This work was supported by the Health Sciences Strategic Investment Fund Faculty Development Grant of Creighton University. The B. burgdorferi RpoC-His10X strain was kindly provided by Dr. D. Scott Samuels of the University of Montana. The E. coli strain harboring the pMAL-C5X plasmid encoding a maltose-binding protein-tagged rpoD allele was kindly provided by Dr. Frank Gherardini of Rocky Mountain Laboratories, NIAID, NIH.

1. Purification of the RNA polymerase and preparation of RNA polymerase stock
<ol>
	<li>Collect the cell pellet from 2-4 L of B. burgdorferi RpoC-His10X cultured in BSKII medium in a microaerophilic environment (34 &#176;C, 5% CO2, 3% O2) to a density of 2-4 x 107 cells/mL using previously described cell collection protocols28,29. Perform centrifugation at 10,000 x g at 4 &#176;C for 30 min in 500 mL centrifugal bottles and pour off the supernatant.</li>
	<li>Resuspend the cells in 30 mL of ice-cold HN buffer (10 mM HEPES, 10 mM NaCl, pH 8.0). Repeat the centrifugation step as described above using a 50 mL centrifuge tube and decant the supernatant. 
	NOTE: Stopping point. Freeze the cell pellet at &#8722;80 &#176;C to store cells for up to 2 weeks or immediately proceed to cell lysis.</li>
	<li>Maintain the cells, lysate, and purified proteins at 4 &#176;C or on ice unless freezing. Keep RNA polymerase in a reducing environment by adding freshly prepared DTT at a concentration of 2 mM to every buffer used in this protocol and maintain pH 8.0.</li>
	<li>Prepare lysate from the B. burgdorferi cell pellet using a commercial bacterial lysis kit following the kit instructions. Resuspend the pellet in 10-15 mL of room temperature (RT) B-PER solution without protease inhibitor and allow lysis to proceed for 5 min on ice. The lysis volume depends on the cell pellet size, as described in the kit instructions.</li>
	<li>Add protease inhibitor cocktail to the lysis solution, followed by three rounds of sonication, as described in the representative results section. 
	NOTE: Alternatively, lyse the cells in a French pressure cell as described previously24. Skip step 1.4. and step 1.5.</li>
	<li>Clarify the cell lysate using centrifugation and filtration using a 50 mL centrifuge tube. Fill the volume of the tube to at least 30 mL total volume by adding cobalt column loading buffer (Table 1) to the solution. Pellet the cell debris by centrifugation at 20,000 x g for 30 min.</li>
	<li>Filter the supernatant using a 0.45 &#181;m syringe filter.</li>
	<li>Dilute the lysate in cobalt column loading buffer (Table 1) using a 1:10 final dilution ratio.</li>
	<li>Perform affinity chromatography on the clarified cell lysate supernatant using a cobalt or nickel-resin column following the manufacturer&#39;s instructions. See the representative results section for an example. Use the buffers listed in Table 1. Collect the flow-through, wash, and elution samples for analysis.</li>
	<li>Immediately exchange the RNA polymerase in the elution buffer solution into RNA polymerase storage buffer solution (Table 1) using a buffer exchange column following the manufacturer&#39;s instructions.</li>
	<li>Prepare the freezer stocks by concentrating the RNA polymerase using 10 kDa-cutoff centrifugal filter units to a concentration of 0.2-0.4 mg/mL determined by spectrophotometry (A280nm without extinction coefficient adjustment [1 abs at 1 cm = 1 mg&#183;mL&#8722;1]).</li>
	<li>Aliquot RNA polymerase freezer stocks in 20-50 &#181;L volumes in PCR tubes and store the RNA polymerase at &#8722;80 &#176;C.</li>
	<li>Determine the quality of the affinity chromatography by SDS-PAGE and measure the total protein yield by spectrophotometry or BCA assay. Run the samples on a 7.5% polyacrylamide gel at 120 V for 50 min for good resolution by SDS-PAGE.</li>
</ol>
2. Purification of recombinant RpoD and preparation of RpoD stock
<ol>
	<li>Culture and induce the expression of Maltose Binding Protein-tagged recombinant B. burgdorferi RpoD (MBP-RpoD) in BL21::MBP-RpoDBb, as described by the protein fusion and purification system instruction manual listed in the Table of Materials, and collect the cells by centrifugation. 
	NOTE: Stopping point. Freeze the cell pellets at &#8722;80 &#176;C to store cells for up to 2 weeks or immediately proceed to cell lysis.</li>
	<li>Perform lysis using a commercial bacterial cell lysis kit or French pressure cell. See the example lysis buffer in Table 1.</li>
	<li>Clarify the cell lysate using centrifugation and filtration. Pellet the cell debris by centrifugation at 20,000 x g for 30 min. Filter the supernatant using a 0.45 &#181;m filter.</li>
	<li>Extract MBP-RpoD using a protein extraction protocol for the expression system. See the representative results section for example implementation details and results.</li>
	<li>Determine the quality of the affinity chromatography process and elution by SDS-PAGE and measure the protein yield by spectrophotometry (A280nm without extinction coefficient adjustment [1 abs at 1 cm = 1 mg&#183;mL&#8722;1]). Use 10% polyacrylamide gel at 200 V for 30 min for separation in SDS-PAGE.</li>
	<li>Concentrate MBP-RpoD using a 10 kDa-cutoff centrifugal filter to a concentration of &#62;2 mg/mL determined by spectrophotometry.</li>
	<li>Cleave MBP from RpoD at the Factor Xa cleavage site. Add CaCl2 to the affinity chromatography elution fractions containing MBP-RpoD at a concentration of 2 mM. Add 200 &#181;g of Factor Xa Protease for every 30 mg of purified protein. Incubate overnight at RT.</li>
	<li>Confirm complete cleavage of MBP from RpoD by SDS-PAGE. Use 10% polyacrylamide gel at 200 V for 30 min for separation in SDS-PAGE.</li>
	<li>Dilute the MBP and RpoD-containing solution at a ratio of 1:10 with a heparin-binding buffer.</li>
	<li>Separate MBP and RpoD on the heparin column by FPLC. See Table 2 for FPLC settings.</li>
	<li>Determine the quality of the affinity chromatography products by SDS-PAGE and measure the protein yield by spectrophotometry. Use 10% polyacrylamide gel at 200 V for 30 min for separation in SDS-PAGE.</li>
	<li>Concentrate the elution fractions containing RpoD using a 10 kDa-cutoff centrifugal filter to a final volume of 2-3 mL.</li>
	<li>Buffer exchange the concentrated RpoD elution fractions to storage buffer using a gel filtration column.</li>
	<li>Concentrate the RpoD stock using a 10 kDa-cutoff centrifugal filter to a concentration of 0.2-0.8 mg/mL as determined by spectrophotometry.</li>
	<li>Aliquot RpoD stock at 20-50 &#181;L volumes in PCR tubes and store at &#8722;80 &#176;C.</li>
</ol>
3. Preparation of DNA template stock
<ol>
	<li>PCR amplify the 500 bp region surrounding an RpoD driven promoter site from B. burgdorferi genomic DNA using a 200 &#181;L reaction (Table 3).</li>
	<li>Purify the PCR products using a commercial PCR purification kit. Elute the DNA in molecular biology grade H2O.</li>
	<li>Adjust the molar concentration of the PCR-generated DNA templates to 100 nM by dilution in molecular biology grade H2O.</li>
</ol>
4. Perform in vitro transcription with the incorporation of radiolabeled nucleotides
<ol>
	<li>Prepare an in vitro transcription experimental plan. Determine the RNA polymerase, RpoD, and DNA template concentrations. Determine the aliquot volumes and pipetting orders for the reaction tubes. See Table 4 and Figure 1 for an example. 
	NOTE: Include controls in the experimental plan, such as reactions containing no RNA polymerase, alternative polymerase, no template, or alternative templates.</li>
	<li>Calculate the volume of &#945;-32P-ATP required for the experiment. Incorporate this into the experimental plan. Typically, include 2 &#181;Ci of activity per in vitro transcription reaction for phosphor screen detection.</li>
	<li>Prepare the work surfaces, including the radiation bench, to reduce the radiation contamination risk.</li>
	<li>Thaw fresh aliquots of RNA polymerase, Rpo, 5x buffer NTP, and template stock solutions on ice. Mix all the thawed frozen stocks thoroughly prior to pipetting.</li>
	<li>Prepare a master reaction mixture and a separate NTP mixture for radiolabeled nucleotides according to the experimental plan.</li>
	<li>Dispense the control reactions and experimental sets into PCR tubes in preparation of adding radiolabel in the reaction. Gently dispense H2O, reaction master mix, RNA polymerase, and RpoD into designated PCR tubes and mix by pipetting.</li>
	<li>Transfer the prepared materials to a radiation bench.</li>
	<li>Add &#945;-32P-ATP to NTP and mix by gentle pipetting. 
	CAUTION: While working with radioactive material, use appropriate protective equipment and handling procedures for radioactive isotopes.</li>
	<li>Add NTP to the tubes containing the in vitro transcription reaction mixtures from step 4.6.</li>
	<li>Start the in vitro transcription reaction with the addition of a DNA template. Mix the reaction volume by gently pipetting.</li>
	<li>Incubate tubes at 37 &#176;C for 5 min in a thermocycler or heat block.</li>
	<li>Remove the reactions from the thermocycler or heat block and add the 2x RNA loading dye containing 50% formamide to an equal reaction volume to stop in vitro transcription reactions.</li>
	<li>Denature the enzymes by incubating reactions at 65 &#176;C for 5 min in a thermocycler or heat block.</li>
	<li>Separate RNA in the in vitro transcription reaction mixture by gel electrophoresis using 10%-15% TBE urea polyacrylamide gels at 180 V for 30-45 min. 
	NOTE: Depending on the gel electrophoresis conditions, the buffer solution may be contaminated with radiolabeled nucleotides or the gel may contain most or all of the radiolabeled nucleotides. Plan for proper radioactivity storage or disposal.</li>
	<li>Image the radiolabeled RNA using phosphoscreen after removing any portion of the gel containing unincorporated radiolabeled ATP. Expose the gel to the phosphoscreen overnight (16 h) and image using a phosphoscreen imager (100 &#181;m resolution, 700 V PMT tube voltage).</li>
	<li>Analyze the data using densitometry methods24.</li>
</ol>

