This method can help answer key questions in the microbiology field such as, how a gene product that is difficult to express in E.Coli purifies. The main advantages of this technique are that no enzymatic reaction, other than PCR, is the carrier, and common applications for protein purification can be used. Though this method can provide insight into protein purification in streptococcus mutans, it can also be a apprised to other microbiota species.
Demonstrating the procedure will be Dr.Mamiko Yamashita, a grad student from our laboratory. After primer design and genomic DNA extraction from streptococcus mutans, perform the first PCR using the wild-type streptococcus mutans and GFC disrupted streptococcus mutans genome as the PCR templates. Amplify the regions harboring the downstream part of the gtfC gene and those harboring the spectinomycin resistance gene using the prepared gtfC-forward and reverse primer and spc r-forward and reverse primer.
Then, electrophorese each PCR product on 1%agarose gel. Use a gel bandcutter to excise the desired DNA fragments of approximately 1, 000 base pairs and 2, 000 base pairs from the gel into microcentrifuge tubes. Add 500 microliters of solubilizing buffer into each tube and incubate them for 10 minutes at 56 degrees Celsius to dissolve the gel slices.
Purify the fragments using silica membrane-based gel extraction method. Perform a second PCR using the products of the first PCR as PCR templates with the nested-forward and nested-reverse primers. When the second PCR product cannot be confirmed by electrophoresis, another nested primer's pair should be designed.
electrophorese five microliters of the PCR mixture on the agarose gel. Confirm the generation of the appropriate amplicon of approximately 3, 000 base pairs by ethidium bromide gel staining image and proceed according to the manuscript. Now, mix five microliters of the concentrated second PCR product to the 50-microliter aloquot of ice-cold, competent wild-type streptococcus mutan cells, frozen previously.
Add the mixture into an electroporation cuvette and place the cuvette into the cuvette chamber of the electroporation apparatus. Give a single electric pulse of 1.8 kilovolts, 600 ohms, and 10 microfarads for 2.5 milliseconds to the cells. Add 500 microliters of brain-heart infusion broth into the cuvette.
Immediately spread 10 to 100 microliters of the suspension onto brain-heart infusion auger plates containing spectinomycin. Incubate the plates for two to six days at 37 degrees Celsius until colonies have grown sufficiently to be picked up. After generation of streptococcus mutans polyhistidine coding sequence incorporated strain and overnight inoculation without spectinomycin according to the manuscript, centrifuge the bacterial culture suspension for 20 minutes at 10, 000 times g at four degrees Celsius.
Transfer the culture supernatant into a three-liter glass beaker. To concentrate the proteins from the culture supernatant, place the two-liter supernatant on a magnetic stirrer and start vigorous stirring. Add 1, 122 grams of ammonium sulfate and allow the precipitate to form with vigorous stirring at four degrees Celsius over a four-hour period or overnight.Next.
centrifuge the ammonium sulfate precipitated solution at 15, 000 times g for 20 minutes at four degrees Celsius. Decant the supernatant. With a spatula, collect the precipitate and transfer it into a 200-milliliter glass beaker.
Resuspend the pellets in 35 milliliters of binding buffer. Then, transfer each approximately 25 milliliters of the suspension into a regenerated cellulose dialysis tubing. Place the dialysis tubing into 2.5 liters of the stirring binding buffer on a stirrer at four degrees Celsius to dialyze the suspension.
Replace the dialysis solution after two hours and continue dialysis overnight. Next transfer the dialyzed suspension from the tubing to centrifuge tubes and place the tubes in the centrifuge at 20, 000 times g for 10 minutes at four degrees Celsius. With section filtration equipment, pour the supernatant over a 0.2-micrometer membrane filter to filter.
Transfer the filtrate to a 75-milliliter flask. To fractionate the polyhistidine-tagged gtfSI from the filtrated suspension, first prepare an immobilized metal affinity chromatography. After equilibrating the resin according to the manuscript, close the Hoffman pinch cock.
Add five milliliters of the filtered suspension into the column to make a slurry. Then, transfer all the slurry to the remaining filtered suspension and swirl the mixture gently for 30 minutes at four degrees Celsius. Load the mixture back into the column.
Open the Hoffman pinch cock to remove the suspension by the gravity flow. Wash the IMAC resin with 20 milliliters of binding buffer and then, elute 20 milliliters of elution buffer to obtain the recombinant gtfSI. After that, load the eluate into a centrifugal ultra filtration tube and centrifuge the solution at 2, 000 times g for one to five minutes to concentrate the solution.
Empty the filtrate container and add 15 milliliters of storage buffer into the tube. Centrifuge the sample again and repeat the process two additional times. The recombinant gtfSI solution is finally concentrated to approximately one milliliter.
Transfer the concentrate into a microcentrifuge tube and store at four degrees Celsius. Continue with the rest functional restoration protocol. In this protocol, the agarose gel electrophoresis shows that the size of each amplicon from the first PCR and the second PCR corresponded with the predicted size.
Streptococcus mutans colonies were transformed with the second PCR product and plated on the brain-heart infusion auger plates containing spectinomycin. Colony PCR products run on the agarose gel shows that each amplicon was of the predicted size. Protein purified with immobilized metal affinity chromatography was observed as a single band by SDS-PAGE.
Western blot, performed using the anti-polyhistidine antibody, confirmed that the observed band was the expected polyhistidine-tagged protein of 160 kilodaltons. The sucrose-derived biofilm forming ability was only seen with streptococcus mutans wild-type and streptococcus mutans His-gtfC, which formed an adherent biofilm on the tube wall in the presence of 1%sucrose. This was not observed in streptococcus mutans delta gtfC.
However, the addition of 25 micrograms of the recombinant gtfSI restored the adherent biofilm formation ability in streptococcus mutans delta gtfC. Since the success of this method depends on the second PCR amplification, the second PCR product must be confirmed by electrophoresis. Already, histidine-type protein obtained by this method is also convening to functional acids, such as protein-protein interactions.
After its development, this technique, in combination with gene disruption, paved the way for researchers in the field of microbiology to explore gene function.
