Name: generation of a gene-disrupted streptococcus mutans strain without gene cloning
Uploaded: 2017-10-23T14:00:00.000+00:00
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Description: Watch this Scientific Journal Video about Generation of a Gene-disrupted Streptococcus mutans Strain Without Gene Cloning at JoVE.com

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/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>

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

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 ...

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7.6K Views.  Tsurumi University School of Dental Medicine. 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.

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

From a Natural Product to Its Biosynthetic Gene Cluster: A Demonstration Using Polyketomycin from Streptomyces diastatochromogenes T&#252;6028

Streptomyces strains are known for their capability to produce a lot of different compounds with various bioactivities. Cultivation under different conditions often leads to the production of new compounds. Therefore, production cultures of the strains are extracted with ethyl acetate and the crude extracts are analyzed by HPLC. Furthermore, the extracts are tested for their bioactivity by different assays. For structure elucidation the compound of interest is purified by a combination of different chromatography methods.
Genome sequencing coupled with genome mining allows the identification of a natural product biosynthetic gene cluster using different computer programs. To confirm that the correct gene cluster has been identified, gene inactivation experiments have to be performed. The resulting mutants are analyzed for the production of the particular natural product. Once the correct gene cluster has been inactivated, the strain should fail to produce the compound.
The workflow is shown for the antibacterial compound polyketomycin produced by Streptomyces diastatochromogenes T&#252;6028. Around ten years ago, when genome sequencing was still very expensive, the cloning and identification of a gene cluster was a very time-consuming process. Fast genome sequencing combined with genome mining accelerates the trial of cluster identification and opens up new ways to explore biosynthesis and to generate novel natural products by genetic methods. The protocol described in this paper can be assigned to any other compound derived from a Streptomyces strain or another microorganism.

Here we present a detailed protocol of (A) the identification of a natural product with antibiotic activity, (B) the purification of the compound, (C) the first model of its biosynthesis, (D) genome sequencing/-mining and the (E) verification of the biosynthetic gene cluster.

Streptomyces strains are known for their capability to produce a lot of different compounds with various bioactivities. Cultivation under different ...

The Green Monster Process for the Generation of Yeast Strains Carrying Multiple Gene Deletions

Phenotypes for a gene deletion are often revealed only when the mutation is tested in a particular genetic background or environmental condition1,2. There are examples where many genes need to be deleted to unmask hidden gene functions3,4. Despite the potential for important discoveries, genetic interactions involving three or more genes are largely unexplored. Exhaustive searches of multi-mutant interactions would be impractical due to the sheer number of possible combinations of deletions. However, studies of selected sets of genes, such as sets of paralogs with a greater a priori chance of sharing a common function, would be informative.
In the yeast Saccharomyces cerevisiae, gene knockout is accomplished by replacing a gene with a selectable marker via homologous recombination. Because the number of markers is limited, methods have been developed for removing and reusing the same marker5,6,7,8,9,10. However, sequentially engineering multiple mutations using these methods is time-consuming because the time required scales linearly with the number of deletions to be generated.
Here we describe the Green Monster method for routinely engineering multiple deletions in yeast11. In this method, a green fluorescent protein (GFP) reporter integrated into deletions is used to quantitatively label strains according to the number of deletions contained in each strain (Figure 1). Repeated rounds of assortment of GFP-marked deletions via yeast mating and meiosis coupled with flow-cytometric enrichment of strains carrying more of these deletions lead to the accumulation of deletions in strains (Figure 2). Performing multiple processes in parallel, with each process incorporating one or more deletions per round, reduces the time required for strain construction.
The first step is to prepare haploid single-mutants termed 'ProMonsters,' each of which carries a GFP reporter in a deleted locus and one of the 'toolkit' loci&#x2014;either Green Monster GMToolkit-a or GMToolkit-&alpha; at the can1&Delta; locus (Figure 3). Using strains from the yeast deletion collection12, GFP-marked deletions can be conveniently generated by replacing the common KanMX4 cassette existing in these strains with a universal GFP-URA3 fragment. Each GMToolkit contains: either the a- or &alpha;-mating-type-specific haploid selection marker1 and exactly one of the two markers that, when both GMToolkits are present, collectively allow for selection of diploids.
The second step is to carry out the sexual cycling through which deletion loci can be combined within a single cell by the random assortment and/or meiotic recombination that accompanies each cycle of mating and sporulation.

The Green Monster method enables the rapid assembly of multiple deletions marked with a reporter gene encoding green fluorescent protein. This method is based on driving yeast strains through repeated cycles of sexual assortment of deletions and fluorescence-based enrichment of cells carrying more deletions.

Phenotypes  for a gene deletion are often revealed only when the mutation is tested in a  particular genetic background or environmental condition1,2. ...

