Name: large-scale production of recombinant rnas on a circular scaffold using a viroid-derived system in escherichia coli
Uploaded: 2018-11-30T14:00:00.000+00:00
Duration: 10 min 38 s
Description: Watch this Scientific Journal Video about Large-scale Production of Recombinant RNAs on a Circular Scaffold Using a Viroid-derived System in Escherichia coli at JoVE.com

This work was supported by grants BIO2017-83184-R and BIO2017-91865-EXP from the Spanish Ministerio de Ciencia, Innovaci&#243;n y Universidades (co-financed FEDER funds).

1. Plasmid Construction
<ol>
	<li>Amplify by PCR (or by reverse transcription PCR if starting from an RNA template) the cDNA corresponding to the RNA of interest using primers with 5&#8217; extension to allow assembly into the expression plasmid.&#160;To avoid undesired mutations, use a high-fidelity DNA polymerase.
	<ol>
		<li>To insert the cDNA in the expression plasmid by Gibson assembly20, add the following 5&#8217; extensions to the PCR primers: forward, 5&#8217;-tctccccctcccaggtactatccccttXXXXXXXXXXXXXXXXXXXXXX-3&#8217;; reverse, 5&#8217;-ccctcctagggaacacatccttgaXXXXXXXXXXXXXXXXXXXXXX-3&#8217;; X represents nucleotides homologous to the terminal ends of the RNA of interest.</li>
		<li>Incubate for 30 s at 98&#160;&#176;C, followed by 30 cycles of 10 s at 98&#160;&#176;C, 30 s at 55&#160;&#176;C and 30 s at 72&#160;&#176;C, and a final extension of 10 min at 72&#160;&#176;C.</li>
	</ol>
	</li>
	<li>Digest 100 ng of plasmid pLELVd-BZB with 10 U of the type-IIS restriction enzyme Bpi I for 1 h at 37&#160;&#176;C in a 20 &#181;L reaction in a 0.5 mL tube in buffer G (10 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 50 mM NaCl, 0.1 mg/mL BSA). 
	NOTE: All plasmids are available on request to the corresponding author. Bpi I is equivalent to Bbs I.</li>
	<li>Separate the PCR and digestion products by electrophoresis in a 1% agarose gel in TAE buffer (40 mM Tris, 20 mM acetic acid, 1 mM EDTA, pH 7.2). Stain the gel for 15 min by shaking in 200-mL of 0.5&#160;&#181;g/mL ethidium bromide. Visualize the DNA using a UV transilluminator and cut the bands corresponding to the amplified cDNA and the Bpi I-digested plasmid (2046 bp) using a scalpel blade. 
	NOTE: Bpi I digestion of pLELVd-BZB also releases a 528 bp product corresponding to the LacZ blue-white reporter.</li>
	<li>Elute the DNAs from the gel fragments using silica gel columns (gel DNA recovery kit in Table of Materials) and quantify the DNA concentration by spectrophotometric analysis.</li>
	<li>Set up a Gibson assembly reaction using the amplified cDNA and the digested plasmid. Use a 3-fold molar excess of insert versus vector20. Incubate for 1 h at 50&#160;&#176;C and purify the reaction products using a silica gel column (DNA clean &#38; concentrator kit in the Table of Materials).</li>
	<li>Use the purified products of the Gibson assembly reaction to electroporate competent E. coli DH5&#945; cells. Using 1-mm electroporation cuvettes, apply the following settings: 1,500 V and 5 ms. Incubate for 1 h at 37&#160;&#176;C in super optimized broth with catabolite repression (SOC; 20 g/L tryptone, 5 g/L yeast extract, 0.5 g/L NaCl, 2.5 mM KCl, 10 mM MgCl2, 20 mM glucose, pH 7.0) liquid medium and then spread onto Luria-Bertani (LB; 10 g/L tryptone, 5 g/L yeast extract, 10 g/L NaCl) agar (1.5%) plates containing 50&#160;&#181;g/mL ampicillin.
	<ol>
		<li>To screen for colonies corresponding to transformed E. coli clones that likely incorporated the insert, 15 min before plating the electroporated cells, spread 30&#160;&#181;L of 50 mg/mL 5-bromo-4-chloro-3-indolyl-&#946;-D-galactopyranoside (X-gal) in dimethylformamide. Incubate plates overnight at 37&#160;&#176;C.</li>
	</ol>
	</li>
	<li>Pick several white colonies and grow overnight at 37&#160;&#176;C in liquid LB media. Purify plasmids using a miniprep kit (see Table of Materials) and analyze their sizes by electrophoresis in a 1% agarose gel in TAE buffer.</li>
	<li>Select the most likely recombinant plasmid based on electrophoretic migration compared to the pLELVd-BZB control. Confirm the sequence of the selected plasmid by sequencing using primers 5&#8217;-CCTTTTTCAATATTATTGAAGC-3&#8217; and 5&#8217;-GATGCTCGTCAGGGGGGCGGAG-3&#8217; that flank the whole expression cassette in pLELVd-BZB. 
	NOTE: Remember that this plasmid contains a 528 bp polylinker with the LacZ marker that is replaced by the cDNA of interest. Restriction mapping may help to select the right recombinant plasmids.</li>
</ol>
2. RNA Expression
<ol>
	<li>Co-electroporate (see conditions in 1.6) the selected E. coli strain (E. coli BL21 or a BL21 derivative) with the pLELVd-BZB-derivative that contains the cDNA corresponding to the RNA of interest and plasmid p15LtRnlSm to co-express the eggplant tRNA ligase. Use the E. coli HT115(DE3), which lacks RNase III21, to express RNAs with long double-stranded regions. 
