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 ...

<ol>
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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 fle...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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&#160;</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>H2O 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&#160;</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 
			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 prepara...

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 ...

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.While working, maintain the cells, lysate, and purified proteins at four degrees Celsius or on ice unless freezing. Keep RNA polymerase in a reducing environment by adding freshly prepared dithiothreitol at a concentration of two-millimolar to every buffer used and maintain pH 8.0. Prepare lysate from the Borreliella burgdorferi cell pellet using a commercial bacterial lysis kit.Resuspend the pellet in 10 to 15 milliliters of B-PER solution without protease inhibitor and allow the lysis to proceed for five minutes on ice. Add a protease inhibitor cocktail to the lysis solution and proceed with three rounds of sonication. Now clarify the cell lysate by centrifugation and filtration using a 50-milliliter centrifuge tube.Add cobalt column loading buffer to the solution, making the total volume up to 30 milliliters. Pellet the cell debris by centrifugation at 20, 000 times G for 30 minutes. Later, filter the supernatant using a 0.45-micrometer syringe filter and dilute the lysate and cobalt column loading buffer to a final dilution ratio of 1:10.Perform affinity chromatography on the clarified cell lysate supernatant using a cobalt or nickel resin column following the manufacturer's instructions. Save the flow-through, wash, and eluted samples for analysis. Immediately exchange the RNA polymerase solution buffer solution with the storage buffer solution using a buffer exchange column following the manufacturer's instructions.Then, concentrate the RNA polymerase to a concentration of 0.2 to 0.4 milligram per milliliter using 10-kilodaltons-cutoff centrifugal filter units. Determine the concentration by spectrophotometer and prepare the freezing stocks. Aliquot 20 to 50 microliters volumes of RNA polymerase freezing stocks in PCR tubes and store the RNA polymerase at minus 80 degrees Celsius.Prepare the work surfaces, including the radiation bench, to reduce radiation contamination. Thaw fresh RNA polymerase and RpoD stocks on ice and mix all the thawed frozen stocks thoroughly prior to pipetting Prepare a master reaction mixture in a separate NTP mixture for radiolabeled nucleotides according to the experimental requirements. Dispense the control reactions in experimental sets into PCR tubes.After dispensing water, reaction mix, RNA polymerase, and RpoD into designated PCR tubes, mix the reagents by pipetting. Transfer the prepared materials to a radiation bench, add alpha-32P-labeled ATP to NTP, and mix by gentle pipetting. Add NTP to the tubes containing the in vitro transcription reaction mixtures and start the in vitro transcription reaction by adding a DNA template.Mix the reaction volume by gently pipetting and incubate the tubes at 37 degrees Celsius for five minutes in a thermocycler or heat block. Remove the reactions from the thermocycler or heat block and stop the in vitro transcription reactions by adding an equal volume of 2X RNA loading dye containing 50%formamide to the reaction mixture. Denature of the enzymes by incubating reactions at 65 degrees Celsius for five minutes in a thermocycler or heat block.Using gel electrophoresis, separate the in vitro transcribed RNA in 10%to 15%TBE urea polyacrylamide gels at 180 volts for 30 to 45 minutes. After removing any portion of the gel containing unincorporated radiolabeled ATP, expose the gel to the phospho screen overnight and image the radiolabeled RNA using a phosphoscreen imager. The SDS-PAGE showed three bands corresponding to three peptides, RpoC, RpoB, and RpoA of RNA polymerase complex.A peptide of 115 kilodaltons matching the size of the MBP-tagged recombinant Borreliella burgdorferi RpoD was also observed. Cleavage of the protein mixture with factor XA protease led to the generation of two significant products, RpoD and MBP. The promoter site of Borreliella burgdorferi FLGB was generated by PCR.A twofold dilution of RpoD concentration gives rise to a lower level of the accumulated RNA product. A low RNA polymerase concentration gives fewer RNA products across the range of RpoD concentrations. The densitometry signal indicated a linear relationship between the amount of RpoD present in the reaction in both experiments.RNA polymerase enzymatic activity is sensitive to a variety of factors during the purification, storage, and experimental process. Fresh enzyme and reagents, along with good planning, give the best possible chance of success in this protocol.

essential-components-borreliella-borrelia-burgdorferi-vitro

Research

JoVE Journal

Immunology and Infection

1.2K visualizações.  Creighton University. O sistema de ensaio de transcrição in vitro para Borreliella burgdorferi fornece uma ferramenta bioquímica para investigadores estudarem mecanismos e fatores regulatórios gênicos que governam a atividade enzimática da RNA polimerase. O sistema nos permite testar como fatores de transcrição, cofatores, concentrações de sal e pH afetam a função da RNA polimerase da Borreliella burgdorferi, o que contribui para nossa compreensão geral dos mecanismos de regulação gênica. Esta téc...