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)<sup class=...

<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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+hybrid+genes+without+the+use+of+restriction+enzymes:+gene+splicing+by+overlap+extension.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+splicing+by+overlap+extension:+tailor-made+genes+using+the+polymerase+chain+reaction.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PCR-mediated+generation+of+a+gene+disruption+construct+without+the+use+of+DNA+ligase+and+plasmid+vectors.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Digalactosyldiacylglycerol+is+required+for+stabilization+of+the+oxygen-evolving+complex+in+photosystem+II.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cloning+of+a+Streptococcus+mutans+glucosyltransferase+gene+coding+for+insoluble+glucan+synthesis.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+characterization+of+the+Streptococcus+mutans+gtfC+gene,+coding+for+synthesis+of+both+soluble+and+insoluble+glucans.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cloning+and+nucleotide+base+sequence+analysis+of+a+spectinomycin+adenyltransferase+AAD(9)+determinant+from+Enterococcus+faecalis.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+cloning+vector+able+to+replicate+in+Escherichia+coli+and+Streptococcus+sanguis.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contribution+of+chloride+channel+permease+to+fluoride+resistance+in+Streptococcus+mutans.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+transformation+of+putative+cariogenic+properties+in+Streptococcus+mutans.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PCR+ligation+mutagenesis+in+transformable+streptococci:+application+and+efficiency.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+gene+deletions+in+Streptococcus+sanguinis+by+high+throughput+PCR.">Genome-wide gene deletions in Streptococcus sanguinis by high throughput PCR.</a> J Vis Exp. (69), (2012).</li></ol>

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>Streptococus mutans</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

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...

Watch this Scientific Journal Video about Generation of a Gene-disrupted Streptococcus mutans Strain Without Gene Cloning at JoVE.com

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

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

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 ...

The overall goal of this procedure is generate targeted gene-disrupted Streptococcus mutans strains using a two-step fusion PCR and electroporation.This method can help answer key questions in the microbiology field such as how a gene of interest works.The main advantage of this technique is that no enzymatic reaction other than PCR is required and competent cell preparation for electroporation is quite simple.Though this method can provide insight into Streptococcus mutans gene disruption technique, it can also be applied to other microbial species such as Staphylococcus aureus.Generally individuals new to this method will struggle because they are not familiar with the effectiveness of nested primers for the second PCR.Demonstrating the procedure will be Doctor Ayako Okada, a research instructor from our laboratory.To begin this procedure streak stock S.mutans UA159 onto a brain heart infusion or BHI agar plate and incubate the plate overnight at 37 degrees Celsius under anaerobic conditions.On the following day, use a sterilized toothpick to pick a single colony and inoculate it into five milliliters of BHI broth.Incubate the S.mutans culture overnight at 37 degrees Celsius under anaerobic conditions.Next, centrifuge the culture at 2000 times G for 15 minutes.Remove the supernatant and resuspend the cell pellet in five milliliters of PBS.Centrifuge again at 2000 times G for 15 minutes.Resuspend the resulting cell pellet in 200 microliters of PBS.Extract genomic DNA from the suspension using a bead-beating based genomic DNA extraction kit.First transfer the cell suspension to a two-milliliter sample tube containing glass beads.Then add 750 microliters of lysis solution to the cell suspension.Cap the tubes tightly and place them symmetrically in the tube holder of the bead-beating disruption apparatus.Process at the maximum speed for five minutes.Centrifuge the bead-beaten samples at 10, 000 times G for one minute.Transfer the supernatant to a spin column in a two-milliliter collection tube and centrifuge at 7000 times G for one minute.Add 1200 microliters of DNA-binding buffer to the filtrate and transfer 800 microliters of the mixture to the spin column in a two-milliliter collection tube.Centrifuge at 10, 000 times G for one minute.Discard the flow-through.Add 200 microliters of prewash buffer to the spin column to wash the column matrix and centrifuge of 10, 000 times G for one minute.Discard the flow-through, add 500 microliters of wash buffer to the spin column to wash the column matrix and centrifuge again.Place each spin column into a new 1.5-milliliter microcentrifuge tube and add 100 microliters of elution buffer to the column matrix.Centrifuge at 10, 000 times G for 30 seconds to elute the genomic DNA.Estimate the DNA concentration and purity by measuring the absorbance at 260 nanometers and 280 nanometers using a spectrophotometer.The first PCR is performed using the S.mutans wild type genome and the synthetic streptomycin resistance gene as PCR templates.Three sets of primers are used to amplify respectively, the upstream flanking region of the glycosyltransferase C or gtfC gene, the downstream flanking region of the gtfC gene and the spectinomycin resistance gene.Fractionate each PCR product on a 1%agarose gel.The size of each product from the first PCR should be approximately one KB.Use a gel band cutter to excise the corresponding bands and purify each amplified DNA product as outlined in the text protocol.The next step is to use the three approximately equal molar fragments as PCR templates to perform the second PCR.These fragments are amplified and spliced using appropriate primers to produce the disruption construct.Since the success of this procedure depends on the amplification of the disruption construct by second PCR, it is important to confirm the second PCR product by electrophoresis.Load 1/10 of the PCR reaction mixture on a 0.8%agarose gel to confirm that the amplicon generated has the predicted band size.In this example, nested primers gave much better amplification.Transfer the remaining PCR mixture to a new 1.5-milliliter microcentrifuge tube and add 55 microliters of ultrapure water to adjust the total volume to 100 microliters.Add 1/10 volume of three molar sodium acetate pH 5.2, followed by 2.5 volumes of 100%ethanol and mix.Freeze at minus 80 degrees Celsius for 30 minutes.Centrifuge at 15, 000 times G at four degrees Celsius for 20 minutes.When the centrifugation is done, check for the pellet at the bottom of the tube.Discard the supernatant.Wash the pellet with one milliliter of 70%ethanol and centrifuge at 15, 000 times G at four degrees Celsius for five minutes.Discard the supernatant and air dry the pellet for approximately 30 minutes.Lastly, dissolve the pellet with 10 microliters of ultrapure water.Competent S.mutans UA159 cells were previously prepared and stored as 50 microliter aliquots at minus 80 degrees Celsius.Mix each 50 microliter aliquot of ice cold competent cells with five microliters of the disruption construct.Add the mixture to electroporation cuvettes with a 0.2 centimeter distance between electrodes.Perform electroporation using the Staphylococcus aureus mode of the electroporation apparatus.Immediately after electroporation, suspend the cells in 500 microliters of BHI broth and add 50 microliters of the suspension to spectinomycin-containing BHI agar plates.Incubate the plates for two to six days at 37

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7.6K vistas.  Tsurumi University School of Dental Medicine. The overall goal of this procedure is generate targeted gene-disrupted Streptococcus mutans strains using a two-step fusion PCR and electroporation.This method can help answer key questions in the microbiology field such as how a gene of interest works.The main advantage of this technique is that no enzymatic reaction other than PCR is required and competent cell preparation for electroporation is quite simple.Though this method can provide insight into Streptococcus mutans gene disruption te...