	Note: Both the RNA of interest and the tRNA ligase mRNA are transcribed by the E. coli RNA polymerase. No DE3 lysogen to express T7 RNA polymerase is required in the E. coli strain, although the presence of this lysogen has no deleterious effects.</li>
	<li>After 1&#160;h incubation at 37&#160;&#176;C in SOC liquid medium, plate electroporated bacteria in LB solid medium containing 50&#160;&#181;g/mL ampicillin and 34&#160;&#181;g/mL chloramphenicol. Incubate overnight at 37&#160;&#176;C.</li>
	<li>Pick a colony and inoculate a 1 L baffled Erlenmeyer flask with 250&#160;mL of liquid Terrific Broth (TB) medium (12&#160;g/L tryptone, 24&#160;g/L yeast extract, 0.4% glycerol, 0.17 M KH2PO4, and 0.72 M K2HPO4), containing 50&#160;&#181;g/mL ampicillin and 34&#160;&#181;g/mL chloramphenicol. Incubate at 37&#160;&#176;C with vigorous shaking at 180 revolutions per minute (rpm). Harvest bacteria between 12 and 16 h after culture inoculation.</li>
</ol>
3. RNA Extraction and Purification
<ol>
	<li>For analytical purposes, take 2 mL aliquots of the culture at the desired time points and centrifuge at 14,000 x g for 2 min. Discard the supernatant and resuspend the cells in 50&#160;&#181;L of TE buffer (10 mM Tris-HCl, pH 8.0, and 1 mM EDTA) by vortexing.
	<ol>
		<li>Add one volume (50&#160;&#181;L) of a 1:1 (v/v) mix of phenol (saturated with water and equilibrated at pH 8.0 with Tris-HCl, pH 8.0) and chloroform. Break the cells by vigorous vortexing and separate the aqueous and organic phases by centrifugation for 5&#160;min at 14,000&#160;x&#160;g.</li>
		<li>Carefully recover the aqueous phases (upper) that contains total bacterial RNA. 
		Note: Preparations can be stored at -20&#160;&#176;C for subsequent analysis.</li>
	</ol>
	</li>
	<li>For preparative purposes, pour culture into a 250 mL centrifuge bottle and spin down cells at 14,000 x g for 10 min. Discard supernatant. Wash the cells by resuspending in 30&#160;mL of water. Transfer to a centrifuge tube and spin down cells again under the same conditions.</li>
	<li>Discard the supernatant and resuspend the cells in 10&#160;mL of chromatography buffer (50 mM Tris-HCl, pH 6.5, 150 mM NaCl, 0.2 mM EDTA) by vortexing. 
	NOTE: At this time, cells can be frozen at -20&#160;&#176;C to proceed with purification at any other moment.</li>
	<li>Using a fresh or thawed bacterial preparation, break cells by adding 1 volume (10&#160;mL) of phenol:chloroform (see step 3.2) and vortexing vigorously.</li>
	<li>Centrifuge for 10 min at 12,000 x g, recover the aqueous phase and re-extract with 1 volume (10&#160;mL) of chloroform under the same conditions. 
	NOTE: The RNA preparation can be stored at -20&#160;&#176;C at this point.</li>
	<li>Further purify total bacterial RNA by anion-exchange chromatography. Filter the RNA preparation through a 45 &#181;m syringe filter and load on a 1 mL diethylethanolamine (DEAE) column.
	<ol>
		<li>For chromatography purification, use a liquid chromatography system at a flow rate of 1&#160;mL/min. Before sample loading, equilibrate the column with 10&#160;mL of chromatography buffer (see step 3.3 for composition).</li>
		<li>Load the sample and wash the column with 10&#160;mL of chromatography buffer. Elute RNA with 20&#160;mL of elution buffer (50 mM Tris-HCl, pH 6.5, 1 M NaCl, 0.2&#160;mM EDTA) and collect 1 mL aliquots. 
		Note: RNA quickly elutes in the initial fractions. The column can be re-used for further purifications. For this, wash the column with 10&#160;mL of water and store at 4&#160;&#176;C in 20% ethanol.</li>
	</ol>
	</li>
	<li>Since a major part of the recombinant RNA accumulates in E. coli in a circular form, this property can be profited for purification to homogeneity12. Separate circular RNAs from the linear counterparts by two-dimensional polyacrylamide gel electrophoresis (2D PAGE) combining non-denaturing and denaturing (8 M urea) conditions12,22.</li>
</ol>
4. RNA Analysis
<ol>
	<li>Prepare a 5% polyacrylamide gel (37.5:1 acrylamyde:N,N&#8217;-methylenebisacrylamide, mass ratio) in TBE buffer (89 mM Tris, 89 mM boric acid, 2 mM EDTA) containing 8 M urea.</li>
	<li>Mix 20&#160;&#181;L of RNA preparations with 1 volume (20&#160;&#181;L) of loading buffer (98% formamide, 10 mM Tris-HCl, pH 8.0, 1 mM EDTA, 0.0025% bromophenol blue, and 0.0025% xylene cyanol), incubate for 1.5 min at 95&#160;&#176;C in a heating block, and snap cool on ice.</li>
	<li>Load the samples in the polyacrylamide gel and run the electrophoresis at appropriate conditions depending on the gel dimensions (e.g., run 140 x 130 x 2 mm gels for 2.5 h at 200 V). Stain the gel for 15 min in 0.5 &#181;g/mL ethidium bromide, wash with water, and visualize RNA under UV light.</li>
</ol>

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The authors declare that the technology described in this protocol has been patented (US Patent No. EP14382177.5, PCT/EP2015/060912).

