Name: Generation of a Gene-disrupted Streptococcus mutans Strain Without Gene Cloning (Video) | JoVE Journal
Uploaded: 2017-10-23T14:00:00.000+00:00
Duration: 12 min 6 s
Description: Learn a rapid gene disruption method for Streptococcus mutans using two-step fusion PCR and electroporation without cloning. This cost-effective technique accelerates microbiology research.

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5 minutes

This work was supported by the Japan Society for the Promotion of Science [Grant Numbers 16K15860 to T.M. and 15K15777 to N.H.].

1. Primer Design
<ol>
	<li>Prepare primers for 2-step fusion PCR and verification of gene disruption. 
	NOTE: Table 1 shows the primer sequences used in this protocol. Figure 1 shows a schematic illustration of the 2-step fusion PCR method used to generate S. mutans &#916;gtfC.
	<ol>
		<li>Design primers for the replacement of the target region in the genome with the spectinomycin-resistance gene (spcr)7. This protocol replaces the entire coding region of the gtfC gene with spcr.</li>
		<li>Include 15 bases complementary to the upper and lower ends of spcr at the 5&#39; regions of up-reverse and down-forward primers, respectively. Similarly, include 15 bases complementary to the lower end of the upstream flanking regions of gtfC and the upper end of the downstream flanking regions of gtfC at the 5&#39; regions of spcr-forward and spcr-reverse primers, respectively (Figure 1A). 
		NOTE: Sequences of up-reverse, down-forward, spcr-forward, and spcr-reverse primers are automatically determined depending on the site of incorporation of the disruption construct.</li>
		<li>Design the up-forward and down-reverse primers to comprise &#62;1-kb upstream and downstream flanking regions of the gtfC gene, respectively. Set the melting temperature (Tm) of up-forward and down-reverse primers in accordance with the melting temperature of up-reverse and down-forward primers, respectively (Figure 1A). 
		NOTE: Refer to the following formula to determine Tm: Tm (&#176;C) = 2(*NA + *NT) + 4(*NC + *NG) - 5, where *N represents the number of primer nucleotides with the specified identity (A, T, C, or G).</li>
		<li>Design nested primers (N_up-forward and N_up-reverse) for the 2nd PCR (Figure 1B). 
		NOTE: Although the outermost primer pair (up-forward and down-reverse) may be used as primers for the 2nd PCR, nested primers (N_up-forward and N_up-reverse) were designed in this protocol. Nested primers are frequently required for the 2nd PCR, as detailed in the Representative Results.</li>
		<li>Design primers specific for spcr. The primers are used for colony PCR to screen for gtfC disruption.</li>
		<li>Design primers for the verification of gtfC disruption. 
		NOTE: The PCR products amplified using these primers straddle the border between gtfC or spcr and upstream or downstream flanking regions of gtfC. The primer sets &#39;gtfC up-forward and gtfC up-reverse&#39; and &#39;gtfC down-forward and gtfC down-reverse&#39; are specific for gtfC, and &#39;gtfC up-forward and spcr2-reverse&#39; and &#39;spcr2-forward and gtfC down-reverse&#39; are specific for spcr.</li>
	</ol>
	</li>
</ol>
2. Genomic DNA Extraction from S. mutans
<ol>
	<li>Streak stock S. mutans UA159 onto a brain heart infusion (BHI) agar plate. Incubate the plate overnight at 37 &#176;C under anaerobic conditions.</li>
