Step</td>
			<td>Temperature</td>
			<td>Time</td>
			<td>Notes</td>
		</tr>
		<tr>
			<td>1.</td>
			<td>94 &#176;C</td>
			<td>3 min</td>
			<td></td>
		</tr>
		<tr>
			<td>2.</td>
			<td>94 &#176;C</td>
			<td>30 s</td>
			<td></td>
		</tr>
		<tr>
			<td>3.</td>
			<td>50 &#176;C</td>
			<td>30 s</td>
			<td></td>
		</tr>
		<tr>
			<td>4.&#160;</td>
			<td>70 &#176;C</td>
			<td>2 min&#160;</td>
			<td>1 min/kb to be amplified, round up</td>
		</tr>
		<tr>
			<td colspan="3">Return to step 2 24 times</td>
			<td></td>
		</tr>
		<tr>
			<td>5.</td>
			<td>70 &#176;C</td>
			<td>5 min</td>
			<td></td>
		</tr>
		<tr>
			<td>6.</td>
			<td>12 &#176;C&#160;</td>
			<td>for ever</td>
			<td>final hold</td>
		</tr>
	</tbody>
</table>
Table 2: Colony PCR program.
Supplementary File 1: An example of colony grid. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58267/Supplementary_file_1.pdf">Please click here to download this file.</a>

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 ...

producing-gene-deletions-escherichia-coli-p1-transduction-with

Nanyang Technological University

Research

JoVE Journal

Genetics

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

Site-specific Bacterial Chromosome Engineering: &#934;C31 Integrase Mediated Cassette Exchange (IMCE)

The bacterial chromosome may be used to stably maintain foreign DNA in the mega-base range1. Integration into the chromosome circumvents issues such as plasmid replication, plasmid stability, plasmid incompatibility, and plasmid copy number variance. This method uses the site-specific integrase from the Streptomyces phage (&Phi;) C312,3. The &Phi;C31 integrase catalyzes a direct recombination between two specific DNA sites: attB and attP (34 and 39 bp, respectively)4. This recombination is stable and does not revert5. A "landing pad" (LP) sequence consisting of a spectinomycin- resistance gene, aadA (SpR), and the E. coli &szlig;-glucuronidase gene (uidA) flanked by attP sites has been integrated into the chromosomes of Sinorhizobium meliloti, Ochrobactrum anthropi, and Agrobacterium tumefaciens in an intergenic region, the ampC locus, and the tetA locus, respectively. S. meliloti is used in this protocol. Mobilizable donor vectors containing attB sites flanking a stuffer red fluorescent protein (rfp) gene and an antibiotic resistance gene have also been constructed. In this example the gentamicin resistant plasmid pJH110 is used. The rfp gene6 may be replaced with a desired construct using SphI and PstI. Alternatively a synthetic construct flanked by attB sites may be sub-cloned into a mobilizable vector such as pK19mob7. The expression of the &Phi;C31 integrase gene (cloned from pHS628) is driven by the lac promoter, on a mobilizable broad host range plasmid pRK78139.
A tetraparental mating protocol is used to transfer the donor cassette into the LP strain thereby replacing the markers in the LP sequence with the donor cassette. These cells are trans-integrants. Trans-integrants are formed with a typical efficiency of 0.5%. Trans-integrants are typically found within the first 500-1,000 colonies screened by antibiotic sensitivity or blue-white screening using 5-bromo-4-chloro-3-indolyl-beta-D-glucuronic acid (X-gluc). This protocol contains the mating and selection procedures for creating and isolating trans-integrants.

A quick and efficient method to integrate foreign DNA of interest into pre-made acceptor strains, termed landing pad strains, is described. The method allows site-specific integration of a DNA cassette into the engineered landing pad locus of a given strain, through conjugation and expression of the &#934;C31 integrase.

The bacterial chromosome may be used to stably maintain foreign DNA in the mega-base range1. Integration into the chromosome circumvents issues such as ...

Bioengineering

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 ...