<ol>
	<li>Brisson, D., Drecktrah, D., Eggers, C. H., Samuels, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetics+of+Borrelia+burgdorferi.">Genetics of Borrelia burgdorferi.</a> Annual Review of Genetics. 46, 515-536 (2012).</li><li>Kingry, L. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surveillance+for+and+discovery+of+Borrelia+Species+in+US+patients+suspected+of+tickborne+illness.">Surveillance for and discovery of Borrelia Species in US patients suspected of tickborne illness.</a> Clinical Infectious Diseases. 66 (12), 1864-1871 (2018).</li><li>Cutler, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relapsing+fever+Borreliae:+A+global+review.">Relapsing fever Borreliae: A global review.</a> Clinics in Laboratory Medicine. 35 (4), 847-865 (2015).</li><li>Barbour, A. G., Hayes, S. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biology+of+Borrelia+species.">Biology of Borrelia species.</a> Microbiological Reviews. 50 (4), 381-400 (1986).</li><li>Tilly, K., Rosa, P. A., Stewart, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biology+of+infection+with+Borrelia+burgdorferi.">Biology of infection with Borrelia burgdorferi.</a> Infectious Disease Clinics of North America. 22 (2), 217-234 (2008).</li><li>Radolf, J. D., Caimano, M. J., Stevenson, B., Hu, L. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Of+ticks,+mice+and+men:+understanding+the+dual-host+lifestyle+of+Lyme+disease+spirochaetes.">Of ticks, mice and men: understanding the dual-host lifestyle of Lyme disease spirochaetes.</a> Nature Reviews Microbiology. 10 (2), 87-99 (2012).</li><li>Caimano, M. J., Drecktrah, D., Kung, F., Samuels, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interaction+of+the+Lyme+disease+spirochete+with+its+tick+vector.">Interaction of the Lyme disease spirochete with its tick vector.</a> Cellular Microbiology. 18 (7), 919-927 (2016).</li><li>Dulebohn, D. P., Richards, C. L., Su, H., Lawrence, K. A., Gherardini, F. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Weak+organic+acids+decrease+Borrelia+burgdorferi+cytoplasmic+pH,+eliciting+an+acid+stress+response+and+impacting+RpoN-+and+RpoS-dependent+gene+expression.">Weak organic acids decrease Borrelia burgdorferi cytoplasmic pH, eliciting an acid stress response and impacting RpoN- and RpoS-dependent gene expression.</a> Frontiers in Microbiology. 8, 1734 (2017).</li><li>Bontemps-Gallo, S., Lawrence, K., Gherardini, F. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+different+virulence-related+regulatory+pathways+in+Borrelia+burgdorferi+are+directly+affected+by+osmotic+fluxes+in+the+blood+meal+of+feeding+ixodes+ticks.">Two different virulence-related regulatory pathways in Borrelia burgdorferi are directly affected by osmotic fluxes in the blood meal of feeding ixodes ticks.</a> PLoS Pathogens. 12 (8), 1005791 (2016).</li><li>Bugrysheva, J. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+the+RelBbu+Regulon+in+Borrelia+burgdorferi+reveals+modulation+of+glycerol+metabolism+by+(p)ppGpp.">Characterization of the RelBbu Regulon in Borrelia burgdorferi reveals modulation of glycerol metabolism by (p)ppGpp.</a> PLoS One. 10 (2), 0118063 (2015).</li><li>Carroll, J. A., Garon, C. F., Schwan, T. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+environmental+pH+on+membrane+proteins+in+Borrelia+burgdorferi.">Effects of environmental pH on membrane proteins in Borrelia burgdorferi.</a> Infection and Immunity. 67 (7), 3181-3187 (1999).</li><li>Drecktrah, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Borrelia+burgdorferi+RelA/SpoT+homolog+and+stringent+response+regulate+survival+in+the+tick+vector+and+global+gene+expression+during+starvation.">The Borrelia burgdorferi RelA/SpoT homolog and stringent response regulate survival in the tick vector and global gene expression during starvation.</a> PLoS Pathogens. 11 (9), 1005160 (2015).</li><li>Hyde, J. A., Trzeciakowski, J. P., Skare, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Borrelia+burgdorferi+alters+its+gene+expression+and+antigenic+profile+in+response+to+CO2+levels.">Borrelia burgdorferi alters its gene expression and antigenic profile in response to CO2 levels.</a> Journal of Bacteriology. 189 (2), 437-445 (2007).</li><li>Lin, Y. H., Chen, Y., Smith, T. C., Karna, S. L. R., Seshu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Short-chain+fatty+acids+alter+metabolic+and+virulence+attributes+of+Borrelia+burgdorferi.">Short-chain fatty acids alter metabolic and virulence attributes of Borrelia burgdorferi.</a> Infection and Immunity. 86 (9), 00217-00218 (2018).</li><li>Seshu, J., Boylan, J. A., Gherardini, F. C., Skare, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissolved+oxygen+levels+alter+gene+expression+and+antigen+profiles+in+Borrelia+burgdorferi.">Dissolved oxygen levels alter gene expression and antigen profiles in Borrelia burgdorferi.</a> Infection and Immunity. 72 (3), 1580-1586 (2004).</li><li>Stevenson, B., Schwan, T. G., Rosa, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temperature-related+differential+expression+of+antigens+in+the+Lyme+disease+spirochete,+Borrelia+burgdorferi.">Temperature-related differential expression of antigens in the Lyme disease spirochete, Borrelia burgdorferi.</a> Infection and Immunity. 63 (11), 4535-4539 (1995).</li><li>Troxell, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Manganese+and+zinc+regulate+virulence+determinants+in+Borrelia+burgdorferi.">Manganese and zinc regulate virulence determinants in Borrelia burgdorferi.</a> Infection and Immunity. 81 (8), 2743-2752 (2013).</li><li>Yang, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interdependence+of+environmental+factors+influencing+reciprocal+patterns+of+gene+expression+in+virulent+Borrelia+burgdorferi.">Interdependence of environmental factors influencing reciprocal patterns of gene expression in virulent Borrelia burgdorferi.</a> Molecular Microbiology. 37 (6), 1470-1479 (2000).</li><li>Samuels, D. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+regulation+and+transcriptomics.">Gene regulation and transcriptomics.</a> Current Issues in Molecular Biology. 42, 223-266 (2021).</li><li>Fraser, C. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomic+sequence+of+a+Lyme+disease+spirochaete,+Borrelia+burgdorferi.">Genomic sequence of a Lyme disease spirochaete, Borrelia burgdorferi.</a> Nature. 390 (6660), 580-586 (1997).</li><li>Iyer, R., Schwartz, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microarray-based+comparative+genomic+and+transcriptome+analysis+of+Borrelia+burgdorferi.">Microarray-based comparative genomic and transcriptome analysis of Borrelia burgdorferi.</a> Microarrays. 5 (2), 9 (2016).</li><li>Iyer, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stage-specific+global+alterations+in+the+transcriptomes+of+Lyme+disease+spirochetes+during+tick+feeding+and+following+mammalian+host+adaptation.">Stage-specific global alterations in the transcriptomes of Lyme disease spirochetes during tick feeding and following mammalian host adaptation.</a> Molecular Microbiology. 95 (3), 509-538 (2015).</li><li>Boyle, W. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DksA-dependent+regulation+of+RpoS+contributes+to+Borrelia+burgdorferi+tick-borne+transmission+and+mammalian+infectivity.">DksA-dependent regulation of RpoS contributes to Borrelia burgdorferi tick-borne transmission and mammalian infectivity.</a> PLoS Pathogen. 17 (2), 1009072 (2021).</li><li>Boyle, W. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+an+in+vitro+RNA+polymerase+transcription+system:+a+new+tool+to+study+transcriptional+activation+in+Borrelia+burgdorferi.">Establishment of an in vitro RNA polymerase transcription system: a new tool to study transcriptional activation in Borrelia burgdorferi.</a> Scientific Reports. 10 (1), 8246 (2020).</li><li>Silar, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+in+vitro+transcription+system+for+analysis+of+Corynebacterium+glutamicum+promoters+recognized+by+two+sigma+factors.">Use of in vitro transcription system for analysis of Corynebacterium glutamicum promoters recognized by two sigma factors.</a> Current Microbiology. 73 (3), 401-408 (2016).</li><li>Maciag, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+transcription+profiling+of+the+sigmaS+subunit+of+bacterial+RNA+polymerase:+re-definition+of+the+sigmaS+regulon+and+identification+of+sigmaS-specific+promoter+sequence+elements.">In vitro transcription profiling of the sigmaS subunit of bacterial RNA polymerase: re-definition of the sigmaS regulon and identification of sigmaS-specific promoter sequence elements.</a> Nucleic Acids Research. 39 (13), 5338-5355 (2011).</li><li>Chang, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BRENDA,+the+ELIXIR+core+data+resource+in+2021:+New+developments+and+updates.">BRENDA, the ELIXIR core data resource in 2021: New developments and updates.</a> Nucleic Acids Research. 49, 498-508 (2021).</li><li>Samuels, D. S., Drecktrah, D., Hall, L. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+transformation+and+complementation.">Genetic transformation and complementation.</a> Methods in Molecular Biology. 1690, 183-200 (2018).</li><li>Hyde, J. A., Weening, E. H., Skare, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+transformation+of+Borrelia+burgdorferi.">Genetic transformation of Borrelia burgdorferi.</a> Current Protocols in Microbiology. 20, 1-17 (2011).</li><li>Ge, Y., Old, I. G., Saint Girons, I., Charon, N. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+characterization+of+a+large+Borrelia+burgdorferi+motility+operon+which+is+initiated+by+a+consensus+sigma70+promoter.">Molecular characterization of a large Borrelia burgdorferi motility operon which is initiated by a consensus sigma70 promoter.</a> Journal of Bacteriology. 179 (7), 2289-2299 (1997).</li><li>Ozaki, M., Wada, A., Fujita, N., Ishihama, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+phase-dependent+modification+of+RNA+polymerase+in+Escherichia+coli.">Growth phase-dependent modification of RNA polymerase in Escherichia coli.</a> Molecular Genetics and Genomics. 230 (1-2), 17-23 (1991).</li><li>Jasinski, D. L., Schwartz, C. T., Haque, F., Guo, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large+scale+purification+of+RNA+nanoparticles+by+preparative+ultracentrifugation.">Large scale purification of RNA nanoparticles by preparative ultracentrifugation.</a> Methods in Molecular Biology. 1297, 67-82 (2015).</li><li>Gierl, A., Zillig, W., Stetter, K. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+the+components+sigma+and+y+of+the+DNA-dependent+RNA+polymerase+of+Lactobacillus+curvatus+in+promotor+selection.">The role of the components sigma and y of the DNA-dependent RNA polymerase of Lactobacillus curvatus in promotor selection.</a> European Journal of Biochemistry. 125 (1), 41-47 (1982).</li><li>Stetter, K. O., Zillig, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcription+in+lactobacillaceae.+DNA-dependent+RNA+polymerase+from+Lactobacillus+curvatus.">Transcription in lactobacillaceae. DNA-dependent RNA polymerase from Lactobacillus curvatus.</a> European Journal of Biochemistry. 48 (2), 527-540 (1974).</li><li>Borbley, G., Schneider, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyanobacteria.">Cyanobacteria.</a> Methods in Enzymology. 167, 3 (1988).</li><li>Svetlov, V., Artsimovitch, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+bacterial+RNA+polymerase:+Tools+and+protocols.">Purification of bacterial RNA polymerase: Tools and protocols.</a> Methods in Molecular Biology. 1276, 13-29 (2015).</li><li>Shimada, T., Yamazaki, Y., Tanaka, K., Ishihama, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+whole+set+of+constitutive+promoters+recognized+by+RNA+polymerase+RpoD+holoenzyme+of+Escherichia+coli.">The whole set of constitutive promoters recognized by RNA polymerase RpoD holoenzyme of Escherichia coli.</a> PLoS One. 9 (3), 90447 (2014).</li><li>Daubendiek, S. L., Ryan, K., Kool, E. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rolling-circle+RNA+synthesis:+Circular+oligonucleotides+as+efficient+substrates+for+T7+RNA+polymerase.">Rolling-circle RNA synthesis: Circular oligonucleotides as efficient substrates for T7 RNA polymerase.</a> Journal of the American Chemical Society. 117 (29), 7818-7819 (1995).</li><li>Marras, S. A., Gold, B., Kramer, F. R., Smith, I., Tyagi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+measurement+of+in+vitro+transcription.">Real-time measurement of in vitro transcription.</a> Nucleic Acids Research. 32 (9), 72 (2004).</li><li>Chamberlin, M. J., Nierman, W. C., Wiggs, J., Neff, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+quantitative+assay+for+bacterial+RNA+polymerases.">A quantitative assay for bacterial RNA polymerases.</a> Journal of Biological Chemistry. 254 (20), 10061-10069 (1979).</li></ol>