While researching the ELVd sequence and structure requirements involved in the recognition by the eggplant tRNA ligase, we noticed that co-expression of both molecules in the non-host E. coli led to an unexpected large accumulation of viroid circular forms in bacterial cells19. We understood that the large accumulation of viroid RNA in E. coli most probably was the consequence of co-expressing a highly stable RNA molecule, such as the relatively small (333 nt), highly based-paire...

In contrast to DNA and proteins, protocols for easy, efficient and cost-effective production of large amounts of RNA are not abundant. However, research and industry demand increasing amounts of these biomolecules to investigate their unique biological properties1, or to be employed in sophisticated biotechnological applications, including their use as highly specific aptamers2, therapeutic agents3, or selective pesticides4. In vitro transcription and chemical synthesis are commonly used in research to produce RNA. However, these methods entail important limitations when large amounts of the products are required. The logical alternative is to use the endogenous transcription machinery of living cells, followed by a purification process to separate the RNAs of interest from the cellular companions. Following this strategy, methods have been developed to produce recombinant RNAs in bacterial cells, such as the lab-friendly Escherichia coli5 or the marine purple phototrophic alpha-proteobacterium Rhodovolum sulfidophilum6. Most methods to produce recombinant RNA in bacteria rely on the expression of a native highly stable RNA scaffold, such as a tRNA or an rRNA, in which the RNA of interest is inserted7. This imposes the necessity of releasing the RNA of interest out of the chimeric molecule, if the presence of extra RNA is a problem for the downstream applications8. Another concept in recombinant RNA biotechnology is the production of recombinant ribonucleoprotein complexes that may be the desired product per se or used as a protective strategy to increase the stability of the RNA of interest9,10. Similarly, the production of circular RNAs has also been suggested as a strategy to generate more stable products11.
We have recently developed a new method to produce large amounts of recombinant RNA in E. coli that participates in three of the above concepts: the insertion of the RNA of interest in a highly stable circular RNA scaffold and the co-expression of the recombinant RNA with an interacting protein to likely produce a stable ribonucleoprotein complex that accumulates in remarkable amounts in bacterial cells12. In contrast to previous developments, we used an RNA scaffold completely alien to E. coli, namely a viroid. Viroids are a very particular type of infectious agents of higher plants that are exclusively constituted by a relatively small (246-401 nt) highly base-paired circular RNA13. Interestingly, viroids are non-coding RNAs and, with no help from their own proteins, they are able to complete complex infectious cycles in the infected hosts14. These cycles include the RNA-to-RNA replication in the nuclei or chloroplasts, depending on the viroid family &#8212;Pospiviroidae or Avsunviroidae, respectively&#8212;&#160;movement through the infected plant and evasion of the host defensive response. Viroids must be ranked among the most stable RNAs in nature, as a consequence of being a naked circular RNA and having to survive in the hostile environment of plant infected cells. This property may make viroids particularly suitable as scaffolds to stabilize recombinant RNA in biotechnological approaches. In addition, the new method is based on co-expression of the viroid scaffold with an interacting plant protein. Viroids replicate through a rolling-circle mechanism in which host enzymes are recruited to catalyze the different steps of the process. Notably some viroids, more specifically those that belong to the family Avsunviroidae15, contain ribozymes that are also involved in replication. Depending on the viroid species, transcription of viroid RNAs is mediated by the host RNA polymerase II or the chloroplastic nuclear-encoded RNA polymerase (NEP). Viroid RNA processing seems to be catalyzed by a host type-III RNase, although in viroids with ribozymes oligomeric RNA intermediates self-cleave during replication. Finally, the resulting viroid monomers are circularized, depending on the viroid family, by the host DNA ligase 1 or the chloroplastic isoform of tRNA ligase16,17. This last enzyme is involved in ligation of the monomeric forms of the viroids in the family Avsunviroidae, such as Eggplant latent viroid (ELVd)18.
In the course of a work to analyze the sequence and structural requirements of ELVd that determine recognition by the eggplant (Solanum melongena L.) tRNA ligase, we set up an experimental system based on co-expression of both molecules in E. coli19. We noticed that longer-than-unit ELVd transcripts self-cleave efficiently in E. coli cells through the embedded hammerhead ribozymes and that the resulting viroid monomers with 5&#39;-hydroxyl and 2&#39;,3&#39;-phosphodiester termini were efficiently circularized by the co-expressed eggplant tRNA ligase. Even more, the resulting circular viroid RNA reached an unexpected high concentration in E. coli, exceeding those of the endogenous rRNAs12. Absence of replication intermediates indicated lack of ELVd RNA-to-RNA amplification in these bacterial cells. Interestingly, insertion of heterologous RNAs in a particular position of the viroid molecule had a moderate effect on accumulation of the circular viroid-derived RNAs12. These observations made us envision a method to produce large amounts of recombinant RNAs in bacteria. In this method, the cDNAs corresponding to the RNAs of interest are inserted in the ELVd cDNA and the resulting chimeric RNA is expressed in E. coli through a strong constitutive promoter. For the system to work, E. coli must be co-transformed with a plasmid to express the eggplant tRNA ligase. The ELVd-derived longer-than-unit transcript is processed by the embedded hammerhead ribozymes and the resulting monomers with the appropriate termini are recognized and circularized by the co-expressed tRNA ligase. This way, the RNA of interest is inserted into a very stable circular scaffold consisting of the viroid circular molecule. This recombinant chimeric RNA is most probably further stabilized inside the E. coli cells by formation of a ribonucleoprotein complex through interaction with tRNA ligase. Using this method (see protocol below), RNA aptamers, hairpin RNAs and other structured RNAs have been easily produced in amounts of tens of milligrams per liter of E. coli culture in regular laboratory conditions and purified to homogeneity taking advantage of circularity12.

