This study details the process of culturing a genetically modified strain of Borrelia burgdorferi that carries engineered bacteriophage DNA. The methodology focuses on inducing antibiotic resistance and producing virions for downstream applications.
Begin with a culture of a genetically modified strain of Borrelia burgdorferi, a pathogenic bacterium.
This strain carries dormant bacteriophage DNA integrated into its genome, which has been engineered to include a target gene conferring antibiotic resistance.
Add the corresponding antibiotic and incubate the culture. The antibiotic resistance gene expresses a protein that inhibits the antibiotic, ensuring only bacteria harboring the gene proliferate.
Once the culture reaches the desired density, centrifuge the cells to pellet them and discard the supernatant.
Resuspend the pellet, introduce a chemical inducer, and incubate to induce DNA damage.
The resulting DNA repair response excises the bacteriophage DNA, initiating its replication, expression of structural proteins, and assembly of new virions carrying the antibiotic-resistance gene.
Centrifuge the culture again, discard the supernatant, and resuspend the cells in a fresh medium.
The bacterial cells containing mature virions are ready for further downstream applications.
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.
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