This protocol is significant because it allows genetic modification of a previously not-tractable serotype, making it possible to research and gain understanding of the serotype. This opens avenues for biological work such as vaccine development. Demonstrating the procedure will be Vanessa S.Terra, an assistant professor from the London School of Hygiene and Tropical Medicine.
To begin generate plasmid pSD1 by performing a ligation following the pGEM-T Easy System I manufacturer instructions. Incubate the reaction overnight at four degree Celsius. On the next day, transform chemically competent E.coli Dh5-alpha with pSD1, incubating 50 microliters of the cells with three microliters of pSD1 ligation reaction for 15 minutes on ice, then expose the cells to thermic shock and place the cells on ice for two minutes.
Remove the cells from ice and add 350 microliters of SOC media then incubate the culture for two hours at 37 degrees Celsius and 120 RPM. Plate the transformation on Luria-Bertani agar supplemented with 0.4 millimolar IPTG, 0.24 milligrams per milliliter X-Gal for blue-white selection, and 100 micrograms per milliliter ampicillin to ensure all colonies growing on the plate have the plasmid backbone. Pick three white colonies which contain pSD1 and set up overnight growths in 10 milliliters of L-B supplemented with 100 micrograms per milliliter ampicillin.
Incubate the cultures overnight at 37 degrees Celsius with shaking. On the next day, centrifuge the cultures at 3, 082 G and use the pellet for plasmid extraction. After extracting the plasmid DNA, set up a BamH1 restriction digestion for both pSD1 plasmid and the spectinomycin cassette.
Incubate the restriction digestion reactions and controls at 37 degree Celsius for three hours. When the restriction is finished, analyze the products by electrophoresis then excise the correct band and purify it using a commercial kit. Next, run a ligation reaction using the BamH1-digested pSD1 and spectinomycin.
This will generate the plasmid pSD2. Transform pSD2 into chemically-competent E.coli DH5-alpha as previously described. Then, select the transformants carrying plasmid pSD2 based on their ability to grow in LBA supplemented with 100 micrograms per milliliter of spectinomycin and ampicillin and extract the plasmid DNA.
Prepare an overnight culture of S.pneumoniae 519/43 in BHI and allow it to grow statically at 37 degrees Celsius and 5%carbon dioxide. On the next day, dilute the cultures 1:50 and 1:100 in 10 milliliters of fresh BHI broth. Incubate the culture statically at 37 degrees Celsius until the OD at 595 nanometers is between 0.05 and 0.1.
Once the appropriate OD is reached, take 860 microliters of culture and transfer it into a microcentrifuge tube. Add 100 microliters of 100-millimolar sodium hydroxide, 10 microliters of 20%BSA, 10 microliters of 100-millimolar calcium chloride, two microliters of 50 nanograms per milliliter CSP1 and 500 nanograms of pSD2. Incubate the reaction statically at 37 degrees Celsius for three hours.
Then, plate 330 microliters onto 5%blood agar plates supplemented with 100 micrograms per milliliter spectinomycin every hour over a three-hour incubation period. Then, incubate the plates overnight at 37 degrees Celsius and 5%carbon dioxide. Patch spectinomycin-resistant colonies onto another blood agar plate supplemented with 100 micrograms per milliliter spectinomycin and onto blood agar plates supplemented with 100 micrograms per milliliter of ampicillin, which tests for the presence of the plasmid backbone.
Incubate both sets of plates overnight. Correct assembly of pSD2 was confirmed by restriction digestion. pSD2 was then used to transform 519.43 wild-type cells.
Colonies growing on spectinomycin after an overnight incubation were considered positive transformants. Phenotypic confirmation of the pneumolysin mutation was performed by assessing hemolytic activity for D39, 519/43 wild-type, and mutant 519/43 delta ply. This was compared to hemolysis of red blood cells by 0.5%saponin, which is considered 100%The mutant lost its ability to lyse red blood cells.
All positive colonies were further confirmed by sequencing to assess the location of the insertion. Primers with binding sites outside of the homology region were used. When performing the transformation reaction, it is important to add the reagents in the correct order.
