Essential Components of Borreliella (Borrelia) burgdorferi In Vitro Transcription Assays

The in vitro transcription assay system for Borreliella burgdorferi provides a biochemical tool for investigators to study gene regulatory mechanisms and factors that govern the enzymatic activity of RNA polymerase. The system allows us to test how transcription factors, co-factors, salt concentrations, and pH impact Borreliella burgdorferi RNA polymerase function, which contributes to our overall understanding of gene regulatory mechanisms. This powerful technique can also allow us to screen for drugs that selectively inhibit RNA polymerase, opening the door to developing novel drugs to treat Lyme borreliosis.

To begin, collect the cell pellet from two to four liters of Borreliella burgdorferi RpoC-His10X cultured in BSKII medium in a microaerophilic environment to a density of two to four times 10 constant per milliliters. Pellet the cells at 10, 000 times G for 30 minutes at four degrees Celsius in 500 milliliters centrifugal bottles and discard the supernatant. Then, resuspend the cells in 30 milliliters of ice-cold HN buffer, repeat the centrifugation step, and decant the supernatant.