The authors declare no conflicts of interest.

In vitro transcription assays constructed using the presented protocol were recently used to study the role of a transcription factor in B. burgdorferi and can be applied to build similar experiments using other transcription factors, sigma factors, and molecules23. Once active RNA polymerase from B. burgdorferi has been obtained and its activity detected, components and conditions within the in vitro transcription assays can be modified. The assay is highly flexible and modifiable. The reactions are modular and built from frozen stocks and require little lead time once the experimental parameters have been chosen. Given sufficient stock, multiple experiments may be carried out in 1 day.
Obtaining active RNA polymerase is the most critical aspect of in vitro transcription reaction preparation. We recommend harvesting RNA polymerase from cells grown to the mid-logarithmic growth phase as described in this protocol. In purifying the RNA polymerase from any bacteria, the rate of growth has been noted as an important factor in purifying active RNA polymerase. RNA polymerases can accumulate post-translational modifications and may be associated with inhibitory transcription factors at the stationary phase of growth31. We have not succeeded in purifying high amounts of active RNA polymerase from B. burgdorferi cultures grown to stationary phase. The affinity chromatography approach, as compared to a physical separation approach32, is also beneficial for purification from B. burgdorferi because the organism reaches low density in culture, and accumulating large amounts of cell culture is relatively material intensive.
During purifications of other bacterial RNA polymerases, sigma factors can co-purify in significant quantities depending on the purification method and conditions33,34,35,36. Co-purification of sigma factors can limit downstream experiments using alternative sigma factors, modified sigma factors, or a lack of sigma factors. Moreover, supplementation with the housekeeping sigma factor RpoD is beneficial for detecting activity regardless of initial co-purification of the sigma factor37. Due to the flexibility of separating the sigma factor from RNA polymerase core, we consider it advantageous that sigma factors do not co-purify under the His-affinity chromatography purification conditions outlined here. It is possible that the purification conditions could be changed to yield more intact and active holoenzymes containing RpoD. For example, salt concentrations are likely to affect the stability of multiple subunit enzymes, and the effect of imidazole concentration on the stability of the B. burgdorferi RNA polymerase complex or its divalent cation incorporation is also unknown.
B. burgdorferi RNA polymerase enzymatic activity is sensitive to the type and quantity of divalent cations present24. We found that manganese is required for maximal RNA polymerase activity, and magnesium appeared to contribute to activity to a lesser extent. Contaminating ions in water add a high amount of variability to RNA polymerase enzymatic activity. Consequently, highly pure water such as molecular biology grade, HPLC grade, and metal analysis grade H2O with few metal ions present is essential for higher RNA polymerase activity in the protocol outlined here. RNA polymerases are also sensitive to pH and require a reducing environment during purification27. Altered pH or the use of BME as a reducing agent has resulted in inactive RNA polymerase. We recommend using commercially available E. coli RNA polymerase as a control to ensure the proper construction of in vitro transcription assays24. E. coli RNA polymerase will allow for testing of the viability of the nucleotides, the template construction method, radiolabel incorporation, RNA separation, and the signal detection method. However, it should be noted that B. burgdorferi RNA polymerase has specific divalent cation, buffer, and promoter requirements compared to E. coli.
There are several detection methods available for in vitro transcription reactions. Dye incorporation and molecular beacon assays are common alternatives to the radiolabel detection method38,39. Radiolabeled nucleotide incorporation is currently the most sensitive RNA detection method. Alternative assays require more RNA polymerase to reach measurable signal levels compared to radiolabel assays. Additionally, dye incorporation and molecular beacon methods typically have more ways for signals to be erroneous. For example, DNase contamination can cause molecular beacons to release a signal in the absence of their target. Using radiolabel incorporation, the relative activity of the B. burgdorferi RNA polymerase enzyme can be detected even at low levels, which suits initial experimentation with RNA polymerase enzymes.
While the concentration of B. burgdorferi RNA polymerase enzyme included in the reaction is standardized by spectrophotometry, this measurement can be a source of variability. The specific activity of RNA polymerases can depend on the number of fully-formed active holoenzymes, which are a portion of the total protein mixture40. For example, the molar quantity of 50 nM of RNA polymerase reported in our results was determined by spectrophotometry, but an analysis of the subunit quantities by quantitative western blotting revealed that the RpoA subunit was a limiting component in the number of potential RNA polymerase holoenzymes, and a maximum of only 21 nM of fully formed RNA polymerase was possible in our mixture24. Experiments should always be conducted to control for batch-to-batch variation in RNA polymerase and other components of the in vitro transcription reaction.
In our representative experiment shown in Figure 5, the RpoD levels were altered, and a linear relationship between RpoD and the detected signal was demonstrated. In vitro transcription is a versatile method that can be easily altered using the experimental template presented (Table 4). For example, alternative sigma factors or alternative DNA templates can be easily tested24. Alternative templates could include different promoter regions generated by PCR or plasmid purified from cells to introduce supercoiled and methylated DNA features. For a given level of RpoD, RNA polymerase, and template, differing levels of a transcription factor may be added to see inhibition or enhancement of activity from a promoter23. The order of component addition and molar ratios within the in vitro transcription reaction can be altered such that transcription initiation complexes are formed prior to reaction initiation; these reactions could measure transcription elongation instead of transcription initiation40. The in vitro transcription assay may be altered to suit a variety of investigations into B. burgdorferi biology.

Lyme disease and relapsing fever are caused by spirochete pathogens in the genera Borrelia and Borreliella1,2,3. Lyme disease is a prominent vector-borne disease in North America, and, consequently, Borreliella burgdorferi is a prominent model organism to study spirochete biology4,5. Investigations into the B. burgdorferi mechanisms of transcriptional regulation aim to better understand its adaptations to changes in the environment as it cycles between its tick vector and mammalian hosts6,7. Changes in pH, temperature, osmolarity, nutrient availability, short-chain fatty acids, organic acids, and dissolved oxygen and carbon dioxide levels modulate the expression of genes that are important for B. burgdorferi to survive in its arthropod vector and to infect animals8,9,10,11,12,13,14,15,16,17,18. Linking these responses to stimuli with regulatory mechanisms has been an important aspect of B. burgdorferi research19.
Transcription factors and sigma factors control the transcription of genes that carry out cellular processes. Lyme and relapsing fever spirochetes harbor a relatively sparse set of transcription factors and alternative sigma factors. Despite this, there are complex transcriptional changes directing B. burgdorferi responses to the environment20,21,22. The specific mechanisms driving transcriptional changes in B. burgdorferi in response to environmental changes remain unclear. In vitro transcription assays are powerful tools for employing a biochemical approach to assay the function and regulatory mechanisms of transcription factors and sigma factors23,24,25,26.
An in vitro transcription assay system using the B. burgdorferi RNA polymerase was recently established24. As bacteria often have unique cellular physiologies, RNA polymerases of different species and genera respond differently to enzyme purification, enzyme storage, and reaction buffer conditions27. B. burgdorferi is also genetically distant from the many bacterial species in which RNA polymerases have been studied20. Aspects of enzyme preparation such as lysis, wash, and elution buffer conditions, storage buffer, in vitro transcription reaction buffer, and the method of assay construction can all alter RNA polymerase activity. Herein, we provide a protocol for the purification of RNA polymerase and sigma factor RpoD, the production of linear double-stranded DNA template, and the construction of in vitro transcription assays to facilitate reproducibility between laboratories using this system. We detail an example reaction to demonstrate the linear range for RpoD-dependent transcription and discuss limitations and alternatives to this approach.

<table><tbody><tr><td>0.45 micron syringe filter</td><td>Thermo Scientific</td><td>726-2545</td><td>Step 1.7 and 2.3</td></tr><tr><td>50 mL conical tubes</td><td>MidSci</td><td>C50B</td><td>Step 1.3, and subsequent steps</td></tr><tr><td>50 mL high-speed centrifuge tubes</td><td>Thermo Scientific</td><td>3119-0050PK</td><td>Step 1.2</td></tr><tr><td>500 mL Centrifuge bottles</td><td>Thermo Scientific</td><td>3120-9500PK</td><td>Step 1.1</td></tr><tr><td>B-PER and instruction manual</td><td>Thermo Scientific</td><td>78248</td><td>Step 1.4 and 2.2</td></tr><tr><td>Calcium chloride</td><td>Fisher Scientific</td><td>10035-04-8</td><td>Step 2.6</td></tr><tr><td>Centrifugal filters 10 Kd cutoff</td><td>Millipore Sigma</td><td>UFC8010</td><td>Step 1.11 and 2.11</td></tr><tr><td>Cobalt resin and instruction manual</td><td>Thermo Scientific</td><td>89969</td><td>Step 1.9</td></tr><tr><td>Dithiothreitol</td><td>Acros Organics</td><td>426380500</td><td>Step 1.4 and subsequent steps</td></tr><tr><td>Dnase (Nuclease)</td><td>Millipore Sigma</td><td>70746</td><td>Step 1.4 and 2.2</td></tr><tr><td>Factor Xa Protease&amp;nbsp;</td><td>Haematologic Technologies</td><td>HCXA-0060</td><td>Step 2.6</td></tr><tr><td>GE Typhoon 5 Phosphoimager</td><td>GE lifesciences</td><td>Multiple</td><td>Step 4.15</td></tr><tr><td>Gel Imager</td><td>Bio-Rad</td><td>Mutiple</td><td>Step 1.13 and subsequent protien quality check steps</td></tr><tr><td>H&lt;sub&gt;2&lt;/sub&gt;O for in vitro transcription</td><td>Fisher Scientific</td><td>7732-18-5</td><td>Step 3.2 and 3.3</td></tr><tr><td>high fidelity PCR kit</td><td>New England Biolabs</td><td>M0530S</td><td>Step 3.1</td></tr><tr><td>High-speed centrifuge</td><td>Eppendorf</td><td></td><td>Step 1.1, and subsequent steps</td></tr><tr><td>HiTrap Heparin HP 5 x 1 mL</td><td>Cytiva Life Sciences</td><td>17040601</td><td>Step 2.8</td></tr><tr><td>Imidazole</td><td>Sigma-Aldrich</td><td>56750-100G</td><td>Step 1.9</td></tr><tr><td>Lysozyme</td><td>Thermo Scientific</td><td>90082</td><td>Step 1.4 and 2.2</td></tr><tr><td>Magnesium chloride&amp;nbsp;</td><td>Fisher Scientific</td><td>S25401</td><td>Step 4.1</td></tr><tr><td>Manganese chloride</td><td>Fisher Scientific</td><td>S25418</td><td>Step 4.1</td></tr><tr><td>Mini protean tetra cell</td><td>Bio-Rad</td><td>Mutiple</td><td>Step 1.13 and subsequent protien quality check steps</td></tr><tr><td>NP-40</td><td>Thermo Scientific</td><td>85124</td><td>Step 4.1</td></tr><tr><td>NTP mixture</td><td>Thermo Scientific</td><td>R0481</td><td>Step 4.1</td></tr><tr><td>PCR purification kit</td><td>Qiagen</td><td>28506</td><td>Step 3.2</td></tr><tr><td>PCR tubes</td><td>MidSci</td><td>PR-PCR28ACF</td><td>Step 1.12</td></tr><tr><td>PD-10 Sephadex buffer exchange column and instruction manual</td><td>Cytiva</td><td>17085101</td><td>Step 1.10 and 2.10 (gel filtration column)</td></tr><tr><td>pMAL Protein Fusion&lt;br/&gt; and Purification System Instruction manual</td><td>New England Biolabs</td><td>E8200S</td><td>Step 2.1</td></tr><tr><td>Polyacrylamide gels AnyKD</td><td>Bio-Rad</td><td>456-8125</td><td>Step 1.13 and subsequent protien quality check steps</td></tr><tr><td>Potassium glutamate</td><td>Sigma-Aldrich</td><td>G1251</td><td>Step 4.1</td></tr><tr><td>Protease inhibitor</td><td>Thermo Scientific</td><td>78425</td><td>Step 1.4 and 2.2</td></tr><tr><td>Radiolabeled ATP</td><td>Perkin Elmer</td><td>BLU503H</td><td>Step 4.2</td></tr><tr><td>RNA Loading Dye, (2x)</td><td>New England Biolabs</td><td>B0363S</td><td>Step 4.13</td></tr><tr><td>Rnase inhibitor</td><td>Thermo Scientific</td><td>EO0381</td><td>Step 4.1</td></tr><tr><td>Spectrophotometer</td><td>Biotek</td><td>Mutiple</td><td>Step 1.13 and subsequent protien quality check steps</td></tr><tr><td>TBE-Urea gels 10 percent</td><td>Bio-Rad</td><td>4566033</td><td>Step 4.14</td></tr></tbody></table>

