The authors would like to thank Jen Sheen of Massachusetts General Hospital for the generous donation of Nicotiana benthamiana seedlings. Jen Bush helped us greatly in advice in growing plants and setting up a growth chamber area. Tom Ferrante of the Wyss Institute offered crucial help with confocal microscopy. The authors would especially like to thank Don Ingber of Children&#8217;s Hospital Boston and the Wyss Institute for the generous donation of a Gene Gun and associated supplies. Funding for this project was provided through a cooperative agreement with the Department of Energy Advanced Research Projects Agency (ARPA- E Award # DE-000079) for PAS, JCW, and MdM, and through Chimerion Biotechnology, Inc. for MJV.

1. Design of Alternatively-spliced Sequences for Multiple Organelle Targeting
<ol>
	<li>Determine the sites for final protein expression. For this work, the interest is in targeting the chloroplast, peroxisome, and cytosol.</li>
	<li>Use the literature to identify protein and DNA sequences known to target proteins to organelles of interest. In this case a chloroplast targeting sequence from Arabidopsis thaliana L-isoaspartate methyltransferase6,15, and a peroxisome targeting sequence from Arabidopsis thaliana transthyretin-like S-allantoin synthase16,17 were determined.</li>
	<li>Identify an alternative-splicing regime that should target the proteins of interest to the correct organelles. For this work, splice sites as observed in a natural Arabidopsis system6. The mRNA sequence was redesigned leaving the natural splice sites, but sequences were placed encoding chloroplast- or peroxisome- targeting tags within the alternatively-spliced introns (Figure 1).</li>
</ol>
2.&#160;Gibson Assembly of DNA Parts
See also Figure 2.
The Gibson assembly method depends on the action of several enzymes in a pre-made mix to 1) partially digest the ends of the double stranded DNA&#8217;s to make single strands and 2) anneal complimentary base pairs of neighboring DNA parts. This allows for cloning of DNA fragments without the limitations of restriction sites.
<ol>
	<li>Choose an order for the elements in the DNA plasmid construct. As an example TriTag-1 has elements of: a) a plasmid vector (pORE, containing a GFP construct known to work in plants, a kanamycin resistance marker, and origins of replication for E. coli and Agrobacterium tumefaciens hosts), b) a promoter sequence, (PENTCUP2, shown to be constitutive in plants), c) a chloroplast targeting sequence (CTS), c) a peroxisome targeting sequence (PTS), and d) a series of splice sites as described previously6.</li>
	<li>Design the construct base-for-base with in silico design software. ApE is an open source freeware package useful for this work. 
	Note: TriTag-1, has the order: plasmid vector, promoter, ATG, splice donor site, chloroplast target sequence, splice donor site, neutral site, splice acceptor site, peroxisome target sequence, splice acceptor site, GFP as contained in plasmid vector.
	<ol>
		<li>Design the in silico DNA construct such that there is a 50-bp overlap between each piece when ordering primers for PCR. It is possible to use the 50 bp overlap as the primers: 25 bp each direction.
		<ol>
			<li>Be sure to keep track of the origin of each of the sequence pieces. Is it as: a plasmid to be grown and miniprepped; a linear sequence that can be used as a PCR template; or a sequence that will need to be synthesized commercially? Several companies offer low-cost DNA synthesis services for fragments &#60;500 bp. In this case, the TriTag itself is 437 bp so it was advantageous to order it commercially.</li>
			<li>The best results will come if all the fragments are PCR-amplified and purified from their template DNA. The resistance marker is a good spot to divide this large DNA into two smaller templates that are more easily PCR-amplified.</li>
			<li>Restriction Restriction enzyme DpnI cuts methylated DNA.&#160; If the PCR template was a miniprep from E. coli, DpnI treatment will remove the contaminating template DNA.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Purify all the DNA sequences separately and elute in water, TE or Buffer EB.
	<ol>
		<li>pORE vector part 1, ~3,000 bp (green arrows). PCR amplified and DpnI treated.</li>
		<li>pORE vector part 2, ~3,000 bp (black arrows). PCR amplified and DpnI treated.</li>
		<li>TriTag insert, 437 bp (blue arrows). Ordered commercially. Resuspend in ddH2O.</li>
		<li>PENTCUP promoter, ~750 bp (orange arrows). PCR-amplified and DpnI treated.</li>
	</ol>
	</li>
	<li>Measure the concentrations of each of the DNA pieces. Aim for 150 ng/&#956;l of the vector pieces.</li>
	<li>Take an aliquot of the Gibson assembly mix out of the freezer and thaw on ice. This is commercially available, but also simple to make in lab12&#8211;14.
