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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.
Virus-bacterial interactions can be important for successful viral infection and stimulation of a host immune response. But currently, highly concentrated and purified stock of human norovirus cannot be produced in the lab. This technique can be used with any virus that binds to bacteria, for which either virus-like particles or live virus are available. It enables us to quantify interactions between these viruses and bacteria. Inoculate five milliliters of liquid medium with a single isolated colony of Enterobacter cloacae from an agar plate directly from frozen glycerol stock. Grow the bacteria overnight.
The following day, transfer 1.3 milliliter aliquots of the culture into two separate 1.5 milliliter centrifuge tubes. Centrifuge the tubes at 10, 000 x g for five minutes. Remove the supernatants, then wash the samples twice using one milliliter of sterile 1X PBS for each wash. Centrifuge the samples again at 10, 000 x g for five minutes. Remove the supernatant and resuspend the pellet in 1.3 milliliters of sterile 1X PBS. Beginning with 0.5 milliliters of washed culture, serially dilute the bacteria in 1X PBS from 10 to the minus one to 10 to the minus four. Using a spectrophotometer, measure the optical density of the washed undiluted culture and each of the four dilutions at 600 nanometers. Perform 10-fold serial dilutions in 1X PBS for each of the previous dilutions. Spread 100 microliters of the last three dilutions for each series onto agar plates to determine the number of colony forming units per milliliter for each sample. Allow the plates to dry at room temperature for five minutes. Invert the plates and incubate them in the incubator. After preparing the reagents, antibodies, and bacteria as described in the manuscript, assemble all the materials in a BSL-2 biosafety cabinet.
Virus-like particles for human norovirus are listed as BSL-2 pathogens and all work perform using VLP should be conducted in a certified biosafety cabinet. Add 10 micrograms of human norovirus VLPs to each tube of bacteria and mix thoroughly by pipetting. Incubate the tubes for one hour at 37 degree Celsius with constant rotation. After incubation, centrifuge the tubes at 10, 000 x g for five minutes. Aspirate and discard the supernatant and resuspend the bacterial pellet in one milliliter of PBS. After repeating the wash steps once, centrifuge the tubes again at 10, 000 X g for five minutes. Discard the supernatant and resuspend the VLP-bacteria pellet in 150 microliters of 5%blocking buffer. A common error that occurs is that tubes are swapped and the wrong treatment is added. To prevent this, arrange all tubes in lines that correspond to the correct treatment and add one treatment all at once to the tubes. For each bacterial sample, prepare 50 microliters of diluted human norovirus GII antibody. Using 5%blocking buffer, dilute the antibody one to 125 for E.cloacae samples. Prepare 50 microliters of diluted isotype antibody for each bacterial sample using the same dilution ratios. Divide each attachment assay sample into 350 microliter aliquots by transferring them into clean 1.5 milliliter centrifuge tubes.
To create unstained controls, add 50 microliters of blocking buffer to the first aliquot from each bacterial sample. For the stained samples, add 50 microliters of the GII antibody dilution to the second set of aliquots. Create the isotype controls by adding 50 microliters of the diluted isotype antibody to the third set of aliquots. Incubate all the samples on ice and in the dark for 30 minutes. Then centrifuge all the samples at 10, 000 X g for five minutes. Discard the supernatants and resuspend each sample in 150 microliters of FCSB. Again, centrifuge all the samples at 10, 000 X g for five minutes. Again, discard the supernatants and resuspend the samples in 100 microliters of FCSB. After centrifuging the samples one final time and discarding the supernatants, resuspend the samples in 150 microliters of FCSB. Transfer each sample to a tube containing 400 microliters of FCSB for a total volume of 550 microliters. Store the samples at 4 degree Celsius until they are analyzed by flow cytometry.
VLP attachment was measured using flow cytometry. First, density plots were used to gate out cellular debris, followed by subsequent dot plot gating to remove bacterial doublets and cellular clumps. Representative histograms demonstrate a lack of PE signal in bacterial only samples and a shift in PE signal intensity in VLP:bacterium samples compared to unstained and isotype controls. After one hour of incubation with 10 micrograms of VLP, flow cytometry detected particle binding to both E.colacae and L.gasseri.
Probiotics powder Quantifying Human Norovirus Viru Paul zhang