Creation of a Dense Transposon Insertion Library Using Bacterial Conjugation in Enterobacterial Strains Such As Escherichia Coli or Shigella flexneri

Transposon mutagenesis is a method that allows gene disruption via the random genomic insertion of a piece of DNA called a transposon. The protocol below outlines a method for high efficiency transfer between bacterial strains of a plasmid harboring a transposon containing a kanamycin resistance marker. The plasmid-borne transposase is encoded by a variant tnp gene that inserts the transposon into the genome of the recipient strain with very low insertional bias. This method thus allows the creation of large mutant libraries in which transposons have been inserted into unique genomic positions in a recipient strain of either Escherichia coli or Shigella flexneri bacteria. By using bacterial conjugation, as opposed to other methods such as electroporation or chemical transformation, large libraries with hundreds of thousands of unique clones can be created. This yields high-density insertion libraries, with insertions occurring as frequently as every 4-6 base pairs in non-essential genes. This method is superior to other methods as it allows for an inexpensive, easy to use, and high efficiency method for the creation of a dense transposon insertion library. The transposon library can be used in downstream applications such as transposon sequencing (Tn-Seq), to infer genetic interaction networks, or more simply, in mutational (forward genetic) screens.

Creation of a Dense Transposon Insertion Library Using Bacterial Conjugation in Enterobacterial Strains Such As Escherichia Coli or Shigella flexneri

Presented here is a simple method for creating a high-density transposon insertion library in Escherichia coli or Shigella flexneri using bacterial conjugation. This protocol allows the creation of a collection of hundreds of thousands of unique mutants in bacteria by the random genomic insertion of a transposon.

Transposon mutagenesis is a method that allows gene disruption via the random genomic insertion of a piece of DNA called a transposon. The protocol below ...

Immunology and Infection

Mapping Bacterial Functional Networks and Pathways in Escherichia Coli using Synthetic Genetic Arrays

Phenotypes are determined by a complex series of physical (e.g. protein-protein) and functional (e.g. gene-gene or genetic) interactions (GI)1. While physical interactions can indicate which bacterial proteins are associated as complexes, they do not necessarily reveal pathway-level functional relationships1. GI screens, in which the growth of double mutants bearing two deleted or inactivated genes is measured and compared to the corresponding single mutants, can illuminate epistatic dependencies between loci and hence provide a means to query and discover novel functional relationships2. Large-scale GI maps have been reported for eukaryotic organisms like yeast3-7, but GI information remains sparse for prokaryotes8, which hinders the functional annotation of bacterial genomes. To this end, we and others have developed high-throughput quantitative bacterial GI screening methods9, 10.
Here, we present the key steps required to perform quantitative E. coli Synthetic Genetic Array (eSGA) screening procedure on a genome-scale9, using natural bacterial conjugation and homologous recombination to systemically generate and measure the fitness of large numbers of double mutants in a colony array format. Briefly, a robot is used to transfer, through conjugation, chloramphenicol (Cm) - marked mutant alleles from engineered Hfr (High frequency of recombination) 'donor strains' into an ordered array of kanamycin (Kan) - marked F- recipient strains. Typically, we use loss-of-function single mutants bearing non-essential gene deletions (e.g. the 'Keio' collection11) and essential gene hypomorphic mutations (i.e. alleles conferring reduced protein expression, stability, or activity9, 12, 13) to query the functional associations of non-essential and essential genes, respectively. After conjugation and ensuing genetic exchange mediated by homologous recombination, the resulting double mutants are selected on solid medium containing both antibiotics. After outgrowth, the plates are digitally imaged and colony sizes are quantitatively scored using an in-house automated image processing system14. GIs are revealed when the growth rate of a double mutant is either significantly better or worse than expected9. Aggravating (or negative) GIs often result between loss-of-function mutations in pairs of genes from compensatory pathways that impinge on the same essential process2. Here, the loss of a single gene is buffered, such that either single mutant is viable. However, the loss of both pathways is deleterious and results in synthetic lethality or sickness (i.e. slow growth). Conversely, alleviating (or positive) interactions can occur between genes in the same pathway or protein complex2 as the deletion of either gene alone is often sufficient to perturb the normal function of the pathway or complex such that additional perturbations do not reduce activity, and hence growth, further. Overall, systematically identifying and analyzing GI networks can provide unbiased, global maps of the functional relationships between large numbers of genes, from which pathway-level information missed by other approaches can be inferred9.

Mapping Bacterial Functional Networks and Pathways in Escherichia Coli using Synthetic Genetic Arrays

Systematic, large-scale synthetic genetic (gene-gene or epistasis) interaction screens can be used to explore genetic redundancy and pathway cross-talk. Here, we describe a high-throughput quantitative synthetic genetic array screening technology, termed eSGA that we developed for elucidating epistatic relationships and exploring genetic interaction networks in Escherichia coli.

