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