<table><tbody><tr><td>Phusion High-Fidelity DNA polymerase</td><td>Thermo Scientific</td><td>F530S</td><td></td></tr><tr><td>&lt;em&gt;Bpi&lt;/em&gt; I</td><td>Thermo Scientific</td><td>ER1011</td><td></td></tr><tr><td>Agarose</td><td>Conda</td><td>8010</td><td>D1 low EEO</td></tr><tr><td>Tris</td><td>PanReac AppliChem</td><td>A1086,1000</td><td></td></tr><tr><td>Acetic acid</td><td>PanReac AppliChem</td><td>131008.1214</td><td></td></tr><tr><td>EDTA</td><td>Sigma-Aldrich</td><td>E5134-500G</td><td></td></tr><tr><td>Ethidium bromide</td><td>PanReac AppliChem</td><td>A1152,0025</td><td>1%</td></tr><tr><td>Zymoclean Gel DNA Recovery</td><td>Zymo Research</td><td>D4001</td><td></td></tr><tr><td>NanoDrop</td><td>ThermoFisher Scientific</td><td>ND-3300</td><td></td></tr><tr><td>NEBuilder HiFi DNA Assembly Master Mix</td><td>New England BioLabs</td><td>E2621S</td><td></td></tr><tr><td>DNA Clean &amp;amp; Concentrator</td><td>Zymo Research</td><td>D4003</td><td></td></tr><tr><td>Eporator</td><td>Eppendorf</td><td>4309000019</td><td></td></tr><tr><td>&lt;em&gt;Escherichia coli&lt;/em&gt; DH5&amp;alpha;</td><td>Invitrogen</td><td>18265-017</td><td></td></tr><tr><td>Tryptone</td><td>Intron Biotechnology</td><td>Ba2014</td><td></td></tr><tr><td>Yeast extract</td><td>Intron Biotechnology</td><td>48045</td><td></td></tr><tr><td>NaCl</td><td>PanReac AppliChem</td><td>131659.1211</td><td></td></tr><tr><td>Agar</td><td>Intron Biotechnology</td><td>25999</td><td></td></tr><tr><td>Ampicillin</td><td>PanReac AppliChem</td><td>A0839,0010</td><td></td></tr><tr><td>X-gal</td><td>Duchefa</td><td>X1402.1000</td><td></td></tr><tr><td>N,N-Dimethylformamide</td><td>PanReac AppliChem</td><td>131785.1611</td><td></td></tr><tr><td>NucloSpin Plasmid</td><td>Macherey-Nagel</td><td>22740588.250</td><td></td></tr><tr><td>&lt;em&gt;Escherichia coli&lt;/em&gt; BL21(DE3)</td><td>Novagen</td><td>69387-3</td><td></td></tr><tr><td>&lt;em&gt;Escherichia coli&lt;/em&gt; HT115(DE3)</td><td></td><td></td><td>Ref. Timmons et al., 2001</td></tr><tr><td>Chloramphenicol</td><td>Duchefa</td><td>C 0113.0025</td><td></td></tr><tr><td>Glycerol</td><td>PanReac AppliChem</td><td>122329.1211</td><td>87%</td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>PanReac AppliChem</td><td>131509.1210</td><td></td></tr><tr><td>K&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>PanReac AppliChem</td><td>122333.1211</td><td></td></tr><tr><td>HCl</td><td>PanReac AppliChem</td><td>131020.1211</td><td></td></tr><tr><td>Phenol</td><td>Scharlau</td><td>FE04791000</td><td>90%</td></tr><tr><td>Chloroform</td><td>PanReac AppliChem</td><td>A3691,1000</td><td></td></tr><tr><td>Filtropur S 0.2</td><td>Sarstedt</td><td>83.1826.001</td><td></td></tr><tr><td>HiTrap DEAE Sepharose FF column</td><td>GE Healthcare Life Sciences</td><td>17-5055-01</td><td></td></tr><tr><td>&amp;Auml;KTAprime plus liquid chromatography system</td><td>GE Healthcare Life Sciences</td><td>11001313</td><td></td></tr><tr><td>Acrylamyde</td><td>PanReac AppliChem</td><td>A1089,1000</td><td>2K</td></tr><tr><td>&lt;em&gt;N,N&amp;rsquo;&lt;/em&gt;-methylenebisacrylamide</td><td>Sigma-Aldrich</td><td>M7279-100G</td><td></td></tr><tr><td>Urea</td><td>PanReac AppliChem</td><td>146392.1211</td><td></td></tr><tr><td>Boric acid</td><td>PanReac AppliChem</td><td>A2940,1000</td><td></td></tr><tr><td>Formamide</td><td>PanReac AppliChem</td><td>A0937,2500</td><td></td></tr><tr><td>Bromophenol blue</td><td>Sigma-Aldrich</td><td>B8026-5G</td><td></td></tr><tr><td>Xylene cyanol</td><td>Sigma-Aldrich</td><td>X4126-10G</td><td></td></tr><tr><td>N,N&amp;prime;-Bis(acryloyl)cystamine</td><td>Sigma-Aldrich</td><td>A4929-5G</td><td></td></tr></tbody></table>

large-scale production of recombinant rnas on a circular scaffold using a viroid-derived system in escherichia coli