	<li>Pick up a single colony using a sterilized toothpick and inoculate it into 5 mL of BHI broth. Incubate the S. mutans culture overnight at 37 &#176;C under anaerobic conditions.</li>
	<li>Centrifuge the bacterial cell culture for 15 min at 2,000 &#215; g. Resuspend the cell pellet in 5 mL of phosphate-buffered saline (PBS).</li>
	<li>Centrifuge for 15 min at 2,000 &#215; g. Resuspend the cell pellet in 200 &#181;L of PBS.</li>
	<li>Extract genomic DNA from the suspension using a bead-beating-base genomic DNA extraction kit according to the instructions provided by the manufacturer.
	<ol>
		<li>Transfer the suspension to a 2.0-mL sample tube containing glass beads (included in the kit). Add 750 &#181;L of lysis solution (included in the kit) to the cell suspension.</li>
		<li>Cap tightly and place the tubes symmetrically in the tube holder of the bead-beating disruption apparatus. Process at the maximum speed for 5 min.Centrifuge the bead-beaten samples for 1 min at 10,000 &#215; g. Transfer the supernatant to the spin filter (included in the kit) in a 2.0-mL collection tube and centrifuge for 1 min at 7,000 &#215; g.</li>
		<li>Add 1,200 &#181;L of DNA binding buffer (included in the kit) to the filtrate. Transfer 800 &#181;L of the mixture to the spin column (included in the kit) to a 2.0-mL collection tube and centrifuge for 1 min at 10,000 &#215;g.</li>
		<li>Discard the flow-through, add 200 &#181;L of pre-wash buffer (included in the kit) to the spin column to wash the column matrix, and centrifuge for 1 min at 10,000 &#215; g. Discard the flow-through, add 500 &#181;L of wash buffer (included in the kit) to the spin column to wash the column matrix, and centrifuge for 1 min at 10,000 &#215; g.</li>
		<li>Place the spin column into a new 1.5-mL microcentrifuge tube and add 100 &#181;L of elution buffer (included in the kit) to the column matrix.</li>
		<li>Centrifuge for 30 s at 10,000 &#215; g to elute the genomic DNA. Estimate the DNA concentration and purity by measuring the absorbance at 260 nm and 280 nmusing a spectrophotometer and confirm that the A260/A280 ratio is greater than 1.8.</li>
	</ol>
	</li>
</ol>
3. PCR Amplification
<ol>
	<li>Perform the 1st PCR using the S. mutans WT genome and the synthetic spcr gene as PCR templates (see Table 1). Amplify the upstream flanking region of the gtfC gene, the downstream flanking region of the gtfC gene, and the spcr gene using the three sets of primers described above (Figure 1A), as per Table 2 and Table 3. 
	NOTE: PCR primers, reagents, and amplification cycles are summarized in Table 1, Table 2, and Table 3, respectively.</li>
	<li>Fractionate each PCR product on a 1% agarose gel and excise the corresponding bands using a gel band cutter.</li>
	<li>Purify each amplified DNA product from the gels using a spin column- and silica membrane-based gel extraction kit.
	<ol>
		<li>Transfer the gel slices to a clean microcentrifuge tube and mix with 500 &#181;L of binding buffer (included in the kit). Incubate the mixture at 55 &#176;C for 15 min until the gel slices are completely dissolved.</li>
		<li>Place the spin column (included in the kit) into a collection tube (included in the kit), load the sample onto the spin column, and centrifuge for 30 s at 11,000 &#215; g. Discard the flow-through, add the 600 &#181;L of Wash Buffer (included in the kit) to the Spin Column to wash the silica membrane, and centrifuge for 30 s at 11,000 &#215; g.</li>
