Phage-Mediated Genetic Manipulation of the Lyme Disease Spirochete Borrelia burgdorferi

This protocol is significant because it describes another important tool for dissecting the molecular biology of the Lyme disease agent Borrelia burgdorferi. The main advantage of this technique is that by using phage transduction, it provides an alternative method for introducing DNA in the Borrelia burgdorferi without requiring the application of an electric pulse, as in electroporation. This method could be used to provide further insight into the molecular mechanisms used by Borrelia burgdorferi to persist in its enzootic cycle and ultimately cause Lyme disease in humans.

To begin, inoculate 150 microliters of the appropriate Borrelia burgdorferi clone into 15 milliliters of BSK in tightly-capped sterile conical centrifuge tubes for the transduction protocol. Then, supplement the medium with the appropriate concentration of antibiotics or a combination of antibiotics for the selection and maintenance of heterologous DNA within the Borrelia burgdorferi clone and incubate the sample at 33 degrees Celsius. After the culture has grown up, centrifuge the appropriate volume of culture at 6, 000 G for 10 minutes.

After decanting the supernatant, resuspend the pellet in four milliliters at fresh BSK and transfer the sample to the smallest sterile tube available to hold the sample with minimal head space. Then, add the appropriate amount of inducing agent to the recommended concentration based on a culture volume of four milliliters to induce phage production, cap the tube tightly, and mix thoroughly. Incubate the sample at 33 degrees Celsius for two to four hours.

Then, transfer the sample to a 15-milliliter centrifuge tube. Centrifuge the sample at 6, 000 G for 10 minutes. After decanting the supernatant, resuspend the cell pellet in 15 milliliters of BSK.

Supplement the prepared culture with the appropriate antibiotic concentration described in the manuscript, while incubating the sample at 33 degrees Celsius for 72 to 96 hours. To prepare solutions for PEG precipitation, prepare 500 milliliters of five-molar sodium chloride, 500 milliliters of 40%PEG, and 100 milliliters of suspension medium as described in the manuscript. To sterilize, autoclave the solution, cool prior to use, and store room temperature or four degrees Celsius.

For PEG precipitation of phage from the donor Borrelia burgdorferi clone, after 72 to 96 hours of incubation, centrifuge the samples at 8, 000 G for 20 minutes at four degrees Celsius. Decant the supernatant into a clean, 50-milliliter conical tube and dispose of the cell pellet. Add five-molar sodium chloride to a final concentration of one molar.

After mixing well, rock gently at room temperature for one hour. Centrifuge the samples at 8, 000 G for 10 minutes at four degrees Celsius. After decanting the supernatant into a clean 50-milliliter conical tube as demonstrated previously, add 40%PEG-8000 solution to the supernatant to a final concentration of 10%Mix well and set on ice for more than one hour, up to overnight.

Centrifuge to the samples at 8, 000 G for 20 minutes at four degrees Celsius as demonstrated previously. Discard the supernatant and remove as much excess liquid as possible without losing any pellet, which contains the phage particles. Resuspend the pellet in a minimal volume of suspension medium using the suspension medium to wash down the side of the bottle and collect any potential phage particles.

Treat the recovered phage sample with an equal volume of chloroform based on the volume of resuspension. Mix the sample well and centrifuge at 8, 000 G for 10 minutes. Then, remove the aqueous layer to a clean tube, avoiding any of the thick interface layers.

After determining the volume recovered, following the first chloroform treatment, again treat the sample with an amount of chloroform equal to 10%of that volume. Transfer the aqueous layer to a clean tube while being careful to avoid any of the interface or organic layers. Use the phage immediately or store it at four degrees Celsius.

After preparing Borrelia burgdorferi cultures to be used as the recipient in transduction assays as demonstrated previously, centrifuge the volume of culture at 6, 000 G for 10 minutes. Decant the supernatant and resuspend the pellet in 14.5 milliliters of fresh BSK. Add less than or equal to 500 microliters of PEG-precipitated phage sample to the culture of the recipient clone.

After mixing well, incubate at 33 degrees Celsius for 72 to 96 hours. Perform the selection of transductants by solid-phase plating after mixing with PEG-precipitated phage as described in the manuscript. Depending on the background of the recipient clone, check that the colonies appear within the agarose on the selection plates after 10 to 21 days of incubation.

Pick at least 5 to 10 colonies that grow on the plate in the presence of both antibiotics using sterilized cotton-plugged 5.75-inches borosilicate pipette and inoculate them into 1.5 milliliters of BSK with the appropriate antibiotic. The PCR amplification of the genes was performed on 10 of the clones encoding kanamycin and gentamycin resistance. The kanamycin resistance gene could be amplified from the donor c1673 and the potential transductants but not the recipient c1706.

Similarly, the gentamycin resistance gene could be amplified from the recipient c1706 and the potential transductants but not the donor, representing transduction events. To demonstrate that the kanamycin resistance cassette was transduced by 5BB1 from the donor into the recipient. The background of the transductants was determined using strain-specific markers.

The c1673 clone and the CA-112A background encodes specific amplicons four, five, and six, whereas c1706, which has a high-passage B31 background, does not. Similarly, the transductants one and two were missing amplicons four, five and six, demonstrating that the clones have the c1706 background and have acquired the kanamycin resistance gene from c1673. For the PEG precipitation of phage, perform all steps carefully to maximize the yield of phage and treat the resuspended phage thoroughly with chloroform to remove as much PEG and culture media contaminants as possible prior to phage use and storage.

Once the transduction assay has been successfully performed and the transductant verified, these clones are then available to answer the questions that require genetically-modified Borrelia burgdorferi. The development of this technique will hopefully allow researchers to genetically manipulate Borrelia burgdorferi strains for which the introduction of DNA via electroporation is currently difficult.