Name: inducible t7 rna polymerase-mediated multigene expression system, pmgx
Uploaded: 2017-06-27T13:00:00.000+00:00
Duration: 10 min 9 s
Description: Watch this Scientific Journal Video about Inducible T7 RNA Polymerase-mediated Multigene Expression System, pMGX at JoVE.com

This work was supported by the Natural Sciences and Engineering Research Council of Canada.

1. Obtaining Genes of Interest
<ol>
	<li>Design synthetic genes.
	<ol>
		<li>Optimize a gene sequence for E. coli expression.</li>
		<li>Remove any problematic restriction sites from the sequence (AvrII, NdeI, EcoRI, and XbaI).</li>
		<li>Incorporate restriction sites for cloning; a 5&#8242;-NdeI site and a 3&#8242;-EcoRI site are recommended. Other sites can be used, if necessary; refer to the multicloning region of the selected plasmid (Figure S1-S4). If desired, include a 5&#8242; or 3&#8242; encoding tag for Western blot detection.</li>
		<li>Commercially order the designed gene.</li>
		<li>Either blunt clone the gene into a blunt vector using a blunt cloning kit, as per the manufacturer&#39;s instructions; design primers to amplify the gene (then proceed to Step 1.2.2); or add additional 5&#8242; and 3&#8242; ends for direct cloning into a pMGX plasmid and proceed to Step 2. Here, the representative results are from blunt cloning the genes of interest.</li>
	</ol>
	</li>
	<li>Amplify the desired gene (from a designed and optimized synthetic gene or from template DNA containing the desired gene)15.
	<ol>
		<li>Design primers with restriction sites for cloning; a 5&#8242;-NdeI site and a 3&#8242;-EcoRI site are recommended. Other sites can be used, if necessary; refer to the multicloning region of the selected plasmid (Figure S1-S4). If desired, include either a 5&#8242; or 3&#8242; encoding tag for Western blot detection.</li>
		<li>PCR amplify the desired gene15.</li>
		<li>Analyze the PCR by agarose gel electrophoresis16.</li>
		<li>If there is unspecific amplification, clean up the amplified gene by gel extraction. If not, use an enzyme clean-up kit.</li>
		<li>Quantify the DNA using a spectrophotometer by checking the absorbance at 260 nm; blank with elution buffer17.</li>
	</ol>
	</li>
</ol>
2. Cloning Genes of Interest into a Multigene Expression System Vector, pMGX18
<ol>
	<li>Restriction digest the obtained gene of interest and the desired vector, pMGX, with NdeI and EcoRI. 
	NOTE: If a large amount of recircularized plasmids are obtained, allow the NdeI reaction to proceed for 1 h before adding EcoRI.
	<ol>
		<li>Use a 40 &#181;L digest reaction containing 0.5-1.5 &#181;g of DNA and 1 &#181;L of each enzyme with 4 &#181;L of the appropriate 10x buffer.</li>
		<li>For an NdeI and EcoRI digest, use EcoRI buffer and first add NdeI endonuclease. Digest for 1 h at 37 &#176;C. Then add EcoRI endonuclease, and allow the digest to proceed for an additional hour. NdeI is sensitive to cleavages close to the end of DNA, so initial digestion by EcoRI can prevent effective digestion by NdeI.</li>
	</ol>
	</li>
	<li>Electrophorese the restriction digest on agarose gel (for a 1 kb gene, use a 0.7% agarose gel at 110 V for 55 min; to select the percent of agarose gel for differing gene sizes, refer to reference16.</li>
	<li>Excise insert and vector bands using a clean scalpel or razor blade and place the excised gel segment into a 1.5 mL tube.</li>
	<li>Extract DNA from the agarose gel using a gel extraction kit according to the manufacturer&#39;s protocol.</li>
	<li>Ligate the gene of interest into the pMGX vector using a 3:1 insert-to-vector ratio; set up a negative ligation of digested pMGX without the insert.
	<ol>
		<li>Set up a 20 &#181;L ligation reaction containing 1 &#181;L of T4 DNA ligase, 0.15-0.5 &#181;g of vector DNA (~5 &#181;L), and an appropriate amount of insert based on a 3:1 insert-to-vector ratio and the size of the gene being inserted; the pMGX backbones are between 5,312 and 5,504 bp in size. Include a negative control reaction that contains everything but the gene being inserted. Ensure that the amount of vector DNA is equivalent in the negative control ligation and the vector plus insert ligation reactions.</li>
	</ol>
	</li>