		<li>Discard the flow-through and centrifuge for 2 min at 11,000 &#215; g to dry the silica membrane.</li>
		<li>Place the spin column into a new 1.5-mL microcentrifuge tube, add 50 &#181;L Elution Buffer (included in the kit), and incubate at room temperature for 2 min.</li>
		<li>Centrifuge for 2 min at 11,000 &#215; g to elute the amplified DNA. Estimate the DNA concentration and purity by measuring the absorbance at 260 nm and 280 nmusing a spectrophotometer and confirm that the A260/A280 ratio is greater than 1.8.</li>
	</ol>
	</li>
	<li>Perform the 2nd PCR of 2-step fusion PCR using the three approximately equimolar fragments as PCR templates, as per Table 2 and Table 3. 
	NOTE: These fragments were amplified and spliced using the nested primers &#39;N_up-forward and N_down-reverse&#39; or the outermost primers &#39;up-forward and down-reverse&#39; (Figure 1B). PCR primers, reagents, and amplification cycles are summarized in Table 1, Table 2, and Table 3, respectively.</li>
	<li>Load one-tenth of the PCR reaction mixture (5 &#181;L) on a 0.8% agarose gel and compare the amplified band size with the expected band size to confirm the generation of the appropriate amplicon (Figure 1C).</li>
	<li>Concentrate the remaining final PCR product by ethanol precipitation without purification.
	<ol>
		<li>Add 55 &#181;L of ultra-pure water to the remaining PCR mixture to adjust the total volume to 100 &#181;L.Add a one-tenth volume of 3 M sodium acetate (10 &#181;L), pH 5.2, followed by 2.5 volumes of 100% ethanol (250 &#181;L). Mix and freeze for 30 min at -80 &#176;C.</li>
		<li>Centrifuge for 20 min at 15,000 &#215; g at 4 &#176;C, check for the pellet on the bottom of tube, and discard the supernatant.Wash the pellet with 1 mL of 70% ethanol, centrifuge for 5 min at 15,000 &#215; g at 4 &#176;C, and discard the supernatant. Air-dry the pellet for approximately 30 min and dissolve with 10 &#181;L of ultra-pure water.</li>
	</ol>
	</li>
</ol>
4. Competent Cell Preparation and Cell Transformation
<ol>
	<li>Streak stock S. mutans UA159 onto a BHI agar plate. Incubate the plate overnight at 37 &#176;C under anaerobic conditions.</li>
	<li>Pick up a single colony using a sterilized toothpick and inoculate it into 5 mL of BHI broth. Incubate the S. mutans culture overnight at 37 &#176;C under anaerobic conditions.</li>
	<li>Transfer 2 mL of the S. mutans culture to 50 mL of BHI broth and incubate for 6-8 h at 37 &#176;C to an optical density at 600 nm (OD600) of 0.2-0.4 under anaerobic conditions.</li>
	<li>Centrifuge the cells for 15 min at 2,000 &#215; g at 4 &#176;C, discard the supernatant, and wash the cells twice with 40 mL of ice-cold water followed by 40 mL of 10% glycerol. 
	NOTE: Residual substances affect subsequent electroporation.</li>
	<li>Aspirate the supernatant carefully with a Pasteur pipette as the adherence of the cell pellet in 10% glycerol is reduced. Resuspend the cells in 1 mL of fresh 10% glycerol, dispense 50 &#181;L of the suspension into each 1.5-mL microcentrifuge tube, and store at -80 &#176;C until just before use.</li>
	<li>Mix the 50 &#181;L aliquot of ice-cold competent cells with 5 &#181;L of the disruption construct described above in section 3. Add the mixture to electroporation cuvettes (with a 0.2 cm distance between electrodes). Employ the Staphylococcus aureus mode (1.8 kV, 600 &#937;, 10 &#181;F, 1 pulse) of the electroporation apparatus to electroporate.</li>