With increasing interest in RNA biology and the use of RNA molecules in sophisticated biotechnological applications, the methods to produce large amounts of recombinant RNAs are limited. Here, we describe a protocol to produce large amounts of recombinant RNA in Escherichia coli based on co-expression of a chimeric molecule that contains the RNA of interest in a viroid scaffold and a plant tRNA ligase. Viroids are relatively small, non-coding, highly base-paired circular RNAs that are infectious to higher plants. The host plant tRNA ligase is an enzyme recruited by viroids that belong to the family Avsunviroidae, such as Eggplant latent viroid (ELVd), to mediate RNA circularization during viroid replication. Although ELVd does not replicate in E. coli, an ELVd precursor is efficiently transcribed by the E. coli RNA polymerase and processed by the embedded hammerhead ribozymes in bacterial cells, and the resulting monomers are circularized by the co-expressed tRNA ligase reaching a remarkable concentration. The insertion of an RNA of interest into the ELVd scaffold enables the production of tens of milligrams of the recombinant RNA per liter of bacterial culture in regular laboratory conditions. A main fraction of the RNA product is circular, a feature that facilitates the purification of the recombinant RNA to virtual homogeneity. In this protocol, a complementary DNA (cDNA) corresponding to the RNA of interest is inserted in a particular position of the ELVd cDNA in an expression plasmid that is used, along the plasmid to co-express eggplant tRNA ligase, to transform E. coli. Co-expression of both molecules under the control of strong constitutive promoters leads to production of large amounts of the recombinant RNA. The recombinant RNA can be extracted from the bacterial cells and separated from the bulk of bacterial RNAs taking advantage of its circularity.

To produce recombinant RNA in E. coli using the ELVd-derived system12, the RNA of interest is grafted into an ELVd RNA scaffold. This chimeric RNA is co-expressed along the eggplant tRNA ligase in E. coli. Once processed, cleaved and circularized, the chimeric circular RNA, from which the RNA of interest protrudes, likely forms a ribonucleoprotein complex with the co-expressed eggplant enzyme that reaches remarkable concentration in the bacterial ...

Watch this Scientific Journal Video about Large-scale Production of Recombinant RNAs on a Circular Scaffold Using a Viroid-derived System in Escherichia coli at JoVE.com

Large-scale Production of Recombinant RNAs on a Circular Scaffold Using a Viroid-derived System in Escherichia coli

Here, we present a protocol to produce large amounts of recombinant RNA in Escherichia coli by co-expressing a chimeric RNA that contains the RNA of interest in a viroid scaffold and a plant tRNA ligase. The main product is a circular molecule that facilitates purification to homogeneity.

Large-scale Production of Recombinant RNAs on a Circular Scaffold Using a Viroid-derived System in Escherichia coli

With increasing interest in RNA biology and the use of RNA molecules in sophisticated biotechnological applications, the methods to produce large amounts ...

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Universidad San Sebastian

Research

JoVE Journal

Biochemistry

9.5K Views.  Consejo Superior de Investigaciones Científicas-Universitat Politècnica de València, Spain. Here, we present a protocol to produce large amounts of recombinant RNA in Escherichia coli by co-expressing a chimeric RNA that contains the RNA of interest in a viroid scaffold and a plant tRNA ligase. The main product is a circular molecule that facilitates purification to homogeneity.

Video: Large-scale Production of Recombinant RNAs on a Circular Scaffold Using a Viroid-derived System in Escherichia coli

Use of Alu Element Containing Minigenes to Analyze Circular RNAs

In addition to linear mRNAs, many eukaryotic genes generate circular RNAs. Most circular RNAs are generated by joining a 5&#39; splice site with an upstream 3&#39; splice site within a pre-mRNA, a process called back-splicing. This circularization is likely aided by secondary structures in the pre-mRNA that bring the splice sites into close proximity. In human genes, Alu elements are thought to promote these secondary RNA structures, as Alu elements are abundant and exhibit base complementarities with each other when present in opposite directions in the pre-mRNA. Here, we describe the generation and analysis of large, Alu element containing reporter genes that form circular RNAs. Through optimization of cloning protocols, reporter genes with up to 20 kb insert length can be generated. Their analysis in co-transfection experiments allows the identification of regulatory factors. Thus, this method can identify RNA sequences and cellular components involved in circular RNA formation.

Use of Alu Element Containing Minigenes to Analyze Circular RNAs

We clone and analyze reporter genes generating circular RNAs. These reporter genes are larger than constructs to analyze linear splicing and contain Alu elements. To investigate the circular RNAs, the constructs are transfected into cells and resulting RNA is analyzed using RT-PCR after removal of linear RNA.

In addition to linear mRNAs, many eukaryotic genes generate circular RNAs. Most circular RNAs are generated by joining a 5&#39; splice site with an ...

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 ...