	<li>Transform ligation reactions into XL1-Blue chemically competent E. coli cells and ligated plasmids pMGX-yfg1 (containing gene of interest 1), pMGX-yfg2 (containing gene of interest 2) &#8230; pMGX-yfgn (containing gene of interest n). Use aseptic technique (inside a biosafety cabinet or under a flame).
	<ol>
		<li>Thaw 100 &#181;L aliquots of chemically competent XL1-Blue E. coli on ice for 5 min and then add 5 &#181;L of the ligation reactions. Incubate for 30 min on ice.</li>
		<li>Heat-shock the cells for 45 s in a water bath held at 42 &#176;C and then add 200 &#181;L of cold LB. Incubate them on ice for 2 min.</li>
		<li>Shake the cells at 37 &#176;C, 220 rpm for 1 h and then spread plate 100 &#181;L onto an LB-agar plate containing an appropriate selectable marker (either ampicillin or kanamycin).</li>
	</ol>
	</li>
	<li>Screen the clones for positive transformants.
	<ol>
		<li>Compare the colony counts on the negative control and ligation plates. A colony count ratio greater than 1:2 is desired. If there are a large number of colonies on the negative control plate, go back to Step 2.1 and review the note.</li>
		<li>Select 4-8 individual colonies (depending on the negative control:ligation ratio) from each ligation reaction of the different genes inserted for screening (1 n) and inoculate a 4 mL LB and the appropriate antibiotic overnight culture/individual colony. Grow cultures overnight with shaking at 37 &#176;C and 220 rpm.</li>
		<li>Isolate plasmid DNA using a plasmid DNA isolation kit according to the manufacturer&#39;s protocol.</li>
		<li>Set up a 20 &#181;L NdeI + EcoRI digest reaction containing 150-500 ng of DNA and 1 &#181;L of each enzyme with 2 &#181;L of the appropriate 10x buffer. In this case, add NdeI and EcoRI at the same time that the gene of interest will be in between the restriction sites. A negative control with the empty pMGX vector is recommended. Digest for 2 h at 37 &#176;C in a water bath.</li>
	</ol>
	</li>
</ol>
3. Inserting Gene 2 into the pMGX Vector Containing Gene 1, pMGX-yfg1
<ol>
	<li>Restriction digest pMGX-yfg1 with AvrII and treat with calf intestinal phosphatase (CIP).
	<ol>
		<li>Use a 40-&#181;L digest reaction containing 0.5-1.5 &#181;g of vector DNA (~5-10 &#181;L of isolated DNA) and 1 &#181;L of AvrII with 4 &#181;L of the appropriate 10x buffer. Digest for 1.5 h at 37 &#176;C and then add 1.5 &#181;L of CIP. Leave it at 37 &#176;C for an additional 30 min.</li>
	</ol>
	</li>
	<li>Restriction digest pMGX-yfg2 with AvrII and XbaI to liberate the gene of interest.
	<ol>
		<li>Use a 40 &#181;L digest reaction containing 0.5-1.5 &#181;g of DNA and 1 &#181;L of each enzyme with 4 &#181;L of the appropriate10x buffer. Digest for 2 h at 37 &#176;C.</li>
	</ol>
	</li>
	<li>Electrophorese restriction digests on an appropriate percent (0.7%) agarose gel and excise the insert and vector bands using a clean scalpel/razor blade (refer to Steps 2.2-2.3).</li>
	<li>Extract the DNA using a gel extraction kit according to the manufacturer&#39;s protocol and quantify the DNA17.</li>
	<li>Ligate gene 2 into pMGX-yfg1 using a 3:1 insert-to-vector ratio. Set up a negative ligation of digested pMGX-yfg1 without the additional insert. Set up as above in Step 2.5.</li>
	<li>Transform 5 &#181;L of the ligation reactions into XL1-Blue chemically competent E. coli cells, ligated plasmids pMGX-yfg1,2 (containing gene of interest 1 and 2), and negative pMGX-yfg1 control, as seen in Step 2.6.</li>
	<li>Compare the colony count on the negative control and ligation plates. A colony count ratio greater than 1:2 is desired. If there are a large number of colonies on the negative control plate, go back to Step 3.1 and review the CIP treatment.</li>
	<li>Select 4-8 individual colonies (depending on the negative control:ligation ratio) from the ligation reaction and inoculate 4 mL of LB plus the appropriate antibiotic per individual colony and grow overnight at 37 &#176;C and 220 rpm.</li>
	<li>Isolate plasmid DNA using a plasmid DNA isolation kit according to the manufacturer&#39;s protocol and quantify the DNA17.</li>
	<li>Screen the effective insertion of the second gene by performing a restriction digest of pMGX-yfg1,2 with EcoRI.