Phenotypes  are determined by a complex series of physical (e.g. protein-protein) and  functional (e.g. gene-gene or genetic) interactions (GI)1. While ...

Biology

Genome-wide Gene Deletions in Streptococcus sanguinis by High Throughput PCR

Transposon mutagenesis and single-gene deletion are two methods applied in genome-wide gene knockout in bacteria 1,2. Although transposon mutagenesis is less time consuming, less costly, and does not require completed genome information, there are two weaknesses in this method: (1) the possibility of a disparate mutants in the mixed mutant library that counter-selects mutants with decreased competition; and (2) the possibility of partial gene inactivation whereby genes do not entirely lose their function following the insertion of a transposon. Single-gene deletion analysis may compensate for the drawbacks associated with transposon mutagenesis. To improve the efficiency of genome-wide single gene deletion, we attempt to establish a high-throughput technique for genome-wide single gene deletion using Streptococcus sanguinis as a model organism. Each gene deletion construct in S. sanguinis genome is designed to comprise 1-kb upstream of the targeted gene, the aphA-3 gene, encoding kanamycin resistance protein, and 1-kb downstream of the targeted gene. Three sets of primers F1/R1, F2/R2, and F3/R3, respectively, are designed and synthesized in a 96-well plate format for PCR-amplifications of those three components of each deletion construct. Primers R1 and F3 contain 25-bp sequences that are complementary to regions of the aphA-3 gene at their 5' end. A large scale PCR amplification of the aphA-3 gene is performed once for creating all single-gene deletion constructs. The promoter of aphA-3 gene is initially excluded to minimize the potential polar effect of kanamycin cassette. To create the gene deletion constructs, high-throughput PCR amplification and purification are performed in a 96-well plate format. A linear recombinant PCR amplicon for each gene deletion will be made up through four PCR reactions using high-fidelity DNA polymerase. The initial exponential growth phase of S. sanguinis cultured in Todd Hewitt broth supplemented with 2.5% inactivated horse serum is used to increase competence for the transformation of PCR-recombinant constructs. Under this condition, up to 20% of S. sanguinis cells can be transformed using ~50 ng of DNA. Based on this approach, 2,048 mutants with single-gene deletion were ultimately obtained from the 2,270 genes in S. sanguinis excluding four gene ORFs contained entirely within other ORFs in S. sanguinis SK36 and 218 potential essential genes. The technique on creating gene deletion constructs is high throughput and could be easy to use in genome-wide single gene deletions for any transformable bacteria.

Genome-wide Gene Deletions in Streptococcus sanguinis by High Throughput PCR

An efficient genome-wide single gene mutation method has been established using Streptococcus sanguinis as a model organism. This method has achieved via high throughput recombinant PCRs and transformations.

Transposon  mutagenesis and single-gene deletion are two methods applied in genome-wide  gene knockout in bacteria 1,2. Although transposon mutagenesis is ...

Generation of In-Frame Gene Deletion Mutants in Pseudomonas aeruginosa and Testing for Virulence Attenuation in a Simple Mouse Model of Infection

Microorganisms are genetically versatile and diverse and have become a major source of many commercial products and biopharmaceuticals. Though some of these products are naturally produced by the organisms, other products require genetic engineering of the organism to increase the yields of production. Avirulent strains of Escherichia coli have traditionally been the preferred bacterial species for producing biopharmaceuticals; however, some products are difficult for E. coli to produce. Thus, avirulent strains of other bacterial species could provide useful alternatives for production of some commercial products. Pseudomonas aeruginosa is a common and well-studied Gram-negative bacterium that could provide a suitable alternative to E. coli. However, P. aeruginosa is an opportunistic human pathogen. Here, we detail a procedure that can be used to generate nonpathogenic strains of P. aeruginosa through sequential genomic deletions using the pEX100T-NotI plasmid. The main advantage of this method is to produce a marker-free strain. This method may be used to generate highly attenuated P. aeruginosa strains for the production of commercial products, or to design strains for other specific uses. We also describe a simple and reproducible mouse model of bacterial systemic infection via intraperitoneal injection of validated test strains to test the attenuation of the genetically engineered strain in comparison to the FDA-approved BL21 strain of E. coli.

Generation of In-Frame Gene Deletion Mutants in Pseudomonas aeruginosa and Testing for Virulence Attenuation in a Simple Mouse Model of Infection

Here, we describe a simple and reproducible protocol of mouse model of infection to evaluate the attenuation of the genetically modified strains of Pseudomonas aeruginosa in comparison to the United States Food and Drug Administration (FDA)-approved Escherichia coli for commercial applications.