	<li>Suspend the cells in 500 &#181;L of BHI broth immediately after electroporation and add 50 &#181;L of the suspension to spectinomycin-containing BHI-agar plates. 
	NOTE: Extra incubation time after electroporation is not required. However, incubation for 1-2 h after electroporation may improve transformation efficiency.</li>
	<li>Incubate the plate for 2-6 days at 37 &#176;C under anaerobic conditions. 
	NOTE: S. mutans &#916;gtfB is constructed as described above. The entire coding region of the gtfB gene is replaced with the erythromycin resistance gene8 in S. mutans &#916;gtfB. Each S. mutans strain is cultured in BHI broth at 37 &#176;C under anaerobic conditions.</li>
</ol>
5. Verification of Gene Disruption and Storage
<ol>
	<li>Pick random colonies and inoculate them into the PCR mixture to perform colony PCR as per Table 2 and Table 3. PCR primers, reagents, and amplification cycles are summarized in Table 1, Table 2, and Table 3, respectively.</li>
	<li>Load the PCR reaction mixture on a 1% agarose gel to confirm the spcr-specific DNA band.</li>
	<li>Pick the positive colony using a sterilized toothpick and subculture cells in 10 mL of spectinomycin-containing BHI broth for 2 days at 37 &#176;C under anaerobic conditions.</li>
	<li>Verify the generation of S. mutans &#916;gtfC.
	<ol>
		<li>Divide the cell suspension equally. Extract genomic DNA from the cells described above (steps 2.3. to 2.5.5.). Perform PCR with genomic DNA as the PCR template, using primers for the verification of gtfC disruption (Table 1) as per Table 2 and Table 3. 
		NOTE: The PCR primers, reagents, and amplification cycles are summarized in Table 1, Table 2, and Table 3, respectively.</li>
		<li>Load the PCR mixture on a 2% agarose gel and compare the amplified band size with the expected band size to confirm the generation of the appropriate amplicon.</li>
		<li>Centrifuge the remaining cell suspension for 15 min at 2,000 &#215; g at 4 &#176;C. Resuspend the cell pellet in 5 mL of PBS.</li>
		<li>Centrifuge for 15 min at 2,000 &#215; g at 4 &#176;C. Resuspend the cell pellet in 1.5 mL of BHI broth containing 25% glycerol and store in -80&#176;Cor -20 &#176;C.</li>
	</ol>
	</li>
</ol>
6. Phenotypic Analysis
<ol>
	<li>Streak each S. mutans strain onto a BHI agar plate. Incubate the plate overnight at 37 &#176;C under anaerobic conditions.</li>
	<li>Pick up a single colony using a sterilized toothpick and inoculate it into 2 mL of BHI broth with or without an appropriate antibiotic. Incubate overnight at 37 &#176;C under anaerobic conditions.</li>
	<li>Inoculate 20 &#181;L of the overnight culture suspension into 2 mL of BHI broth containing 1% or 5% sucrose without antibiotics in a glass test tube; culture the cells with the test tube in an inclined position overnight at 37 &#176;C under anaerobic conditions.</li>
	<li>Vortex the glass test tubes for 10 s and decant culture suspensions. Wash the test tubes three times with distilled water. Add 1 mL of 0.25% Coomassie brilliant blue (CBB) to stain the biofilms on the tube wall, and then incubate for 1 min.</li>
	<li>Decant the staining solution, wash the test tubes three times with distilled water, and dry the test tubes.</li>
	<li>Evaluate both color density and extension of the CBB stained-biofilm visually to determine the biofilm-forming ability in a phenotypic analysis.</li>
</ol>