	<ol>
		<li>Use a 20 &#181;L digest reaction containing 150-500 ng of DNA and 1 &#181;L of EcoRI enzyme with 2 &#181;L of EcoRI 10x buffer. Digest for 2 h at 37 &#176;C.</li>
	</ol>
	</li>
	<li>Electrophorese the restriction digest on an appropriate percent agarose gel; look for a band that corresponds to the size of gene 2 (see Step 2.2). A gene may insert into the vector in the undesired, reversed orientation.</li>
</ol>
4. Adding a Third Gene into the pMGX Vector Containing Genes 1 and 2, pMGX-yfg1,2
<ol>
	<li>Restriction digest pMGX-yfg1,2 with AvrII and treat with CIP, as seen in Step 3.1.</li>
	<li>Restriction digest pMGX-yfg3 with AvrII and XbaI, as seen in Step 3.2.</li>
	<li>Electrophorese the restriction digests on an appropriate percent agarose gel and excise insert and vector bands using a clean scalpel/razor blade; refer to Steps 2.2-2.3.</li>
	<li>Extract the DNA from the agarose gel using a gel extraction kit and quantify the DNA17.</li>
	<li>Ligate gene 3 into pMGX-yfg1,2 using a 3:1 insert-to-vector ratio and set up a negative ligation of digested pMGX-yfg1,2 without an additional insert, as seen in Step 3.5.</li>
	<li>Transform 5 &#181;L of the ligation reactions into XL1-Blue chemically competent E. coli cells, ligated plasmid pMGX-yfg1,2,3 (containing genes of interest 1, 2, and 3), and negative pMGX-yfg1,2 control, as seen in Step 2.6.</li>
	<li>Compare the colony count on the negative control and ligation plates. If there are a large number of colonies on the negative control plate, go back to Step 4.1 and review the CIP treatment.</li>
	<li>Select 4-8 individual colonies (depending on the negative control:ligation ratio) from the ligation reaction and inoculate 4 mL of LB plus the appropriate antibiotic per individual colony; grow overnight at 37 &#176;C and 220 rpm.</li>
	<li>Isolate the plasmid DNA using a plasmid DNA isolation kit according to the manufacturer&#39;s protocol.</li>
	<li>Screen the effective insertion of the third gene by performing a restriction digest of pMGX-yfg1,2,3 with EcoRI, as seen in Step 3.10.</li>
	<li>Electrophorese the restriction digest on an appropriate percent agarose gel; look for bands that correspond to the sizes of gene 2 and gene 3 (see Step 2.2). Note: gene 3 may insert into the vector in the undesired, reversed orientation. If genes 2 and 3 are the same size, another appropriate restriction digest site must be selected for screening. 
	NOTE: Repeat as needed for each new gene.</li>
</ol>
5. Producing Proteins of Interest Using a Multigene Expression System and Assessing Production by Western Blotting
<ol>
	<li>Transform the positive clone containing all genes of interest into chemically competent, protein-production E. coli, such as BL21-(&#955;DE3).
	<ol>
		<li>Thaw 100 &#181;L aliquots of chemically competent BL21-(&#955;DE3) E. coli on ice for 5 min and then add 1 &#181;L of the positive cloned plasmid DNA; incubate for 30 min on ice.</li>
		<li>Heat shock the cells for 45 s in a water bath held at 42 &#176;C and then add 200 &#181;L of cold LB. Incubate on ice for 2 min.</li>
		<li>Shake the cells at 37 &#176;C and 220 rpm for 1 h and then spread plate 100 &#181;L onto an LB-agar plate containing the appropriate selectable marker (either ampicillin or kanamycin).</li>
	</ol>
	</li>
	<li>Express the protein by isopropyl-&#946;-D-1-thiogalactopyranoside (IPTG) induction.
	<ol>
		<li>Select an isolated colony from the B21-(&#955;DE3) transformation plate and inoculate 4 mL of LB plus the appropriate antibiotic; grow overnight, shaking at 37 &#176;C and 220 rpm.</li>
		<li>Inoculate a 100 mL LB plus the appropriate antibiotic culture using 1 mL of overnight culture.</li>
		<li>Grow at 37 &#176;C with shaking at 220 rpm to an OD600 of 0.6.</li>
		<li>Induce the culture with 100 &#181;M IPTG and grow for 15 h at 25 &#176;C and 220 rpm.</li>
		<li>Remove 1 mL of the culture and centrifuge it at 13,000 x g for 1 min; discard the supernatant.</li>
	</ol>
	</li>
	<li>Lyse the cells using lysis solution as per the manufacturer&#39;s instructions and preform a Western blot of the soluble cell lysate to determine if all proteins were successfully produced19.</li>
</ol>