Immunology and Infection

CFPS Protocols for Micro-Compartmentalized Synthetic Cells

Cell-Free Protein Synthesis System for Building Synthetic Cells

The Cell-Free Protein Synthesis (CFPS) system has been widely employed to facilitate the bottom-up assembly of synthetic cells. It serves as the host for the core machinery of the Central Dogma, standing as an optimal chassis for the integration and assembly of diverse artificial cellular mimicry systems. Despite its frequent use in the fabrication of synthetic cells, establishing a tailored and robust CFPS system for a specific application remains a nontrivial challenge. In this methods paper, we present a comprehensive protocol for the CFPS system, routinely employed in constructing synthetic cells. This protocol encompasses key stages in the preparation of the CFPS system, including the cell extract, template preparation, and routine expression optimization utilizing a fluorescent reporter protein. Additionally, we show representative results by encapsulating the CFPS system within various micro-compartments, such as monolayer droplets, double-emulsion vesicles, and chambers situated atop supported lipid bilayers. Finally, we elucidate the critical steps and conditions necessary for the successful assembly of these CFPS systems in distinct environments. We expect that our approach will facilitate the establishment of good working practices among various laboratories within the continuously expanding synthetic cell community, thereby accelerating progress in the field of synthetic cell development.

Author Spotlight: Optimizing CFPS Systems for Synthetic Cell Construction

This protocol describes the Cell-Free Protein Synthesis (CFPS) system used in constructing synthetic cells. It outlines key stages with representative results in different micro-compartments. The protocol aims to establish best practices for diverse laboratories in the synthetic cell community, advancing progress in synthetic cell development.

The Cell-Free Protein Synthesis (CFPS) system has been widely employed to facilitate the bottom-up assembly of synthetic cells. It serves as the host for ...

Cell-free Protein Expression Using the Rapidly Growing Bacterium Vibrio natriegens

The marine bacterium Vibrio natriegens has garnered considerable attention as an emerging microbial host for biotechnology due to its fast growth rate. A general protocol is described for the preparation of V. natriegens crude cell extracts using common laboratory equipment. This high yielding protocol has been specifically optimized for user accessibility and reduced cost. Cell-free protein synthesis (CFPS) can be carried out in small scale 10 &#956;L batch reactions in either a 96- or 384-well format and reproducibly yields concentrations of &#62; 260 &#956;g/mL super folder GFP (sfGFP) within 3 h. Overall, crude cell extract preparation and CFPS can be achieved in 1&#8722;2 full days by a single user. This protocol can be easily integrated into existing protein synthesis pipelines to facilitate advances in bio-production and synthetic biology applications.

Cell-free Protein Expression Using the Rapidly Growing Bacterium Vibrio natriegens

Cell-free expression systems are powerful and cost-efficient tools for the high-throughput synthesis and screening of important proteins. Here, we describe the preparation of cell-free protein expression system using Vibrio natriegens for the rapid protein production using plasmid DNA, linear DNA, and mRNA template.

The marine bacterium Vibrio natriegens has garnered considerable attention as an emerging microbial host for biotechnology due to its fast growth rate. A ...

Bioengineering

An Efficient Method for Adenovirus Production

Adenoviral transduction has the advantage of a strong and transient induction of the expression of the gene of interest into a broad variety of cell types and organs. However, recombinant adenoviral technology is laborious, time-consuming, and expensive. Here, we present an improved protocol using the pAdEasy system to obtain purified adenoviral particles that can induce a strong green fluorescent protein (GFP) expression in transduced cells. The advantages of this improved method are faster preparation and decreased production cost compared to the original method developed by Bert Vogelstein. The main steps of the adenoviral technology are: (1) the recombination of pAdTrack-GFP with the pAdEasy-1 plasmid in BJ5183 bacteria; (2) the packaging of the adenoviral particles; (3) the amplification of the adenovirus in AD293 cells; (4) the purification of the adenoviral particles from cell lysate and culture medium; and (5) the viral titration and functional testing of the adenovirus. The improvements to the original method consist of (i) the recombination in BJ5183-containing pAdEasy-1 by chemical transformation of bacteria; (ii) the selection of recombinant clones by &#8220;negative&#8221; and &#8220;positive&#8221; PCR; (iii) the transfection of AD293 cells using the K2 transfection system for adenoviral packaging; (iv) the precipitation with ammonium sulfate of the viral particles released by AD293 cells in cell culture medium; and (v) the purification of the virus by one-step cesium chloride discontinuous gradient ultracentrifugation. A strong expression of the gene of interest (in this case, GFP) was obtained in different types of transduced cells (such as hepatocytes, endothelial cells) from various sources (human, bovine, murine). Adenoviral-mediated gene transfer represents one of the main tools for developing modern gene therapies.

Here, we present a protocol for adenovirus production using the pAdEasy system. The technology includes the recombination of the pAdTrack and pAdEasy-1 plasmids, the adenovirus packaging and amplification, the purification of the adenoviral particles from cell lysate and culture medium, the viral titration, and the functional testing of the adenovirus.

Adenoviral transduction has the advantage of a strong and transient induction of the expression of the gene of interest into a broad variety of cell types ...

