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

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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 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><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 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 ...

essential-components-borreliella-borrelia-burgdorferi-vitro

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

Methods for Rapid Transfer and Localization of Lyme Disease Pathogens Within the Tick Gut

Lyme disease is caused by infection with the spirochete pathogen Borrelia burgdorferi, which is maintained in nature by a tick-rodent infection cycle 1. A tick-borne murine model 2 has been developed to study Lyme disease in the laboratory. While na&iacute;ve ticks can be infected with B. burgdorferi by feeding them on infected mice, the molting process takes several weeks to months to complete. Therefore, development of more rapid and efficient tick infection techniques, such as a microinjection-based procedure, is an important tool for the study of Lyme disease 3,4. The procedure requires only hours to generate infected ticks and allows control over the delivery of equal quantities of spirochetes in a cohort of ticks. This is particularly important as the generation of B. burgdorferi infected ticks by the natural feeding process using mice fails to ensure 100% infection rate and potentially results in variation of pathogen burden amongst fed ticks. Furthermore, microinjection can be used to infect ticks with B. burgdorferi isolates in cases where an attenuated strain is unable to establish infection in mice and thus can not be naturally acquired by ticks 5. This technique can also be used to deliver a variety of other biological materials into ticks, for example, specific antibodies or double stranded RNA 6. In this article, we will demonstrate the microinjection of nymphal ticks with in vitro-grown B. burgdorferi. We will also describe a method for localization of Lyme disease pathogens in the tick gut using confocal immunofluorescence microscopy.

Lyme disease research studies often require generation of ticks infected with the pathogen Borrelia burgdorferi, a process that typically takes several weeks. Here we demonstrate a microinjection-based tick infection procedure that can be accomplished within hours. We also demonstrate an immunofluorescence method for in situ localization of B. burgdorferi within ticks.

Lyme disease is caused by infection with the spirochete pathogen Borrelia burgdorferi, which is maintained in nature by a tick-rodent infection cycle 1.  ...

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 ...

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 ...

In Vitro SUMOylation Assay to Study SUMO E3 Ligase Activity

Small ubiquitin-like modifier (SUMO) modification is an important post-translational modification (PTM) that mediates signal transduction primarily through modulating protein-protein interactions. Similar to ubiquitin modification, SUMOylation is directed by a sequential enzyme cascade including E1-activating enzyme (SAE1/SAE2), E2-conjugation enzyme (Ubc9), and E3-ligase (i.e., PIAS family, RanBP2, and Pc2). However, different from ubiquitination, an E3 ligase is non-essential for the reaction but does provide precision and efficacy for SUMO conjugation. Proteins modified by SUMOylation can be identified by in vivo assay via immunoprecipitation with substrate-specific antibodies and immunoblotting with SUMO-specific antibodies. However, the demonstration of protein SUMO E3 ligase activity requires in vitro reconstitution of SUMOylation assays using purified enzymes, substrate, and SUMO proteins. Since in the in vitro reactions, usually SAE1/SAE2 and Ubc9, alone are sufficient for SUMO conjugation, enhancement of SUMOylation by a putative E3 ligase is not always easy to detect. Here, we describe a modified in vitro SUMOylation protocol that consistently identifies SUMO modification using an in vitro reconstituted system. A step-by-step protocol to purify catalytically active K-bZIP, a viral SUMO-2/3 E3 ligase, is also presented. The SUMOylation activities of the purified K-bZIP are shown on p53, a well-known target of SUMO. This protocol can not only be employed for elucidating novel SUMO E3 ligases, but also for revealing their SUMO paralog specificity.

In Vitro SUMOylation Assay to Study SUMO E3 Ligase Activity

Unlike ubiquitin ligases, few E3 SUMO ligases have been identified. This modified in vitro SUMOylation protocol is able to identify novel SUMO E3 ligases by an in vitro reconstitution assay.

Small ubiquitin-like modifier (SUMO) modification is an important post-translational modification (PTM) that mediates signal transduction primarily ...

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

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In Vitro SUMOylation Assay to Study SUMO E3 Ligase Activity