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The authors have nothing to disclose.

Co-expression of multiple genes is increasingly essential, particularly in characterizing and reconstituting complex, multigene metabolic pathways3,4,5. The pMGX system makes multigene co-expression in E. coli routine6,7,8 and accessible to diverse researchers. In this study, five proteins of interest were shown to be simultane...

Co-expression of multiple proteins is increasingly essential, particularly in synthetic biology applications, where multiple functional modules must be expressed1; in studying protein-protein complexes, where expression and function often require co-expression2,3; and in characterizing and harnessing biosynthetic pathways, where each gene in the pathway must be expressed4,5,6,7,8. A number of systems have been developed for co-expression, particularly in the host organism Escherichia coli, the work horse for laboratory recombinant protein expression9. For example, multiple plasmids with differing selectable markers can be used to express individual proteins using a wealth of different expression vectors10,11. Single plasmid systems for multiple protein expression have used either multiple promoters to control the expression of each gene10,12; synthetic operons, where multiple genes are encoded on a single transcript2,13; or, in some cases, a single gene encoding a polypeptide that is ultimately proteolytically processed, yielding the desired proteins of interest14.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55187/55187fig1.jpg" /> 
Figure 1: pMGX workflow showing the construction of a polycistronic vector. The pMGX system provides a flexible, easy-to-use strategy for the construction of synthetic operons under the control of an inducible T7 promoter. <a href="//cloudflare.jove.com/files/ftp_upload/55187/55187fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
In this manuscript, the use of a highly effective system for the construction of multigene synthetic operons under the control of an inducible T7 RNA polymerase (Figure 1) is described. This system allows many genes to be expressed simultaneously from one plasmid. It is based on a plasmid system, originally called pKH22, that has been used successfully for a number of different applications6,7,8. Here, this plasmid set is expanded to include four related vectors: pMGX-A, an expression vector lacking any C- or N-terminal tags and with the ampicillin resistance marker; pMGX-hisA, an expression vector encoding an N-terminal hexahistidine tag and with the ampicillin resistance marker; pMGX-K, an expression vector lacking any C- or N-terminal tags and with the kanamycin resistance marker; and pMGX-hisK, an expression vector encoding an N-terminal hexahistidine tag and with the kanamycin resistance marker. In this study, the method for generating a polycistronic vector containing five genes using the pMGX system, specifically pMGX-A, is demonstrated along with the successful production of each individual protein in Escherichia coli.

<table><tbody><tr><td>&lt;strong&gt;Enzymes&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Alkaline Phosphatase, Calf Intestinal (CIP)</td><td>New England Biolabs</td><td>M0290S</td><td></td></tr><tr><td>AvrII</td><td>New England Biolabs</td><td>R0174S</td><td></td></tr><tr><td>EcoRI</td><td>New England Biolabs</td><td>R0101S</td><td></td></tr><tr><td>NdeI</td><td>New England Biolabs</td><td>R0111S</td><td></td></tr><tr><td>XbaI</td><td>New England Biolabs</td><td>R0145S</td><td></td></tr><tr><td>Herculase II Fusion DNA Polymerase</td><td>Agilent Technologies</td><td>600677</td><td></td></tr><tr><td>T4 DNA Ligase</td><td>New England Biolabs</td><td>M0202S</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>1 kb DNA ladder</td><td>New England Biolabs</td><td>N3232L</td><td></td></tr><tr><td>4-20% Mini-PROTEAN TGX Stain-Free Protein Gels</td><td>Bio-Rad</td><td>456-8095</td><td></td></tr><tr><td>50x TAE</td><td>Fisher Thermo Scientific</td><td>BP1332-4</td><td></td></tr><tr><td>Agar</td><td>Fisher Thermo Scientific</td><td>BP1423-500</td><td></td></tr><tr><td>Agarose</td><td>Fisher Thermo Scientific</td><td>BP160-500</td><td></td></tr><tr><td>Ampicilin</td><td>Sigma-Alrich</td><td>A9518-5G</td><td></td></tr><tr><td>BL21 (DE3) chemically comeptent cells</td><td></td><td></td><td>Comeptent cell prepared in house</td></tr><tr><td>B-PER Bacterial Protein Extraction Reagent</td><td>Fisher Thermo Scientific</td><td>PI78243</td><td></td></tr><tr><td>dNTP mix</td><td>Agilent Technologies</td><td></td><td>Supplied with polymerase</td></tr><tr><td>Gel Extraction Kit</td><td>Omega</td><td>D2500-02</td><td>E.Z.N.A Gel Extraction, supplied by VWR Cat 3: CA101318-972</td></tr><tr><td>Glycine</td><td>Fisher Thermo Scientific</td><td>BP381-1</td><td></td></tr><tr><td>His Tag Antibody [HRP], mAb, Mouse</td><td>GenScript</td><td>A00612</td><td></td></tr><tr><td>Immobilon Western Chemiluminescent HRP Substrate</td><td>EMD Millipore</td><td>WBKLS0100</td><td></td></tr><tr><td>IPTG</td><td>Sigma-Alrich</td><td>15502-10G</td><td></td></tr><tr><td>LB</td><td>Fisher Thermo Scientific</td><td>BP1426-500</td><td></td></tr><tr><td>Methanol</td><td>Fisher Thermo Scientific</td><td>A411-20</td><td></td></tr><tr><td>Pasteurized instant skim milk powder</td><td>Local grocery store</td><td></td><td>No-name grocery store milk is adequate</td></tr><tr><td>Nitrocellulose membrane</td><td>Amersham Protran (GE Healthcare Life Sciences)</td><td>10600007</td><td>Membrane PT 0.45 &amp;micro;m 200 mm x 4 m, supplied by VWR Cat #: CA10061-086</td></tr><tr><td>Plasmid DNA Isolation Kit</td><td>Omega</td><td>D6943-02</td><td>E.Z.N.A Plasmid DNA MiniKit I, supplied by VWR Cat #: CA101318-898</td></tr><tr><td>pMGX</td><td>Boddy Lab</td><td></td><td>Request from the Boddy Lab Contact cboddy@uottawa.ca</td></tr><tr><td>Primers</td><td>Intergrated DNA Technologies</td><td></td><td>Design primers as needed for desired gene</td></tr><tr><td>Synthetic Gene</td><td>Life Technologies</td><td></td><td>Design and optimize as needed</td></tr><tr><td>Thick Blot Filter Paper</td><td>Bio-Rad</td><td>1703932</td><td></td></tr><tr><td>Tris base</td><td>BioShop</td><td>TRS001.1</td><td></td></tr><tr><td>Tween-20</td><td>Sigma-Alrich</td><td>P9416-50ML</td><td></td></tr><tr><td>XL1-Blue chemically competent cells</td><td></td><td></td><td>Comeptent cell prepared in house</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>BioSpectrometer</td><td>Eppendorf</td><td>RK-83600-07</td><td></td></tr><tr><td>Gel box - PAGE</td><td>Bio-Rad</td><td>1658005</td><td>Mini-PROTEIN Tetra Vertical Electrophoresis Cell</td></tr><tr><td>Gel Imager</td><td>Alpha Innotech</td><td></td><td>AlphaImager EC</td></tr><tr><td>Incubator-oven</td><td>Fisher Thermo Scientific</td><td>11-690-650D</td><td>Isotemp</td></tr><tr><td>Incubator-shaker</td><td>Fisher Thermo Scientific</td><td>SHKE6000-7</td><td>MaxQ 6000</td></tr><tr><td>Personna Razors</td><td>Fisher Thermo Scientific</td><td>S04615</td><td></td></tr><tr><td>Power Pack</td><td>Bio-Rad</td><td>S65533Q</td><td>FB300</td></tr><tr><td>Transilluminator</td><td>VWR International</td><td>M-10E,6W</td><td></td></tr><tr><td>Thermocylcer</td><td>Eppendorf</td><td>Z316091</td><td>Mastercycler Personal, supplied by Sigma</td></tr><tr><td>UV Face-Shield</td><td></td><td>18-999-4542</td><td></td></tr><tr><td>Waterbath</td><td>Fisher Thermo Scientific</td><td>15-460-2SQ</td><td></td></tr><tr><td>Western Transfer Apparatus</td><td>Bio-Rad</td><td>1703935</td><td>Mini-Trans Blot Cell</td></tr></tbody></table>