essential components of borreliella (borrelia) burgdorferi in vitro transcription assays

Borreliella burgdorferi is a bacterial pathogen with limited metabolic and genomic repertoires. B. burgdorferi transits extracellularly between vertebrates and ticks and dramatically remodels its transcriptional profile to survive in disparate environments during infection. A focus of B. burgdorferi studies is to clearly understand how the bacteria responds to its environment through transcriptional changes. In vitro transcription assays allow for the basic mechanisms of transcriptional regulation to be biochemically dissected. Here, we present a detailed protocol describing B. burgdorferi RNA polymerase purification and storage, sigma factor purification, DNA template generation, and in vitro transcription assays. The protocol describes the use of RNA polymerase purified from B. burgdorferi 5A4 RpoC-His (5A4-RpoC). 5A4-RpoC is a previously published strain harboring a 10XHis-tag on the rpoC gene encoding the largest subunit of the RNA polymerase. In vitro transcription assays consist of the RNA polymerase purified from strain 5A4-RpoC, a recombinant version of the housekeeping sigma factor RpoD, and a PCR-generated double-stranded DNA template. While the protein purification techniques and approaches to assembling in vitro transcription assays are conceptually well understood and relatively common, handling considerations for RNA polymerases often differ from organism to organism. The protocol presented here is designed for enzymatic studies on the B. burgdorferi RNA polymerase. The method can be adapted to test the role of transcription factors, promoters, and post-translational modifications on the activity of the RNA polymerase.