Microorganisms are genetically versatile and diverse and have become a major source of many commercial products and biopharmaceuticals. Though some of ...

Markerless Gene Deletion by Floxed Cassette Allelic Exchange Mutagenesis in Chlamydia trachomatis

Chlamydia trachomatis is an obligate intracellular pathogen that has been historically difficult to genetically manipulate. Definitive progress in elucidating the mechanisms that C. trachomatis use to create and maintain a privileged intracellular niche has been limited due to a lack of genetic tools. Fortunately, there have recently been several new advances in genetic manipulation techniques. Among these is the development of fluorescence-reported allelic exchange mutagenesis (FRAEM). This method allows targeted gene deletion coupled with insertion of a selection cassette encoding antibiotic resistance and green fluorescent protein (GFP). Reliance on this strategy can be complicated when targeting genes within polycistronic operons due to the potential of polar effects on downstream genes. Floxed cassette allelic exchange mutagenesis (FLAEM), the protocol for which is described here, was developed to alleviate cassette-induced polar effects. FLAEM utilizes Cre-loxP genome editing to remove the selection cassette after targeted deletion by allelic exchange. The resulting strains contain markerless gene deletions of one or more coding sequences. This technique facilitates direct assessment of gene function and expands the repertoire of tools for genetic manipulation in C. trachomatis.

Markerless Gene Deletion by Floxed Cassette Allelic Exchange Mutagenesis in Chlamydia trachomatis

Described here is a method for targeted, markerless gene deletion in Chlamydia trachomatis using floxed cassette allelic exchange mutagenesis, FLAEM.

Chlamydia trachomatis is an obligate intracellular pathogen that has been historically difficult to genetically manipulate. Definitive progress in ...

Generation of Enterobacter sp. YSU Auxotrophs Using Transposon Mutagenesis

Prototrophic bacteria grow on M-9 minimal salts medium supplemented with glucose (M-9 medium), which is used as a carbon and energy source. Auxotrophs can be generated using a transposome. The commercially available, Tn5-derived transposome used in this protocol consists of a linear segment of DNA containing an R6K&#947; replication origin, a gene for kanamycin resistance and two mosaic sequence ends, which serve as transposase binding sites. The transposome, provided as a DNA/transposase protein complex, is introduced by electroporation into the prototrophic strain, Enterobacter sp. YSU, and randomly incorporates itself into this host&#8217;s genome. Transformants are replica plated onto Luria-Bertani agar plates containing kanamycin, (LB-kan) and onto M-9 medium agar plates containing kanamycin (M-9-kan). The transformants that grow on LB-kan plates but not on M-9-kan plates are considered to be auxotrophs. Purified genomic DNA from an auxotroph is partially digested, ligated and transformed into a pir+ Escherichia coli (E. coli) strain. The R6K&#947; replication origin allows the plasmid to replicate in pir+ E. coli strains, and the kanamycin resistance marker allows for plasmid selection. Each transformant possesses a new plasmid containing the transposon flanked by the interrupted chromosomal region. Sanger sequencing and the Basic Local Alignment Search Tool (BLAST) suggest a putative identity of the interrupted gene. There are three advantages to using this transposome mutagenesis strategy. First, it does not rely on the expression of a transposase gene by the host. Second, the transposome is introduced into the target host by electroporation, rather than by conjugation or by transduction and therefore is more efficient. Third, the R6K&#947; replication origin makes it easy to identify the mutated gene which is partially recovered in a recombinant plasmid. This technique can be used to investigate the genes involved in other characteristics of Enterobacter sp. YSU or of a wider variety of bacterial strains.

Generation of Enterobacter sp. YSU Auxotrophs Using Transposon Mutagenesis

Enterobacter sp. YSU grows in glucose minimal salts medium. Auxotrophs are generated by transforming it with a transposome which randomly inserts itself into the host genome. Mutants are found by replica plating from complex medium to minimal medium. Interrupted genes are identified by gene rescue and sequencing.

Prototrophic bacteria grow on M-9 minimal salts medium supplemented with glucose (M-9 medium), which is used as a carbon and energy source. Auxotrophs can ...