inducible t7 rna polymerase-mediated multigene expression system, pmgx

Co-expression of multiple proteins is increasingly essential for synthetic biology, studying protein-protein complexes, and characterizing and harnessing biosynthetic pathways. In this manuscript, the use of a highly effective system for the construction of multigene synthetic operons under the control of an inducible T7 RNA polymerase is described. This system allows many genes to be expressed simultaneously from one plasmid. Here, a set of four related vectors, pMGX-A, pMGX-hisA, pMGX-K, and pMGX-hisK, with either the ampicillin or kanamycin resistance selectable marker (A and K) and either possessing or lacking an N-terminal hexahistidine tag (his) are disclosed. Detailed protocols for the construction of synthetic operons using this vector system are provided along with the corresponding data, showing that a pMGX-based system containing five genes can be readily constructed and used to produce all five encoded proteins in Escherichia coli. This system and protocol enables researchers to routinely express complex multi-component modules and pathways in E. coli.

In this study, the goal was to co-express five proteins from a single plasmid. The five-codon optimized synthetic gene fragments encoding either N- or C-terminal hexahistidine tags were purchased commercially. The synthetic genes were amplified by PCR and individually cloned into a PCR-blunt vector and sequenced. To generate the polycistronic plasmid, the five genes of interest were first cloned into a suitable pMGX plasmid, pMGX-A. Figure 2 shows PCRBlunt-<e...

Watch this Scientific Journal Video about Inducible T7 RNA Polymerase-mediated Multigene Expression System, pMGX at JoVE.com

Inducible T7 RNA Polymerase-mediated Multigene Expression System, pMGX

This study describes methods for the T7-mediated co-expression of multiple genes from a single plasmid in Escherichia coli using the pMGX plasmid system.