<ol><li>Horton, R. M., Hunt, H. D., Ho, S. N., Pullen, J. K., Pease, L. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Engineering+hybrid+genes+without+the+use+of+restriction+enzymes%3A+gene+splicing+by+overlap+extension%5BTitle%5D)+AND+%22Gene%22%5BJournal%5D)">Engineering hybrid genes without the use of restriction enzymes: gene splicing by overlap extension.</a> Gene. 77 (1), 61-68 (1989).</li>
<li>Horton, R. M., Cai, Z., Ho, S. M., Pease, L. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Gene+splicing+by+overlap+extension%3A+tailor-made+genes+using+the+polymerase+chain+reaction%5BTitle%5D)+AND+%22BioTechniques%22%5BJournal%5D)">Gene splicing by overlap extension: tailor-made genes using the polymerase chain reaction.</a> BioTechniques. 54 (3), 129-133 (2013).</li>
<li>Kuwayama, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((PCR-mediated+generation+of+a+gene+disruption+construct+without+the+use+of+DNA+ligase+and+plasmid+vectors%5BTitle%5D)+AND+%22Nucleic+Acids+Res%22%5BJournal%5D)">PCR-mediated generation of a gene disruption construct without the use of DNA ligase and plasmid vectors.</a> Nucleic Acids Res. 30 (2), E2(2002).</li>
<li>Sakurai, I., Mizusawa, N., Wada, H., Sato, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Digalactosyldiacylglycerol+is+required+for+stabilization+of+the+oxygen-evolving+complex+in+photosystem+II%5BTitle%5D)+AND+%22Plant+Physiol%22%5BJournal%5D)">Digalactosyldiacylglycerol is required for stabilization of the oxygen-evolving complex in photosystem II.</a> Plant Physiol. 145 (4), 1361-1370 (2007).</li>
<li>Aoki, H., Shiroza, T., Hayakawa, M., Sato, S., Kuramitsu, H. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cloning+of+a+Streptococcus+mutans+glucosyltransferase+gene+coding+for+insoluble+glucan+synthesis%5BTitle%5D)+AND+%22Infect+Immun%22%5BJournal%5D)">Cloning of a Streptococcus mutans glucosyltransferase gene coding for insoluble glucan synthesis.</a> Infect Immun. 53 (3), 587-594 (1986).</li>
<li>Hanada, N., Kuramitsu, H. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Isolation+and+characterization+of+the+Streptococcus+mutans+gtfC+gene%2C+coding+for+synthesis+of+both+soluble+and+insoluble+glucans%5BTitle%5D)+AND+%22Infect+Immun%22%5BJournal%5D)">Isolation and characterization of the Streptococcus mutans gtfC gene, coding for synthesis of both soluble and insoluble glucans.</a> Infect Immun. 56 (8), 1999-2005 (1988).</li>
<li>LeBlanc, D. J., Lee, L. N., Inamine, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cloning+and+nucleotide+base+sequence+analysis+of+a+spectinomycin+adenyltransferase+AAD%289%29+determinant+from+Enterococcus+faecalis%5BTitle%5D)+AND+%22Antimicrob+Agents+Chemother%22%5BJournal%5D)">Cloning and nucleotide base sequence analysis of a spectinomycin adenyltransferase AAD(9) determinant from Enterococcus faecalis.</a> Antimicrob Agents Chemother. 35 (9), 1804-1810 (1991).</li>
<li>Macrina, F. L., Tobian, J. A., Jones, K. R., Evans, R. P., Clewell, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+cloning+vector+able+to+replicate+in+Escherichia+coli+and+Streptococcus+sanguis%5BTitle%5D)+AND+%22Gene%22%5BJournal%5D)">A cloning vector able to replicate in Escherichia coli and Streptococcus sanguis.</a> Gene. 19 (3), 345-353 (1982).</li>
<li>Murata, T., Hanada, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Contribution+of+chloride+channel+permease+to+fluoride+resistance+in+Streptococcus+mutans%5BTitle%5D)+AND+%22FEMS+Microbiol+Lett%22%5BJournal%5D)">Contribution of chloride channel permease to fluoride resistance in Streptococcus mutans.</a> FEMS Microbiol Lett. 363 (11), (2016).</li>
<li>Perry, D., Wondrack, L. M., Kuramitsu, H. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Genetic+transformation+of+putative+cariogenic+properties+in+Streptococcus+mutans%5BTitle%5D)+AND+%22Infect+Immun%22%5BJournal%5D)">Genetic transformation of putative cariogenic properties in Streptococcus mutans.</a> Infect Immun. 41 (2), 722-727 (1983).</li>
<li>Lau, P. C., Sung, C. K., Lee, J. H., Morrison, D. A., Cvitkovitch, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((PCR+ligation+mutagenesis+in+transformable+streptococci%3A+application+and+efficiency%5BTitle%5D)+AND+%22J+Microbiol+Methods%22%5BJournal%5D)">PCR ligation mutagenesis in transformable streptococci: application and efficiency.</a> J Microbiol Methods. 49 (2), 193-205 (2002).</li>
<li>Ge, X., Xu, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Genome-wide+gene+deletions+in+Streptococcus+sanguinis+by+high+throughput+PCR%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Genome-wide gene deletions in Streptococcus sanguinis by high throughput PCR.</a> J Vis Exp. (69), (2012).</li>
</ol>