Phage-mediated Delivery of Targeted sRNA Constructs to Knock Down Gene Expression in E. coli

RNA-mediated knockdowns are widely used to control gene expression. This versatile family of techniques makes use of short RNA (sRNA) that can be synthesized with any sequence and designed to complement any gene targeted for silencing. Because sRNA constructs can be introduced to many cell types directly or using a variety of vectors, gene expression can be repressed in living cells without laborious genetic modification. The most common RNA knockdown technology, RNA interference (RNAi), makes use of the endogenous RNA-induced silencing complex (RISC) to mediate sequence recognition and cleavage of the target mRNA. Applications of this technique are therefore limited to RISC-expressing organisms, primarily eukaryotes. Recently, a new generation of RNA biotechnologists have developed alternative mechanisms for controlling gene expression through RNA, and so made possible RNA-mediated gene knockdowns in bacteria. Here we describe a method for silencing gene expression in E. coli that functionally resembles RNAi. In this system a synthetic phagemid is designed to express sRNA, which may designed to target any sequence. The expression construct is delivered to a population of E. coli cells with non-lytic M13 phage, after which it is able to stably replicate as a plasmid. Antisense recognition and silencing of the target mRNA is mediated by the Hfq protein, endogenous to E. coli. This protocol includes methods for designing the antisense sRNA, constructing the phagemid vector, packaging the phagemid into M13 bacteriophage, preparing a live cell population for infection, and performing the infection itself. The fluorescent protein mKate2 and the antibiotic resistance gene chloramphenicol acetyltransferase (CAT) are targeted to generate representative data and to quantify knockdown effectiveness.

Phage-mediated Delivery of Targeted sRNA Constructs to Knock Down Gene Expression in E. coli

We describe a method to knock down gene expression in a growing population of E. coli cells using sequence-targeted sRNA expression cassettes delivered by an M13 phagemid vector.

RNA-mediated knockdowns are widely used to control gene expression. This versatile family of techniques makes use of short RNA (sRNA) that can be ...

Generation of Marked and Markerless Mutants in Model Cyanobacterial Species

Cyanobacteria are ecologically important organisms and potential platforms for production of biofuels and useful industrial products. Genetic manipulation of cyanobacteria, especially model organisms such as Synechocystis sp. PCC6803 and Synechococcus sp. PCC7002, is a key tool for both basic and applied research. Generation of unmarked mutants, whereby chromosomal alterations are introduced into a strain via insertion of an antibiotic resistance cassette (a manipulatable fragment of DNA containing one or more genes), followed by subsequent removal of this cassette using a negative selectable marker, is a particularly powerful technique. Unmarked mutants can be repeatedly genetically manipulated, allowing as many alterations to be introduced into a strain as desired. In addition, the absence of genes encoding antibiotic resistance proteins in the mutated strain is desirable, as it avoids the possibility of 'escape' of antibiotic resistant organisms into the environment. However, detailed methods for repeated rounds of genetic manipulation of cyanobacteria are not well described in the scientific literature. Here we provide a comprehensive description of this technique, which we have successfully used to generate mutants with multiple deletions, single point mutations within a gene of interest and insertion of novel gene cassettes.

Introducing multiple genomic alterations into cyanobacteria is an essential tool in the development of strains for industrial and basic research purposes. We describe a system for generating unmarked mutants in the model cyanobacterial species Synechocystis sp. PCC6803 and marked mutants in Synechococcus sp. PCC7002.

Cyanobacteria are ecologically important organisms and potential platforms for production of biofuels and useful industrial products. Genetic manipulation ...

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

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Site-specific Bacterial Chromosome Engineering: ΦC31 Integrase Mediated Cassette Exchange (IMCE)

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

Creation of a Dense Transposon Insertion Library Using Bacterial Conjugation in Enterobacterial Strains Such As Escherichia Coli or Shigella flexneri

Mapping Bacterial Functional Networks and Pathways in Escherichia Coli using Synthetic Genetic Arrays

Genome-wide Gene Deletions in Streptococcus sanguinis by High Throughput PCR

Generation of In-Frame Gene Deletion Mutants in Pseudomonas aeruginosa and Testing for Virulence Attenuation in a Simple Mouse Model of Infection

Markerless Gene Deletion by Floxed Cassette Allelic Exchange Mutagenesis in Chlamydia trachomatis

Generation of Enterobacter sp. YSU Auxotrophs Using Transposon Mutagenesis

Phage-mediated Delivery of Targeted sRNA Constructs to Knock Down Gene Expression in E. coli

Generation of Marked and Markerless Mutants in Model Cyanobacterial Species