Co-expression of multiple proteins is increasingly essential for synthetic biology, studying protein-protein complexes, and characterizing and harnessing ...

inducible-t7-rna-polymerase-mediated-multigene-expression-system-pmgx

Research

JoVE Journal

Genetics

13.2K Views.  University of Ottawa. This study describes methods for the T7-mediated co-expression of multiple genes from a single plasmid in Escherichia coli using the pMGX plasmid system.

Video: Inducible T7 RNA Polymerase-mediated Multigene Expression System, pMGX

Transient Gene Expression in Tobacco using Gibson Assembly and the Gene Gun

In order to target a single protein to multiple subcellular organelles, plants typically duplicate the relevant genes, and express each gene separately using complex regulatory strategies including differential promoters and/or signal sequences. Metabolic engineers and synthetic biologists interested in targeting enzymes to a particular organelle are faced with a challenge: For a protein that is to be localized to more than one organelle, the engineer must clone the same gene multiple times. This work presents a solution to this strategy: harnessing alternative splicing of mRNA. This technology takes advantage of established chloroplast and peroxisome targeting sequences and combines them into a single mRNA that is alternatively spliced. Some splice variants are sent to the chloroplast, some to the peroxisome, and some to the cytosol. Here the system is designed for multiple-organelle targeting with alternative splicing. In this work, GFP was expected to be expressed in the chloroplast, cytosol, and peroxisome by a series of rationally designed 5&#8217; mRNA tags. These tags have the potential to reduce the amount of cloning required when heterologous genes need to be expressed in multiple subcellular organelles. The constructs were designed in previous work11, and were cloned using Gibson assembly, a ligation independent cloning method that does not require restriction enzymes. The resultant plasmids were introduced into Nicotiana benthamiana epidermal leaf cells with a modified Gene Gun protocol. Finally, transformed leaves were observed with confocal microscopy.

This work describes a novel method for selectively targeting subcellular organelles in plants, assayed using the BioRad Gene Gun.

In order to target a single protein to multiple subcellular organelles, plants typically duplicate the relevant genes, and express each gene separately ...

Environment

Controllable Ion Channel Expression through Inducible Transient Transfection

Transfection, the delivery of foreign nucleic acids into a cell, is a powerful tool in protein research. Through this method, ion channels can be investigated through electrophysiological analysis, biochemical characterization, mutational studies, and their effects on cellular processes. Transient transfections offer a simple protocol in which the protein becomes available for analysis within a few hours to days. Although this method presents a relatively straightforward and time efficient protocol, one of the critical components is calibrating the expression of the gene of interest to physiological relevant levels or levels that are suitable for analysis. To this end, many different approaches that offer the ability to control the expression of the gene of interest have emerged. Several stable cell transfection protocols provide a way to permanently introduce a gene of interest into the cellular genome under the regulation of a tetracycline-controlled transcriptional activation. While this technique produces reliable expression levels, each gene of interest requires a few weeks of skilled work including calibration of a killing curve, selection of cell colonies, and overall more resources. Here we present a protocol that uses transient transfection of the Transient Receptor Potential cation channel subfamily V member 1 (TRPV1) gene in an inducible system as an efficient way to express a protein in a controlled manner which is essential in ion channel analysis. We demonstrate that using this technique, we are able to perform calcium imaging, whole cell, and single channel analysis with controlled channel levels required for each type of data collection with a single transfection. Overall, this provides a replicable technique that can be used to study ion channels structure and function.

Studying ion channels through a heterologously expressing system has become a core technique in biomedical research. In this manuscript, we present a time efficient method to achieve tightly controlled ion channel expression by performing transient transfection under the control of an inducible promoter.

Transfection, the delivery of foreign nucleic acids into a cell, is a powerful tool in protein research. Through this method, ion channels can be ...

Constitutive and Inducible Systems for Genetic In Vivo Modification of Mouse Hepatocytes Using Hydrodynamic Tail Vein Injection

In research models of liver cancer, regeneration, inflammation, and fibrosis, flexible systems for in vivo gene expression and silencing are highly useful. Hydrodynamic tail vein injection of transposon-based constructs is an efficient method for genetic manipulation of hepatocytes in adult mice. In addition to constitutive transgene expression, this system can be used for more advanced applications, such as shRNA-mediated gene knock-down, implication of the CRISPR/Cas9 system to induce gene mutations, or inducible systems. Here, the combination of constitutive CreER expression together with inducible expression of a transgene or miR-shRNA of choice is presented as an example of this technique. We cover the multi-step procedure starting from the preparation of sleeping beauty-transposon constructs, to the injection and treatment of mice, and the preparation of liver tissue for analysis by immunostaining. The system presented is a reliable and efficient approach to achieve complex genetic manipulations in hepatocytes. It is specifically useful in combination with Cre/loxP-based mouse strains and can be applied to a variety of models in the research of liver disease.