In an in vitro transcription reaction in which the limiting step of the reaction is sigma factor-mediated transcription initiation, the transcription activity should increase linearly with the amount of sigma factor. We present the preparation of in vitro transcription experiments testing a range of RpoD concentrations along with two concentrations of RNA polymerase to observe the resulting varying signal from radiolabeled nucleotide incorporation into RNA products. Representative results of the preparation of RNA polymerase, RpoD, and double-stranded DNA template stocks are shown. The representative in vitro transcription reactions serve as an example of RNA polymerase activity levels acceptable for analysis.
For the purification of RNA polymerase, a 4 L culture of B. burgdorferi RpoC-His10X was grown at 34 &#730;C under microaerophilic conditions (5% CO2, 3% O2). The cultures were closely monitored for spirochete density using dark-field microscopy and collected prior to reaching densities higher than 4 x 107 spirochetes/mL. The cells were resuspended in B-PER mixture for lysis (Table 1). The cell pellet was homogenized by pipetting, followed by 5 min incubation at RT, and subsequent rounds of sonication performed for 3 s in triplicate. Clarified lysates were diluted with cobalt column loading buffer and 5 mL of cobalt resin to a total volume of 180 mL. RpoC-His10X was allowed to bind to the cobalt resin by nutating the mixture for 1 h at 4 &#176;C. The mixture was transferred to a gravity flow column and washed five times with 40 mL of column wash buffer. Bound protein was released by passing 5 mL of elution buffer through the column, and elution was repeated seven times. Samples of the flow-through, washes, and elution fractions were separated by SDS-PAGE and imaged by stain incorporation (Figure 1). The RNA polymerase core complex is composed of RpoC, RpoB, and RpoA, sized 154 kDa, 129 kDa, and 38.5 kDa, respectively. The representative result displayed in Figure 1 shows three prominent bands at the sizes of the three peptides composing the RNA polymerase complex. The flow-through from affinity chromatography contained 0.5-1 mg/mL of protein with a 260/280 ratio between 1.0 and 1.7, indicating a higher concentration of nucleic acids. The RNA polymerase protein yield following the concentration of the protein mixture as determined by spectrophotometry was 0.3 mg/mL.
To prepare RpoD protein stocks, recombinant RpoD with a C-terminal Maltose Binding Protein tag was expressed from a pMAL-C5X plasmid in BL21 (DE3) pLysS E. coli. 2 L of E. coli was grown at 32 &#176;C to an OD600nm of 0.5. The culture was then treated with 0.3 mM IPTG to induce the expression of RpoD for 1 h. The cells were pelleted by centrifugation and lysed in B-PER. The resulting 200 mL of diluted cell lysate was filtered through 10 mL of amylose resin. The resin was washed with 40 mL of binding buffer eight times to remove residual DNA and peptides. MBP-tagged RpoD was eluted with 5 mL of elution buffer into the resin column five times. The flow-through from the binding and washing steps and the elution fractions were analyzed by SDS-PAGE (Figure 2A). The major component in the elution fraction is a peptide of ~115 kDa, matching the size of the MBP-tagged recombinant B. burgdorferi RpoD. The elution fractions were combined, the CaCl2 concentration was adjusted to 2 mM, and 100 &#181;g of FactorXa protease was added to 40 mg of purified protein. Following overnight digestion at room temperature, the reaction was analyzed by SDS-PAGE (Figure 2B). Following cleavage with Factor Xa protease, the two major products in the mixture were RpoD (73.6 kDa) and MBP (45 kDa). The mixture of RpoD and MBP proteins was separated using heparin affinity chromatography. The mixture was diluted at a 1:10 ratio in heparin-binding buffer and loaded into a heparin-bound resin column in an FPLC. RpoD was eluted with an increasing gradient of NaCl to a concentration of 1 M. When the fractions were analyzed by SDS-PAGE, a major band of 73.6 kDa size was apparent in the elution fraction in the range of 400-600 mM NaCl (Figure 2C). Some degraded RpoD fragments were co-purified. RpoD-containing fractions were combined, buffer exchanged into RpoD storage buffer, and then concentrated using a 10 kDa-cutoff centrifugal filter. The final RpoD concentration was 0.67 mg/mL (9.1 mM) as determined by spectrophotometry.
A double-stranded DNA template encompassing the promoter site of B. burgdorferi flgB was generated by PCR. The flgB promoter was amplified by primers that corresponded to 250 bp upstream and downstream of the promoter (Table 6) using a 200 &#181;L reaction containing 30 ng of B. burgdorferi DNA24,30. The PCR product was analyzed by gel electrophoresis in a 1% agarose gel (Figure 3). The PCR product was roughly 500 bp in size with apparent homogeneity. Importantly, salts and cell component contamination can contribute to the variability of in vitro transcription reaction outcomes; we ensure that the DNA template is thoroughly desalted on the PCR purification column.
An experimental scheme in which transcription is initiated by the addition of template DNA can test the rate of promoter recognition and transcription initiation. In our representative experiments, a dilution series of RpoD protein was prepared by twofold dilutions in water ranging from 16 nM-1 &#181;M. A master mix was created containing RNA polymerase in a reaction buffer containing divalent cations required for activity (Table 4 and Table 5). The RpoD and master mix were aliquoted together on ice. The master mix containing RNA polymerase and RpoD was allowed to incubate while other steps of the in vitro transcription were prepared. We incorporated three control reactions: a positive control reaction prepared separately from the master mix, a negative control reaction containing no template DNA to ensure the in vitro transcription signal was template dependent, and a negative control reaction without RNA polymerase to ensure the signal was RNA polymerase dependent. Radiolabeled nucleotides were added to the reaction by mixing 5 &#956;L of (&#945;-32P-ATP) to 20 &#956;L of 10x NTP stock mixture to create enough aliquot for 20 reactions to compensate for unrecoverable volumes. Finally, template DNA encoding the flgB promoter was introduced to the reaction tubes by pipetting following the previously planned pipetting order (Figure 4).
The signal from the RNA fragments generated by in vitro transcription was detected by phosphor screen following a 16 h exposure period of the TBE-urea gel containing RNA separated by gel electrophoresis. An experiment containing 50 nM of RNA polymerase and RpoD in the 500 nM to 16 nM range generated RNA products of 250 bp (Figure 5). For each twofold dilution of RpoD concentration, there was a lower level of accumulated RNA product. There was a series of lower bp products that appeared below the prominent band, indicating incomplete RNA fragments due to transcription halting or fragmented DNA template. RNA products were absent from the no template control, indicating that there were no exogenous DNA fragments contributing to the signal in this assay. No signal was detected in the no RNA polymerase control, indicating the B. burgdorferi RNA polymerase was the only polymerase contributing to the production of RNA in this assay.
In a second experiment, we used a lower concentration of 25 nM RNA polymerase (Figure 6). Fewer RNA products were detected across the range of RpoD concentrations tested compared to Figure 5, and the background signal intensity was higher. This is problematic for downstream analysis by densitometry. When the phosphor image was analyzed by densitometry (Figure 7), the densitometry signal obtained from the phosphoscreen indicated a linear relationship between the amount of RpoD present in the reaction in both experiments. However, in the experiment with high background signal, the reaction containing 25 nM RNA polymerase and less than 100 nM of RpoD was unreliable for relative transcription level comparisons.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64134/64134fig01.jpg" /> 