The authors have nothing to disclose.

In the present protocol, the primers for the 1st PCR must be designed to amplify approximately 1 kb upstream and downstream flanking regions of the target region in the genome. Such long flanking sequences are necessary to improve the efficiency of homologous recombination.
The sequences of the primers (up-reverse, down-forward, spcr-forward, and spcr-reverse) in this protocol were automatically determined based on the site of incorporation of...

Gene function analysis typically involves a phenotypic comparison between a wild-type strain and a strain in which a particular gene of interest has been disrupted. This gene-disruption technique has been utilized for gene function analyses in a wide range of taxa, from bacteria to mammals. A gene-disruption construct is necessary for the generation of the gene-disrupted strain; this deoxyribonucleic acid (DNA) construct consists of the antibiotic resistance marker gene fused to the upstream and downstream flanking regions of the target gene, and is incorporated into the genome via homologous recombination. The gene-disrupted strain may be isolated using selective media containing the antibiotic. The conventional method used for the construction of the gene-disrupted strain requires complex steps, such as the amplification of the target gene by polymerase chain reaction (PCR), ligation of the gene into a suitable plasmid, digestion with restriction enzymes, and religation to insert the antibiotic-resistance marker gene. This process is time-consuming, taking several days to several weeks, depending on the success of each step. In addition, the design of disruption constructs is dependent on the restriction enzyme sites of the target gene. To resolve these issues, PCR-based DNA splicing methods1,2 for the design of gene-disrupted mutants of Dictyostelium discoideum3 and Synechocystis sp. PCC68034 have been reported. In the present study, a method was developed to generate a gene-disrupted streptococcal strain in a relatively rapid, facile, and inexpensive manner, utilizing a modified PCR-based method (designated 2-step fusion PCR) for the construction of the disruption construct and electroporation for genetic transformation.
The 2-step fusion PCR requires genomic DNA, the antibiotic resistance marker gene as a PCR template, and four pairs of appropriately designed oligonucleotide primers. In the first step (1st PCR), a 1-kb upstream flanking region of the target gene, a 1-kb downstream flanking region of the target gene, and the antibiotic-resistance marker gene are amplified by PCR. The 5&#39; regions of both the reverse primer and the forward primer, for amplification of the upstream and downstream flanking regions, respectively, include 15 bases complementary to the ends of the marker gene. The 5&#39; regions of both forward and reverse primers for marker gene amplification similarly include 15 bases complementary to the lower end of the upstream flanking region of the target gene and the upper end of the downstream flanking region of the target gene, respectively. Therefore, the three amplified fragments contain overlapping 30-base pair regions at the fusion site. In the second step (2nd PCR), PCR is performed with nested PCR primers using the three fragments amplified by the 1st PCR as templates. The complementary regions of the templates are additionally linked to each other via the 2nd PCR. Finally, the construct for homologous recombination is introduced to the wild-type strain by electroporation. Successful gene disruption may be verified by PCR using specific primers. Although the disruption construct is amplified using high-fidelity DNA polymerase, additional enzymatic reactions, such as DNA ligation or DNA digestion, are not necessary to generate the construct. In addition, a deleted or preserved region on the genome may be flexibly chosen according to primer design.
In the present work, deletion of the entire coding region of the gtfC gene, which encodes glucosyltransferase in Streptococcus mutans, was performed to demonstrate the rapid, easy gene disruption method in a streptococcal species. Additionally, a gtfB-disrupted strain was generated in the same manner. The glucosyltransferases encoded by both gtfC and gtfB contribute to cariogenic dental biofilm development5,6. The biofilm-forming abilities of the wild-type strain (S. mutans WT), gtfC-disrupted strain (S. mutans &#916;gtfC), and gtfB-disrupted strain (S. mutans &#916;gtfB) were evaluated for comparative phenotypic analyses. This protocol extends the application of a two-step method for gene disruption to an additional species and may be modified for studies of gene function in a wider range of taxa.