Constitutive and Inducible Systems for Genetic In Vivo Modification of Mouse Hepatocytes Using Hydrodynamic Tail Vein Injection

Hydrodynamic tail vein injection of transposon-based integration vectors enables stable transfection of murine hepatocytes in vivo. Here, we present a practical protocol for transfection systems that enables the long-term constitutive expression of a single transgene or combined constitutive and doxycycline-inducible expression of a transgene or miR-shRNA in the liver.

In research models of liver cancer, regeneration, inflammation, and fibrosis, flexible systems for in vivo gene expression and silencing are highly ...

An Ecdysone Receptor-based Singular Gene Switch for Deliberate Expression of Transgene with Robustness, Reversibility, and Negligible Leakiness

Precise control of transgene expression is desirable in biological and clinical studies. However, because the binary feature of currently employed gene switches requires the transfer of two therapeutic expression units concurrently into a single cell, the practical application of the system for gene therapy is limited. To simplify the transgene expression system, we generated a gene switch designated as pEUI(+) encompassing a complete set of transgene expression modules in a single vector. Comprising of the GAL4 DNA-binding domain and modified EcR (GvEcR), a minimal VP16 activation domain fused with a GAL4 DNA-binding domain, as well as a modified Drosophila ecdysone receptor (EcR), the newly developed singular gene switch is highly responsive to the administration of a chemical inducer in a time- and dosage-dependent manner. The pEUI(+) vector is a potentially powerful tool for improving the control of transgene expression in both biological research and pre-clinical studies. Here, we present a detailed protocol for modulation of a transient and stable transgene expression using pEUI(+) vector by the treatment of tebufenozide (Teb). Additionally, we share important guidelines for the use of Teb as a chemical inducer.

Here, we present a protocol for the modulation of transgene expression using pEUI(+) singular gene switch by tebufenozide treatment.

Precise control of transgene expression is desirable in biological and clinical studies. However, because the binary feature of currently employed gene ...

Inducible, Cell Type-Specific Expression in Arabidopsis thaliana Through LhGR-Mediated Trans-Activation

Inducible, tissue-specific expression is an important and powerful tool to study the spatio-temporal dynamics of genetic perturbation. Combining the flexible and efficient GreenGate cloning system with the proven and benchmarked LhGR system (here termed GR-LhG4) for the inducible expression, we have generated a set of transgenic Arabidopsis lines that can drive the expression of an effector cassette in a range of specific cell types in the three main plant meristems. To this end, we chose the previously developed GR-LhG4 system based on a chimeric transcription factor and a cognate pOp-type promoter ensuring tight control over a wide range of expression levels. In addition, to visualize the expression domain where the synthetic transcription factor is active, an ER-localized mTurquoise2 fluorescent reporter under control of the pOp4 or pOp6 promoter is encoded in driver lines. Here, we describe the steps necessary to generate a driver or effector line and demonstrate how cell type specific expression can be induced and followed in the shoot apical meristem, the root apical meristem and the cambium of Arabidopsis. By using several or all driver lines, the context specific effect of expressing one or multiple factors (effectors) under control of the synthetic pOp promoter can be assessed rapidly, for example in F1 plants of a cross between one effector and multiple driver lines. This approach is exemplified by the ectopic expression of VND7, a NAC transcription factor capable of inducing ectopic secondary cell wall deposition in a cell autonomous manner.

Inducible, Cell Type-Specific Expression in Arabidopsis thaliana Through LhGR-Mediated Trans-Activation

Here, we describe the generation and application of a set of transgenic Arabidopsis thaliana lines enabling inducible, tissue-specific expression in the three main meristems, the shoot apical meristem, the root apical meristem, and the cambium.

Inducible, tissue-specific expression is an important and powerful tool to study the spatio-temporal dynamics of genetic perturbation. Combining the ...

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.

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.

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

Biochemistry

Applying an Inducible Expression System to Study Interference of Bacterial Virulence Factors with Intracellular Signaling

The technique presented here allows one to analyze at which step a target protein, or alternatively a small molecule, interacts with the components of a signaling pathway. The method is based, on the one hand, on the inducible expression of a specific protein to initiate a signaling event at a defined and predetermined step in the selected signaling cascade. Concomitant expression, on the other hand, of the gene of interest then allows the investigator to evaluate if the activity of the expressed target protein is located upstream or downstream of the initiated signaling event, depending on the readout of the signaling pathway that is obtained. Here, the apoptotic cascade was selected as a defined signaling pathway to demonstrate protocol functionality. Pathogenic bacteria, such as Coxiella burnetii, translocate effector proteins that interfere with host cell death induction in the host cell to ensure bacterial survival in the cell and to promote their dissemination in the organism. The C. burnetii effector protein CaeB effectively inhibits host cell death after induction of apoptosis with UV-light or with staurosporine. To narrow down at which step CaeB interferes with the propagation of the apoptotic signal, selected proteins with well-characterized pro-apoptotic activity were expressed transiently in a doxycycline-inducible manner. If CaeB acts upstream of these proteins, apoptosis will proceed unhindered. If CaeB acts downstream, cell death will be inhibited. The test proteins selected were Bax, which acts at the level of the mitochondria, and caspase 3, which is the major executioner protease. CaeB interferes with cell death induced by Bax expression, but not by caspase 3 expression. CaeB, thus, interacts with the apoptotic cascade between these two proteins.