Figure 1: Separation of His-tagged B. burgdorferi RNA polymerase purified by affinity chromatography. The flow-through fraction of cell lysate (lane 1), first wash fraction (lane 2), last wash fraction (lane 3), and five elution fractions (lane 4-8) were diluted in 2x sample loading buffer, boiled, and separated by SDS-PAGE in a gradient polyacrylamide gel at 200 V for 30 min. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64134/64134fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64134/64134fig02.jpg" /> 
Figure 2: Analysis of RpoD purification by gel electrophoresis. (A) Pooled flow-through fractions (lane 1 and lane 2), wash fractions (lanes 3-5), and elution fractions (lanes 6-10) from the purification of MBP-tagged RpoD by amylose resin affinity chromatography. (B) Protein mixture following the cleavage of MBP and RpoD using Factor Xa protease. (C) Flowthrough solution (lane 1) and elution fractions with an increasing gradient of NaCl (lanes 2-9) from heparin affinity chromatography to separate MBP and RpoD. In each gel presented, the samples were mixed with 2x loading buffer, boiled, and separated in a gradient polyacrylamide gel using SDS-PAGE at 200 V for 30 min. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64134/64134fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64134/64134fig03.jpg" /> 
Figure 3: PCR generation of in vitro transcription templates. 500 bp sized amplicon containing flgB promoter (lane 1 and lane 2) and control PCR reactions containing amplicons of other promoter sites (lanes 3-4) are shown. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64134/64134fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/64134/64134fig04.jpg" /> 
Figure 4: Experimental arrangement and pipetting sequence plan. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64134/64134fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/64134/64134fig05.jpg" /> 
Figure 5: RpoD-dependent variability in phosphor image signal from RNA generated by in vitro transcription using B. burgdorferi RNA polymerase. In vitro transcription reactions contained 50 nM of RNA polymerase and varying levels of RpoD indicated above the bands24. RNA was separated by a 10% TBE-urea gel, and a phosphor screen was used to capture the signal from radioactive decay over 16 h. This figure has been modified from Boyle&#160;et al.24. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64134/64134fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/64134/64134fig06.jpg" /> 
Figure 6: RpoD-dependent variability in phosphor image signal from RNA generated by in vitro transcription. In vitro transcription contained 25 nM of RNA polymerase and varying levels of RpoD indicated above the bands. RNA was separated by a 15% TBE-urea gel, and a phosphor screen was used to capture the signal from radioactive decay over 16 h. The lower RNA polymerase concentration and higher background signal increased the difficulty of accurate measurement. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64134/64134fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/64134/64134fig07.jpg" /> 
Figure 7: Increasing radiolabeled RNA accumulation in in vitro transcription reactions with increasing RpoD levels. The signals detected in the phosphor screen imaging were analyzed by densitometry with no background subtraction. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64134/64134fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Table 1: Media recipes. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64134/Table 1 Recipe list-64134R1.zip" target="_blank">Please click here to download this Table.</a>
Table 2: FPLC settings. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64134/Table 2 FPLC settings-64134R1.zip" target="_blank">Please click here to download this Table.</a>
Table 3: PCR reactions. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64134/Table 3 PCR-64134R1.zip" target="_blank">Please click here to download this Table.</a>
Table 4: 50 nM RNA polymerase experimental plan and volume calculation. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64134/Table 4 example IVT plan-64134R1.zip" target="_blank">Please click here to download this Table.</a>
Table 5: 25 nM RNA polymerase experimental plan and volume calculation. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64134/Table 5 example IVT plan 2-64134R1.zip" target="_blank">Please click here to download this Table.</a>
Table 6: Strains and oligonucleotides used in this protocol. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64134/Table 6 strains and oligonucleotides-64134R1.zip" target="_blank">Please click here to download this Table.</a>

Watch this Scientific Journal Video about Essential Components of Borreliella Borrelia burgdorferi In Vitro Transcription Assays at JoVE.com

Essential Components of Borreliella Borrelia burgdorferi In Vitro Transcription Assays

In vitro transcription assays can decipher the mechanisms of transcriptional regulation in Borreliella burgdorferi. This protocol describes the steps to purify B. burgdorferi RNA polymerase and perform in vitro transcription reactions. Experimental approaches using in vitro transcription assays require reliable purification and storage of active RNA polymerase.

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

Borreliella burgdorferi is a bacterial pathogen with limited metabolic and genomic repertoires. B. burgdorferi transits extracellularly between ...

essential-components-borreliella-borrelia-burgdorferi-vitro

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

1.2K Views.  Creighton University. In vitro transcription assays can decipher the mechanisms of transcriptional regulation in Borreliella burgdorferi. This protocol describes the steps to purify B. burgdorferi RNA polymerase and perform in vitro transcription reactions. Experimental approaches using in vitro transcription assays require reliable purification and storage of active RNA polymerase. 

Video: Essential Components of Borreliella Borrelia burgdorferi In Vitro Transcription Assays

In Vitro Transcription Assays and Their Application in Drug Discovery

In vitro transcription assays have been developed and widely used for many years to study the molecular mechanisms involved in transcription. This process requires multi-subunit DNA-dependent RNA polymerase (RNAP) and a series of transcription factors that act to modulate the activity of RNAP during gene expression. Sequencing gel electrophoresis of radiolabeled transcripts is used to provide detailed mechanistic information on how transcription proceeds and what parameters can affect it. In this paper we describe the protocol to study how the essential elongation factor NusA regulates transcriptional pausing, as well as a method to identify an antibacterial agent targeting transcription initiation through inhibition of RNAP holoenzyme formation. These methods can be used a as platform for the development of additional approaches to explore the mechanism of action of the transcription factors which still remain unclear, as well as new antibacterial agents targeting transcription which is an underutilized drug target in antibiotic research and development.

In Vitro Transcription Assays and Their Application in Drug Discovery

In this manuscript, we describe a protocol to functionally examine transcription and the inhibitory activity of antibacterial agents targeting bacterial transcription.

In vitro transcription assays have been developed and widely used for many years to study the molecular mechanisms involved in transcription. This process ...