Generation, Amplification, and Titration of Recombinant Respiratory Syncytial Viruses

The use of recombinant viruses has become crucial in basic or applied virology. Reverse genetics has been proven to be an extremely powerful technology, both to decipher viral replication mechanisms and to study antivirals or provide development platform for vaccines. The construction and manipulation of a reverse genetic system for a negative-strand RNA virus such as a respiratory syncytial virus (RSV), however, remains delicate and requires special know-how. The RSV genome is a single-strand, negative-sense RNA of about 15 kb that serves as a template for both viral RNA replication and transcription. Our reverse genetics system uses a cDNA copy of the human RSV long strain genome (HRSV). This cDNA, as well as cDNAs encoding viral proteins of the polymerase complex (L, P, N, and M2-1), are placed in individual expression vectors under T7 polymerase control sequences. The transfection of these elements in BSR-T7/5 cells, which stably express T7 polymerase, allows the cytoplasmic replication and transcription of the recombinant RSV, giving rise to genetically modified virions. A new RSV, which is present at the cell surface and in the culture supernatant of BSRT7/5, is gathered to infect human HEp-2 cells for viral amplification. Two or three rounds of amplification are needed to obtain viral stocks containing 1 x 106 to 1 x 107 plaque-forming units (PFU)/mL. Methods for the optimal harvesting, freezing, and titration of viral stocks are described here in detail. We illustrate the protocol presented here by creating two recombinant viruses respectively expressing free green fluorescent protein (GFP) (RSV-GFP) or viral M2-1 fused to GFP (RSV-M2-1-GFP). We show how to use RSV-GFP to quantify RSV replication and the RSV-M2-1-GFP to visualize viral structures, as well as viral protein dynamics in live cells, by using video microscopy techniques.

We describe a method for generating and amplifying genetically modified respiratory syncytial viruses (RSVs) and an optimized plaque assay for RSVs. We illustrate this protocol by creating two recombinant viruses that respectively allow quantification of RSV replication and live analysis of RSV inclusion bodies and inclusion bodies-associated granules dynamics.

The use of recombinant viruses has become crucial in basic or applied virology. Reverse genetics has been proven to be an extremely powerful technology, ...

A Convenient and General Expression Platform for the Production of Secreted Proteins from Human Cells

Recombinant protein expression in bacteria, typically E. coli, has been the most successful strategy for milligram quantity expression of proteins. However, prokaryotic hosts are often not as appropriate for expression of human, viral or eukaryotic proteins due to toxicity of the foreign macromolecule, differences in the protein folding machinery, or due to the lack of particular co- or post-translational modifications in bacteria. Expression systems based on yeast (P. pastoris or S. cerevisiae) 1,2, baculovirus-infected insect (S. frugiperda or T. ni) cells 3, and cell-free in vitro translation systems 2,4 have been successfully used to produce mammalian proteins. Intuitively, the best match is to use a mammalian host to ensure the production of recombinant proteins that contain the proper post-translational modifications. A number of mammalian cell lines (Human Embryonic Kidney (HEK) 293, CV-1 cells in Origin carrying the SV40 larget T-antigen (COS), Chinese Hamster Ovary (CHO), and others) have been successfully utilized to overexpress milligram quantities of a number of human proteins 5-9. However, the advantages of using mammalian cells are often countered by higher costs, requirement of specialized laboratory equipment, lower protein yields, and lengthy times to develop stable expression cell lines. Increasing yield and producing proteins faster, while keeping costs low, are major factors for many academic and commercial laboratories.
Here, we describe a time- and cost-efficient, two-part procedure for the expression of secreted human proteins from adherent HEK 293T cells. This system is capable of producing microgram to milligram quantities of functional protein for structural, biophysical and biochemical studies. The first part, multiple constructs of the gene of interest are produced in parallel and transiently transfected into adherent HEK 293T cells in small scale. The detection and analysis of recombinant protein secreted into the cell culture medium is performed by western blot analysis using commercially available antibodies directed against a vector-encoded protein purification tag. Subsequently, suitable constructs for large-scale protein production are transiently transfected using polyethyleneimine (PEI) in 10-layer cell factories. Proteins secreted into litre-volumes of conditioned medium are concentrated into manageable amounts using tangential flow filtration, followed by purification by anti-HA affinity chromatography. The utility of this platform is proven by its ability to express milligram quantities of cytokines, cytokine receptors, cell surface receptors, intrinsic restriction factors, and viral glycoproteins. This method was also successfully used in the structural determination of the trimeric ebolavirus glycoprotein 5,10.
In conclusion, this platform offers ease of use, speed and scalability while maximizing protein quality and functionality. Moreover, no additional equipment, other than a standard humidified CO2 incubator, is required. This procedure may be rapidly expanded to systems of greater complexity, such as co-expression of protein complexes, antigens and antibodies, production of virus-like particles for vaccines, or production of adenoviruses or lentiviruses for transduction of difficult cell lines.

In the post-human genomics era, the availability of recombinant proteins in native conformations is crucial to structural, functional and therapeutic research and development. Here, we describe a test- and large-scale protein expression system in human embryonic kidney 293T cells that can be used to produce a variety of recombinant proteins.

Recombinant protein expression in bacteria, typically E. coli, has been the most successful strategy for milligram quantity expression of proteins. ...

Biology

Recombinant Protein Expression for Structural Biology in HEK 293F Suspension Cells: A Novel and Accessible Approach

The expression and purification of large amounts of recombinant protein complexes is an essential requirement for structural biology studies. For over two decades, prokaryotic expression systems such as E. coli have dominated the scientific literature over costly and less efficient eukaryotic cell lines. Despite the clear advantage in terms of yields and costs of expressing recombinant proteins in bacteria, the absence of specific co-factors, chaperones and post-translational modifications may cause loss of function, mis-folding and can disrupt protein-protein interactions of certain eukaryotic multi-subunit complexes, surface receptors and secreted proteins. The use of mammalian cell expression systems can address these drawbacks since they provide a eukaryotic expression environment. However, low protein yields and high costs of such methods have until recently limited their use for structural biology. Here we describe a simple and accessible method for expressing and purifying milligram quantities of protein by performing transient transfections of suspension grown HEK (Human Embryonic Kidney) 293F cells.