The method described here is used to induce the apoptotic signaling cascade at defined steps in order to dissect the activity of an anti-apoptotic bacterial effector protein. This method can also be used for inducible expression of pro-apoptotic or toxic proteins, or for dissecting interference with other signaling pathways.

The technique presented here allows one to analyze at which step a target protein, or alternatively a small molecule, interacts with the components of a ...

Immunology and Infection

DNA-Tethered RNA Polymerase for Programmable In vitro Transcription and Molecular Computation

DNA nanotechnology enables programmable self-assembly of nucleic acids into user-prescribed shapes and dynamics for diverse applications. This work demonstrates that concepts from DNA nanotechnology can be used to program the enzymatic activity of the phage-derived T7 RNA polymerase (RNAP) and build scalable synthetic gene regulatory networks. First, an oligonucleotide-tethered T7 RNAP is engineered via expression of an N-terminally SNAP-tagged RNAP and subsequent chemical coupling of the SNAP-tag with a benzylguanine (BG)-modified oligonucleotide. Next, nucleic-acid strand displacement is used to program polymerase transcription on-demand. In addition, auxiliary nucleic acid assemblies can be used as &#34;artificial transcription factors&#34; to regulate the interactions between the DNA-programmed T7 RNAP with its DNA templates. This in vitro transcription regulatory mechanism can implement a variety of circuit behaviors such as digital logic, feedback, cascading, and multiplexing. The composability of this gene regulatory architecture facilitates design abstraction, standardization, and scaling. These features will enable the rapid prototyping of in vitro&#160;genetic devices for applications such as bio-sensing, disease detection, and data storage.

We describe the engineering of a novel DNA-tethered T7 RNA polymerase to regulate in vitro transcription reactions. We discuss the steps for protein synthesis and characterization, validate proof-of-concept transcriptional regulation, and discuss its applications in molecular computing, diagnostics, and molecular information processing.

DNA nanotechnology enables programmable self-assembly of nucleic acids into user-prescribed shapes and dynamics for diverse applications. This work ...

Bioengineering

The MultiBac Protein Complex Production Platform at the EMBL

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

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

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

Biology

Inducible Tet-On Regulatable System-Based Gene Expression In Vivo: A Tetracycline-Induced Gene Expression System for Target Gene Transcription Activation in Mouse Model

Source: Hubner, E. K. et al.&#160; <a href="https://www.jove.com/v/56613">Constitutive and Inducible Systems for Genetic In Vivo Modification of Mouse Hepatocytes Using Hydrodynamic Tail Vein Injection.</a> J. Vis. Exp. (2018)
This video demonstrates the tetracycline, or Tet-On, gene expression system in a mouse model. In the presence of doxycycline, a tetracycline derivative, the Tet-On system consisting of reverse tetracycline-controlled transactivator protein becomes activated and expresses the gene of interest.

Indukowalna regulacyjna ekspresja genów oparta na systemie Tet-On in vivo: indukowany tetracykliną system ekspresji genów do aktywacji transkrypcji genu docelowego w modelu mysim

Source: Hubner, E. K. et al.&#160; Constitutive and Inducible Systems for Genetic In Vivo Modification of Mouse Hepatocytes Using Hydrodynamic Tail Vein ...

Inducible T7 RNA Polymerase-mediated Multigene Expression System, pMGX

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Rozdziały w tym wideo

Transient Gene Expression in Tobacco using Gibson Assembly and the Gene Gun

Controllable Ion Channel Expression through Inducible Transient Transfection

Constitutive and Inducible Systems for Genetic In Vivo Modification of Mouse Hepatocytes Using Hydrodynamic Tail Vein Injection

An Ecdysone Receptor-based Singular Gene Switch for Deliberate Expression of Transgene with Robustness, Reversibility, and Negligible Leakiness

Inducible, Cell Type-Specific Expression in Arabidopsis thaliana Through LhGR-Mediated Trans-Activation

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

Applying an Inducible Expression System to Study Interference of Bacterial Virulence Factors with Intracellular Signaling

DNA-Tethered RNA Polymerase for Programmable In vitro Transcription and Molecular Computation

The MultiBac Protein Complex Production Platform at the EMBL

Indukowalna regulacyjna ekspresja genów oparta na systemie Tet-On in vivo: indukowany tetracykliną system ekspresji genów do aktywacji transkrypcji genu docelowego w modelu mysim