Genetics

Isolation of Active Caenorhabditis elegans Nuclear Extract and Reconstitution for In Vitro Transcription

Caenorhabditis elegans has been an important model system for biological research since it was introduced in 1963. However, C. elegans has not been fully utilized in the biochemical study of biological reactions using its nuclear extracts such as in vitro transcription and DNA replication. A significant hurdle for using C. elegans in biochemical studies is disrupting the nematode&#39;s thick outer cuticle without sacrificing the activity of the nuclear extract. While several methods are used to break the cuticle, such as Dounce homogenization or sonication, they often lead to protein instability. There are no established protocols for isolating active nuclear proteins from larva or adult C. elegans for in vitro reactions. Here, the protocol describes in detail the homogenization of larval stage 4 C. elegans using a Balch homogenizer. The Balch homogenizer uses pressure to slowly force the animals through a narrow gap breaking the cuticle in the process. The uniform design and precise machining of the Balch homogenizer allow for consistent grinding of animals between experiments. Fractionating the homogenate obtained from the Balch homogenizer yields functionally active nuclear extract that can be used in an in vitro method for assaying transcription activity of C. elegans.

Isolation of Active Caenorhabditis elegans Nuclear Extract and Reconstitution for In Vitro Transcription

Here, we describe a detailed protocol for isolating active nuclear extract from larval stage 4 C. elegans and visualizing transcription activity in an in vitro system.

Caenorhabditis elegans has been an important model system for biological research since it was introduced in 1963. However, C. elegans has not been fully ...

Biochemistry

Bacterial Artificial Chromosomes: A Functional Genomics Tool for the Study of Positive-strand RNA Viruses

Reverse genetics, an approach to rescue infectious virus entirely from a cloned cDNA, has revolutionized the field of positive-strand RNA viruses, whose genomes have the same polarity as cellular mRNA. The cDNA-based reverse genetics system is a seminal method that enables direct manipulation of the viral genomic RNA, thereby generating recombinant viruses for molecular and genetic studies of both viral RNA elements and gene products in viral replication and pathogenesis. It also provides a valuable platform that allows the development of genetically defined vaccines and viral vectors for the delivery of foreign genes. For many positive-strand RNA viruses such as Japanese encephalitis virus (JEV), however, the cloned cDNAs are unstable, posing a major obstacle to the construction and propagation of the functional cDNA. Here, the present report describes the strategic considerations in creating and amplifying a genetically stable full-length infectious JEV cDNA as a bacterial artificial chromosome (BAC) using the following general experimental procedures: viral RNA isolation, cDNA synthesis, cDNA subcloning and modification, assembly of a full-length cDNA, cDNA linearization, in vitro RNA synthesis, and virus recovery. This protocol provides a general methodology applicable to cloning full-length cDNA for a range of positive-strand RNA viruses, particularly those with a genome of &gt;10 kb in length, into a BAC vector, from which infectious RNAs can be transcribed in vitro with a bacteriophage RNA polymerase.

Here, a protocol is presented for creating an infectious bacterial artificial chromosome containing a full-length cDNA of the positive-strand genomic RNA of Japanese encephalitis virus. This protocol can be used to construct a functional cDNA of other positive-strand RNA viruses, making it a powerful genomic tool for studying virus biology.

Reverse genetics, an approach to rescue infectious virus entirely from a cloned cDNA, has revolutionized the field of positive-strand RNA viruses, whose ...

Whole Mount in Situ Hybridization of E8.5 to E11.5 Mouse Embryos

Whole mount in situ hybridization is a very informative approach for defining gene expression patterns in embryos. The in situ hybridization procedures are lengthy and technically demanding with multiple important steps that collectively contribute to the quality of the final result. This protocol describes in detail several key quality control steps for optimizing probe labeling and performance. Overall, our protocol provides a detailed description of the critical steps necessary to reproducibly obtain high quality results. First, we describe the generation of digoxygenin (DIG) labeled RNA probes via in vitro transcription of DNA templates generated by PCR. We describe three critical quality control assays to determine the amount, integrity and specific activity of the DIG-labeled probes. These steps are important for generating a probe of sufficient sensitivity to detect endogenous mRNAs in a whole mouse embryo. In addition, we describe methods for the fixation and storage of E8.5-E11.5 day old mouse embryos for in situ hybridization. Then, we describe detailed methods for limited proteinase K digestion of the rehydrated embryos followed by the details of the hybridization conditions, post-hybridization washes and RNase treatment to remove non-specific probe hybridization. An AP-conjugated antibody is used to visualize the labeled probe and reveal the expression pattern of the endogenous transcript. Representative results are shown from successful experiments and typical suboptimal experiments.

Whole Mount in Situ Hybridization of E8.5 to E11.5 Mouse Embryos

This whole mount in situ hybridization protocol discusses critical steps that ensure reproducible high quality results for gene expression studies in E8.5-E11.5 day old mouse embryos.

Whole mount in situ hybridization is a very informative approach for defining gene expression patterns in embryos. The in situ hybridization procedures ...

Biology

In Vitro Transcribed RNA-based Luciferase Reporter Assay to Study Translation Regulation in Poxvirus-infected Cells

Every poxvirus mRNA transcribed after viral DNA replication has an evolutionarily conserved, non-templated 5&#39;-poly(A) leader in the 5&#39;-UTR. To dissect the role of 5&#39;-poly(A) leader in mRNA translation during poxvirus infection we developed an in vitro transcribed RNA-based luciferase reporter assay. This reporter assay comprises of four core steps: (1) PCR to amplify the DNA template for in vitro transcription; (2) in vitro transcription to generate mRNA using T7 RNA polymerase; (3) Transfection to introduce in vitro transcribed mRNA into cells; (4) Detection of luciferase activity as the indicator of translation. The RNA-based luciferase reporter assay described here circumvents issues of plasmid replication in poxvirus-infected cells and cryptic transcription from the plasmid. This protocol can be used to determine translation regulation by cis-elements in an mRNA including 5&#39;-UTR and 3&#39;-UTR in systems other than poxvirus-infected cells. Moreover, different modes of translation initiation like cap-dependent, cap-independent, re-initiation, and internal initiation can be investigated using this method.

We present a protocol to study mRNA translation regulation in poxvirus-infected cells using in vitro Transcribed RNA-based luciferase reporter assay. The assay can be used for studying translation regulation by cis-elements of an mRNA, including 5&#8217;-untranslated region (UTR) and 3&#8217;-UTR. Different translation initiation modes can also be examined using this method.

Every poxvirus mRNA transcribed after viral DNA replication has an evolutionarily conserved, non-templated 5&#39;-poly(A) leader in the 5&#39;-UTR. To ...

The MultiBac Protein Complex Production Platform at the EMBL

Proteomics research revealed the impressive complexity of eukaryotic proteomes in unprecedented detail. It is now a commonly accepted notion that proteins in cells mostly exist not as isolated entities but exert their biological activity in association with many other proteins, in humans ten or more, forming assembly lines in the cell for most if not all vital functions.1,2 Knowledge of the function and architecture of these multiprotein assemblies requires their provision in superior quality and sufficient quantity for detailed analysis. The paucity of many protein complexes in cells, in particular in eukaryotes, prohibits their extraction from native sources, and necessitates recombinant production. The baculovirus expression vector system (BEVS) has proven to be particularly useful for producing eukaryotic proteins, the activity of which often relies on post-translational processing that other commonly used expression systems often cannot support.3 BEVS use a recombinant baculovirus into which the gene of interest was inserted to infect insect cell cultures which in turn produce the protein of choice. MultiBac is a BEVS that has been particularly tailored for the production of eukaryotic protein complexes that contain many subunits.4 A vital prerequisite for efficient production of proteins and their complexes are robust protocols for all steps involved in an expression experiment that ideally can be implemented as standard operating procedures (SOPs) and followed also by non-specialist users with comparative ease. The MultiBac platform at the European Molecular Biology Laboratory (EMBL) uses SOPs for all steps involved in a multiprotein complex expression experiment, starting from insertion of the genes into an engineered baculoviral genome optimized for heterologous protein production properties to small-scale analysis of the protein specimens produced.5-8 The platform is installed in an open-access mode at EMBL Grenoble and has supported many scientists from academia and industry to accelerate protein complex research projects.

Protein complexes catalyze key cellular functions. Detailed functional and structural characterization of many essential complexes requires recombinant production. MultiBac is a baculovirus/insect cell system particularly tailored for expressing eukaryotic proteins and their complexes. MultiBac was implemented as an open-access platform, and standard operating procedures developed to maximize its utility.

Proteomics  research revealed the impressive complexity of eukaryotic proteomes in  unprecedented detail. It is now a commonly accepted notion that ...