The expression of recombinant proteins by mammalian systems is becoming an attractive method for producing protein complexes for structural biology. Here we present a simple yet highly efficient expression system using suspension grown mammalian cells to purify protein complexes for structural studies.

The expression and purification of large amounts of recombinant protein complexes is an essential requirement for structural biology studies. For over two ...

Production of Double-stranded DNA Ministrings

We constructed linear covalently closed (LCC) DNA minivectors as a non-viral gene-delivery vector alternative produced via a simple platform in vivo. DNA ministrings possess a heightened safety profile and also efficiently deliver DNA cargo to targeted cells. Conventional DNA vectors carry undesirable prokaryotic sequences, including antibiotic resistance genes, CpG motifs, and bacterial origins of replication, which may lead to the stimulation of host immunological responses. The bioavailability of conventional DNA vectors is also compromised due to their larger molecular size. Their circular nature may also impart chromosomal integration, leading to insertional mutagenesis.
Bacterial sequences are excised from DNA minivectors, leaving only the gene of interest (GOI) and necessary eukaryotic expression elements. Our LCC DNA minivectors, or DNA ministrings, are devoid of immunogenic bacterial sequences; therefore improving their bioavailability and GOI expression. In the event of vector integration into the chromosome, the LCC DNA ministring will lethally disrupt the host chromosome, thereby removing the potentially dangerous mutant from the proliferating cell population. Consequently, DNA ministrings offer the benefits of &#39;minicircle&#39; DNA while eliminating the potential for undesirable vector integration events. In comparison to conventional plasmids and their isogenic circular covalently closed (CCC) counterparts, DNA ministrings demonstrate superior bioavailability, transfection efficiency, and cytoplasmic kinetics - they thus require lower amounts of cationic surfactants for effective transfection of target cells.
We have constructed a one-step inducible in vivo system for the production of DNA ministrings in Escherichia coli that is simple to use, rapid, and scalable.

The linear covalently closed (LCC) DNA minivector (DNA ministring) is a non-viral gene delivery vector offering high transfection efficiency and is relevant to any DNA delivery application. The production system is simple, rapid, and scalable in vivo. The following protocol provides visual step-by-step instructions for the production of DNA ministrings.

We constructed linear covalently closed (LCC) DNA minivectors as a non-viral gene-delivery vector alternative produced via a simple platform in vivo. DNA ...

Phage-mediated Delivery of Targeted sRNA Constructs to Knock Down Gene Expression in E. coli

RNA-mediated knockdowns are widely used to control gene expression. This versatile family of techniques makes use of short RNA (sRNA) that can be synthesized with any sequence and designed to complement any gene targeted for silencing. Because sRNA constructs can be introduced to many cell types directly or using a variety of vectors, gene expression can be repressed in living cells without laborious genetic modification. The most common RNA knockdown technology, RNA interference (RNAi), makes use of the endogenous RNA-induced silencing complex (RISC) to mediate sequence recognition and cleavage of the target mRNA. Applications of this technique are therefore limited to RISC-expressing organisms, primarily eukaryotes. Recently, a new generation of RNA biotechnologists have developed alternative mechanisms for controlling gene expression through RNA, and so made possible RNA-mediated gene knockdowns in bacteria. Here we describe a method for silencing gene expression in E. coli that functionally resembles RNAi. In this system a synthetic phagemid is designed to express sRNA, which may designed to target any sequence. The expression construct is delivered to a population of E. coli cells with non-lytic M13 phage, after which it is able to stably replicate as a plasmid. Antisense recognition and silencing of the target mRNA is mediated by the Hfq protein, endogenous to E. coli. This protocol includes methods for designing the antisense sRNA, constructing the phagemid vector, packaging the phagemid into M13 bacteriophage, preparing a live cell population for infection, and performing the infection itself. The fluorescent protein mKate2 and the antibiotic resistance gene chloramphenicol acetyltransferase (CAT) are targeted to generate representative data and to quantify knockdown effectiveness.

Phage-mediated Delivery of Targeted sRNA Constructs to Knock Down Gene Expression in E. coli

We describe a method to knock down gene expression in a growing population of E. coli cells using sequence-targeted sRNA expression cassettes delivered by an M13 phagemid vector.

Large-scale Production of Recombinant RNAs on a Circular Scaffold Using a Viroid-derived System in Escherichia coli

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Rozdziały w tym wideo

Use of Alu Element Containing Minigenes to Analyze Circular RNAs

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

Cell-Free Protein Synthesis System for Building Synthetic Cells

Cell-free Protein Expression Using the Rapidly Growing Bacterium Vibrio natriegens

An Efficient Method for Adenovirus Production

Generation, Amplification, and Titration of Recombinant Respiratory Syncytial Viruses

A Convenient and General Expression Platform for the Production of Secreted Proteins from Human Cells

Recombinant Protein Expression for Structural Biology in HEK 293F Suspension Cells: A Novel and Accessible Approach

Production of Double-stranded DNA Ministrings

Phage-mediated Delivery of Targeted sRNA Constructs to Knock Down Gene Expression in E. coli