<table><tbody><tr><td>&lt;em&gt;Streptococus mutans&lt;/em&gt;</td><td>Stock strain in the lab.</td><td></td><td>Wild-type strain (Strain UA159)</td></tr><tr><td>Genomic DNA extraction kit</td><td>Zymo Research</td><td>D6005</td><td></td></tr><tr><td>Bead-beating disruption apparatus</td><td>Taitec</td><td>GM-01</td><td></td></tr><tr><td>Synthetic genes</td><td>Eurofins Genomics</td><td>Custom-ordered</td><td>Spectinomycin- and erythromycin-resistance gene</td></tr><tr><td>Thermal cycler</td><td>Astec</td><td>PC-708</td><td></td></tr><tr><td>PCR primers</td><td>Eurofins Genomics</td><td>Custom-ordered</td><td>35 bases in length</td></tr><tr><td>DNA polymerase</td><td>Takara</td><td>R045A</td><td>High-fidelity DNA polymerase</td></tr><tr><td>Gel extraction kit</td><td>Nippon Genetics</td><td>FG-91202</td><td>DNA extraction from agarose gel</td></tr><tr><td>Gel band cutter</td><td>Nippon Genetics</td><td>FG-830</td><td></td></tr><tr><td>Spectrophotometer</td><td>Beckman Coulter</td><td>DU 800</td><td></td></tr><tr><td>Electroporator</td><td>Bio-rad</td><td>1652100</td><td></td></tr><tr><td>Electroporation cuvette</td><td>Bio-rad</td><td>1652086</td><td>0.2-cm gap</td></tr><tr><td>Anaeropack</td><td>Mitsubishi Gas Chemical</td><td>A-03</td><td>Anaerobic culture system</td></tr><tr><td>Brain heart infusion broth</td><td>Becton, Dickinson</td><td>237500</td><td>Bacterial culture media</td></tr><tr><td>Spectinomycin</td><td>Wako</td><td>195-11531</td><td>Antibiotics</td></tr><tr><td>Erythromycin</td><td>Wako</td><td>057-07151</td><td>Antibiotics</td></tr><tr><td>Sucrose</td><td>Wako</td><td>196-00015</td><td>For biofilm development</td></tr><tr><td>CBB R-250</td><td>Wako</td><td>031-17922</td><td>For biofilm staining</td></tr></tbody></table>

generation of a gene-disrupted streptococcus mutans strain without gene cloning

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.

Figure 2 shows that the size of each product from the 1st PCR, as observed on the 1% agarose gel, was in good agreement with the predicted size of approximately 1 kb. Figure 3 shows the gel electrophoresis analysis of the products of the 2nd PCR. Figure 4 shows S. mutans colonies transformed with the disruption construct, and a gel electrophoresis analysis of the colony...

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 ...

generation-gene-disrupted-streptococcus-mutans-strain-without-gene

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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.

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

Source: Lea-Smith, D. J., et al. Generation of Marked and Markerless Mutants in Model Cyanobacterial Species. J. Vis. Exp. (2016)The video demonstrates a method for generating antibiotic-marked mutants in cyanobacteria. The protocol begins with culturing cyanobacteria, followed by centrifugation and washing steps that preserve surface pili. A plasmid carrying an antibiotic resistance gene flanked by homologous sequences is then introduced. During incubation, the plasmid is internalized via pili...

Generation of Antibiotic-Marked Mutants in Cyanobacteria via Homologous Recombination

Source: Saragliadis, A., et al. Producing Gene Deletions in Escherichia coli by P1 Transduction with Excisable Antibiotic Resistance Cassettes. J. Vis. Exp. (2018)This video demonstrates the phage-mediated transfer of donor DNA carrying an excisable antibiotic resistance cassette into Escherichia coli. The integrated cassette replaces the target gene via homologous recombination, resulting in a stable gene deletion.

Generating Gene Deletions in E. coli via Phage-Mediated Transfer of an Excisable Antibiotic Cassette

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.

Generation of Enterobacter sp. YSU Auxotrophs Using Transposon Mutagenesis

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.

Generation of Marked and Markerless Mutants in Model Cyanobacterial Species

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

Described here is a simple method for the purification of a gene product in Streptococcus mutans. This technique may be advantageous in the purification of proteins, especially membrane proteins and high molecular mass proteins, and can be used with various other bacterial species.

Purification of a High Molecular Mass Protein in Streptococcus mutans

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.

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

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

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

Here, we describe the construction of null mutants of Aeromonas in specific glycosyltransferases or regions containing glycosyltransferases, the motility assays, and flagella purification performed to establish the involvement and function of their encoded enzymes in the biosynthesis of a glycan, as well as the role of this glycan in bacterial pathogenesis.

Generation of Null Mutants to Elucidate the Role of Bacterial Glycosyltransferases in Bacterial Motility

Learn a rapid gene disruption method for Streptococcus mutans using two-step fusion PCR and electroporation without cloning. This cost-effective technique accelerates microbiology research.

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

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