Quantitation of Rabies Virus in Various Bovine Brain Structures

Bovine paralytic rabies (BPR) is a form of viral encephalitis that is of substantial economic importance throughout Latin America, where it poses a major zoonotic risk. Here, our objective was to utilize a laboratory protocol to determine the relative copy number of the rabies virus (RABV) genome in different bovine brain anatomical structures using quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR). qRT-PCR quantifies the specific number of gene copies present in a sample based on fluorescence emitted after amplification that is directly proportional to the amount of target nucleic acid present in the sample. This method is advantageous owing to its short duration, reduced risk of contamination, and potential to detect viral nucleic acids in different samples more easily compared to other techniques. The brains of six rabid animals were divided into six anatomical structures, namely the Ammon&#8217;s horn, cerebellum, cortex, medulla, pons, and thalamus. All brains were identified as positive for RABV antigens based on a direct immunofluorescence test. The same anatomical structures from the brains of four RABV-negative bovines were also assessed. RNA was extracted from each structure and used for qRT-PCR. An assay was performed to determine the copy numbers of RABV genes using an in vitro transcribed nucleoprotein gene. The standard curve used to quantify viral RNA exhibited an efficiency of 100% and linearity of 0.99. Analysis revealed that the cortex, medulla, and thalamus were the ideal CNS portions for use in RABV detection, based on the observation that these structures possessed the highest levels of RABV. The test specificity was 100%. All samples were positive, no false positives were detected. This method can be used to detect RABV in samples that contain low levels of RABV during diagnosis of BPR.

This protocol presents a qRT-PCR-based approach for determining the rabies virus nucleoprotein (N) gene copy number within various bovine brain anatomical structures using in vitro transcription.

Bovine paralytic rabies (BPR) is a form of viral encephalitis that is of substantial economic importance throughout Latin America, where it poses a major ...

Cultivation Methods of Spirochetes from Borrelia burgdorferi Sensu Lato Complex and Relapsing Fever Borrelia

The Borrelia consists of three groups of species, those of the Lyme borreliosis (LB) group, also known as B. burgdorferi sensu lato (s.l.) and recently reclassified into Borreliella, the relapsing fever (RF) group Borrelia, and a third reptile-associated group of spirochetes. Culture-based methods remain the gold standard for the laboratory detection of bacterial infections for both research and clinical work, as the culture of pathogens from bodily fluids or tissues directly detects replicating pathogens and provides source material for research. Borrelia and&#160;Borreliella spirochetes&#160;are fastidious and slow growing, and thus are not commonly cultured for clinical purposes; however, culture is necessary for research. This protocol demonstrates the methodology and recipes required to successfully culture LB and RF spirochetes, including all recognized species from B. burgdorferi s.l. complex including&#160;B. afzelii, B. americana, B. andersonii, B. bavariensis, B. bissettii/bissettiae, B. burgdorferi sensu stricto&#160;(s.s.), B. californiensis, B. carolinensis, B. chilensis, B. finlandensis, B. garinii, B. japonica, B. kurtenbachii, B. lanei, B. lusitaniae, B. maritima, B. mayonii, B. spielmanii, B. tanukii, B. turdi, B. sinica, B. valaisiana, B. yangtzensis, and RFspirochetes, B. anserina, B. coriaceae, B. crocidurae, B. duttonii, B. hermsii, B. hispanica, B. persica, B. recurrentis, and B. miyamotoi. The basic medium for growing LB and RF spirochetes is the Barbour-Stoenner-Kelly (BSK-II or BSK-H) medium, which reliably supports the growth of spirochetes in established cultures. To be able to grow newly isolated Borrelia isolates from tick- or host-derived samples where the initial spirochete number is low in the inoculum, modified Kelly-Pettenkofer (MKP) medium is preferred. This medium also supports the growth of B. miyamotoi. The success of the cultivation of RF spirochetes also depends critically on the quality of ingredients.

Cultivation Methods of Spirochetes from Borrelia burgdorferi Sensu Lato Complex and Relapsing Fever Borrelia

In vitro culture is a direct detection method for the presence of living bacteria. This protocol describes methods for the culture of diverse Borrelia spirochetes,&#160;including those of the Borrelia burgdorferi sensu lato complex, relapsing fever Borrelia species, and Borrelia miyamotoi. These species are fastidious and slow growing but can be cultured.

The Borrelia consists of three groups of species, those of the Lyme borreliosis (LB) group, also known as B. burgdorferi sensu lato (s.l.) and recently ...

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

Introducing foreign DNA into the spirochete Borrelia burgdorferi has been almost exclusively accomplished by transformation using electroporation. This process has notably lower efficiencies in the Lyme disease spirochete relative to other, better-characterized Gram-negative bacteria. The rate of success of transformation is highly dependent upon having concentrated amounts of high-quality DNA from specific backgrounds and is subject to significant strain-to-strain variability. Alternative means for introducing foreign DNA (i.e., shuttle vectors, fluorescent reporters, and antibiotic-resistance markers) into B. burgdorferi could be an important addition to the armamentarium of useful tools for the genetic manipulation of the Lyme disease spirochete. Bacteriophage have been well-recognized as natural mechanisms for the movement of DNA among bacteria in a process called transduction. In this study, a method has been developed for using the ubiquitous borrelial phage &#966;BB-1 to transduce DNA between B. burgdorferi cells of both the same and different genetic backgrounds. The transduced DNA includes both borrelial DNA and heterologous DNA in the form of small shuttle vectors. This demonstration suggests a potential use of phage-mediated transduction as a complement to electroporation for the genetic manipulation of the Lyme disease spirochete. This report describes methods for the induction and purification of phage &#966;BB-1 from B. burgdorferi, the use of this phage in transduction assays, and the selection and screening of potential transductants.

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

The ability of bacteriophage to move DNA between bacterial cells makes them effective tools for the genetic manipulation of their bacterial hosts. Presented here is a methodology for inducing, recovering, and using &#966;BB-1, a bacteriophage of Borrelia burgdorferi, to transduce heterologous DNA between different strains of the Lyme disease spirochete.

Introducing foreign DNA into the spirochete Borrelia burgdorferi has been almost exclusively accomplished by transformation using electroporation. This ...

Detecting the Lyme Disease Spirochete, Borrelia Burgdorferi, in Ticks Using Nested PCR

Lyme disease is a serious vector-borne infection that is caused by the Borrelia burgdorferi sensu lato family of spirochetes, which are transmitted to humans through the bite of infected Ixodes ticks. The primary etiological agent in North America is Borrelia burgdorferi sensu stricto. As geographic risk regions expand, it is prudent to support robust surveillance programs that can measure tick infection rates, and communicate findings to clinicians, veterinarians, and the general public. The molecular technique of nested polymerase chain reaction (nPCR) has long been used for this purpose, and it remains a central, inexpensive, and robust approach in the detection of Borrelia in both ticks and wildlife. 
This article demonstrates the application of nPCR to tick DNA extracts to identify infected specimens. Two independent B. burgdorferi targets, genes encoding Flagellin B (FlaB) and Outer surface protein A (OspA), have been used extensively with this technique. The protocol involves tick collection, DNA extraction, and then an initial round of PCR to detect each of the two Borrelia-specific loci. Subsequent polymerase chain reaction (PCR) uses the product of the first reaction as a new template to generate smaller, internal amplification fragments. The nested approach improves upon both the specificity and sensitivity of conventional PCR. A tick is considered positive for the pathogen when inner amplicons from both Borrelia genes can be detected by agarose gel electrophoresis.

Detecting the Lyme Disease Spirochete, Borrelia Burgdorferi, in Ticks Using Nested PCR

Nested PCR is a sensitive, specific, and straightforward technique that can be applied to tick DNA extracts to probe for Borrelia burgdorferi, the causative agent of Lyme disease. The initial PCR experiment uses gene-specific primers to generate long amplicons, which then become templates for a subsequent reaction using internal primers.

Lyme disease is a serious vector-borne infection that is caused by the Borrelia burgdorferi sensu lato family of spirochetes, which are transmitted to ...

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

Podsumowanie

Przeglądaj więcej filmów

Rozdziały w tym wideo

In Vitro Transcription Assays and Their Application in Drug Discovery

Isolation of Active Caenorhabditis elegans Nuclear Extract and Reconstitution for In Vitro Transcription

Bacterial Artificial Chromosomes: A Functional Genomics Tool for the Study of Positive-strand RNA Viruses

Whole Mount in Situ Hybridization of E8.5 to E11.5 Mouse Embryos

In Vitro Transcribed RNA-based Luciferase Reporter Assay to Study Translation Regulation in Poxvirus-infected Cells

The MultiBac Protein Complex Production Platform at the EMBL

Quantitation of Rabies Virus in Various Bovine Brain Structures

Cultivation Methods of Spirochetes from Borrelia burgdorferi Sensu Lato Complex and Relapsing Fever Borrelia

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

Detecting the Lyme Disease Spirochete, Borrelia Burgdorferi, in Ticks Using Nested PCR