Biology

Purification of a High Molecular Mass Protein in Streptococcus mutans

Elucidation of a gene&#39;s function typically involves comparison of phenotypic traits of wild-type strains and strains in which the gene of interest has been disrupted. Loss of function following gene disruption is subsequently restored by exogenous addition of the product of the disrupted gene. This helps to determine the function of the gene. A method previously described involves generating a gtfC gene-disrupted Streptococcus mutans strain. Here, an undemanding method is described for purifying the gtfC gene product from the newly generated S. mutans strain following the gene disruption. It involves the addition of a polyhistidine-coding sequence at the 3&#8242; end of the gene of interest, which allows simple purification of the gene product using immobilized metal affinity chromatography. No enzymatic reactions other than PCR are required for the genetic modification in this method. The restoration of the gene product by exogenous addition after gene disruption is an efficient method for determining gene function, which may also be adapted to different species.

Purification of a High Molecular Mass Protein in Streptococcus mutans

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.

Elucidation of a gene&#39;s function typically involves comparison of phenotypic traits of wild-type strains and strains in which the gene of interest has ...

Immunology and Infection

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 ...

Constructing Mutants in Serotype 1 Streptococcus pneumoniae strain 519/43

Streptococcus pneumoniae serotype 1 remains a huge problem in low-and-middle income countries, particularly in sub-Saharan Africa. Despite its importance, studies in this serotype have been hindered by the lack of genetic tools to modify it. In this study, we describe a method to genetically modify a serotype 1 clinical isolate (strain 519/43). Interestingly, this was achieved by exploiting the Pneumococcus&#8217; ability to naturally acquire DNA. However, unlike most pneumococci, the use of linear DNA was not successful; to mutate this important strain, a suicide plasmid had to be used. This methodology has provided the means for a deeper understanding of this elusive serotype, both in terms of its biology and pathogenicity. To validate the method, the major known pneumococcal toxin, pneumolysin, was mutated because it has a well-known and easy to follow phenotype. We showed that the mutant, as expected, lost its ability to lyse red blood cells. By being able to mutate an important gene in the serotype of interest, we were able to observe different phenotypes for loss of function mutants upon intraperitoneal and intranasal infections from the ones observed for other serotypes. In summary, this study proves that strain 519/43 (serotype 1) can be genetically modified.

Constructing Mutants in Serotype 1 Streptococcus pneumoniae strain 519/43

Here, we describe a S. pneumoniae serotype 1 strain 519/43 that can be genetically modified by using its ability to naturally acquire DNA and a suicide-plasmid. As proof of principle, an isogenic mutant in the pneumolysin (ply) gene was made.

Streptococcus pneumoniae serotype 1 remains a huge problem in low-and-middle income countries, particularly in sub-Saharan Africa. Despite its importance, ...

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

The study of glycosylation in prokaryotes is a rapidly growing area. Bacteria harbor different glycosylated structures on their surface whose glycans constitute a strain-specific barcode. The associated glycans show higher diversity in sugar composition and structure than those of eukaryotes and are important in bacterial-host recognition processes and interaction with the environment. In pathogenic bacteria, glycoproteins have been involved in different stages of the infectious process, and glycan modifications can interfere with specific functions of glycoproteins. However, despite the advances made in the understanding of glycan composition, structure, and biosynthesis pathways, understanding of the role of glycoproteins in pathogenicity or interaction with the environment remains very limited. Furthermore, in some bacteria, the enzymes required for protein glycosylation are shared with other polysaccharide biosynthetic pathways, such as lipopolysaccharide and capsule biosynthetic pathways. The functional importance of glycosylation has been elucidated in several bacteria through mutation of specific genes thought to be involved in the glycosylation process and the study of its impact on the expression of the target glycoprotein and the modifying glycan. Mesophilic Aeromonas have a single and O-glycosylated polar flagellum. Flagellar glycans show diversity in carbohydrate composition and chain length between Aeromonas strains. However, all strains analyzed to date show a pseudaminic acid derivative as the linking sugar that modifies serine or threonine residues. The pseudaminic acid derivative is required for polar flagella assembly, and its loss has an impact on adhesion, biofilm formation, and colonization. The protocol detailed in this article describes how the construction of null mutants can be used to understand the involvement of genes or genome regions containing putative glycosyltransferases in the biosynthesis of a flagellar glycan. This includes the potential to understand the function of the glycosyltransferases involved and the role of the glycan. This will be achieved by comparing the glycan deficient mutant to the wild-type strain.

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.

The study of glycosylation in prokaryotes is a rapidly growing area. Bacteria harbor different glycosylated structures on their surface whose glycans ...

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 ...

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 ...

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

Summary

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Chapters in this video

From a Natural Product to Its Biosynthetic Gene Cluster: A Demonstration Using Polyketomycin from Streptomyces diastatochromogenes Tü6028

The Green Monster Process for the Generation of Yeast Strains Carrying Multiple Gene Deletions

Purification of a High Molecular Mass Protein in Streptococcus mutans

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

Constructing Mutants in Serotype 1 Streptococcus pneumoniae strain 519/43

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

Generation of Enterobacter sp. YSU Auxotrophs Using Transposon Mutagenesis

Generation of Marked and Markerless Mutants in Model Cyanobacterial Species