	<ol>
		<li>There are two steps to this recipe: a viscous 5x master mix and 15 &#956;l reaction-size 1.33x aliquots. The 5x master mix contains: 3 ml 1 M Tris-HCl pH 7.5, 300 &#956;l 1 M MgCl2, 600 &#956;l 10 mM of each dNTP, 300 &#956;l 1 M DTT, 1.5 g PEG-8000, 20 mg NAD, and ddH2O to 6 ml. Prepare 320 &#956;l aliquots (18) and freeze all but one at -20 &#176;C. The solution will be quite viscous. Label these &#8220;5X isotherm buffer&#8221;</li>
		<li>To the one remaining (320 &#956;l), add 1.2 &#956;l T5 Exonuclease, 20 &#956;l Phusion High-Fidelity DNA Polymerase, 160 &#956;l Taq DNA Ligase and 700 &#956;l ddH2O. Prepare 15 &#956;l aliquots (~80) on ice in PCR tubes and store at -20 &#176;C.</li>
	</ol>
	</li>
	<li>Determine volumes for equimolar amounts of each of the fragments. There are a few online calculators, including Gibthon.org. There should be a total of 5 &#956;l of all the DNA pieces. Add them to the 15 &#956;l Gibson mix aliquot. If this requires volumes too small to pipette, dilute the DNA fragments and/or use more than one aliquot.
	<ol>
		<li>Do not forget to prepare a control of the vector DNA alone (e.g. the DNA part containing the antibiotic resistance marker) and make up the volume with water.</li>
	</ol>
	</li>
	<li>Incubate the tubes in a thermal cycler at 50-55 &#176;C for 30-60 minutes. Replace on ice and transform into E. coli via heat-shock chemically competent cells. Do not use electrocompetent cells. The high salt concentration and the relatively low DNA concentration may cause the cells to arc.
	<ol>
		<li>Apply all 20 &#956;l to a commercial preparation of 200 &#956;l calcium-chloride competent E. coli. 
		Incubate on ice 30 min and heat shock 60 sec at 42 &#176;C</li>
		<li>Return to ice 2 min, apply 750 &#956;l SOC or LB medium and recover shaking in a microcentrifuge tube 30 min at 37 &#176;C. Plate the mixture and appropriate dilutions on LB+Kan (or other appropriate antibiotic). This may result in higher background (and a higher number of non-target recombination events) than traditional ligation-based cloning but it is worthwhile to screen about 10 colonies.</li>
	</ol>
	</li>
	<li>Confirm clone via sequence and store an aliquot at -80 &#176;C</li>
</ol>
3.&#160;Biolistic Transfection of Tobacco with the Gene Gun
This is a technique that is established in JoVE18,19. Key steps and differences are described below.
<ol>
	<li>Grow 50-100 ml E. coli or 200 ml Agrobacterium. Maxi-prep the culture. A concentration of about 1,500 ng/&#956;l is required to continue.</li>
	<li>Prepare gene gun bullets as previously described. Load them into the Gene Gun.</li>
	<li>For transfection of Nicotiana benthamiana tobacco leaf epidermal tissue, choose a large leaf close to the base of a 3 month old plant. Carefully cut it and place it bottom side up on wet paper towels in a 15 cm Petri dish.</li>
	<li>Set the He pressure for the Gene Gun to 200-250 psi.</li>
	<li>Carefully aim the Gene Gun between ribs on the bottom side of a leaf, about 3-5 cm above it. Release the safety and deliver the particles to the leaf. Each bullet may be used twice in the same location on the leaf. If the leaf explodes, discard and start a new leaf&#8212;there is some variance in leaf toughness due to growth conditions such as age, moisture, etc. For a 12-cm leaf, expect to fit about 6 shots.</li>
	<li>Store the leaf, covered with the top of a Petri dish for 2-3 days in low light on the bench.</li>
	<li>Examine the leaf in a dissecting microscope equipped with UV fluorescence, and search for individual cells expressing GFP. Dissect 5-10 mm sections of leaf.</li>
	<li>Submerge the leaf in a deep well microscope slide under ~200 &#956;l 0.1% Triton X-100. The detergent helps prevent air bubbles from forming on the surface, and the deep well microscope slide allows for a larger tissue imaging area.</li>
</ol>
4. Confocal Microscopy of Transfected Tissue
These instructions vary for every instrument, so it is essential to get properly trained.
<ol>
	<li>For fluorescence detection experiments, set the excitation laser to 489 nm, and set the photomultiplier detectors to 500-569 nm for GFP fluorescence and 630-700 nm for chlorophyll auto-fluorescence. Image cells using a 40x water-immersion objective.</li>
</ol>

<ol>
	<li>Que, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trait+stacking+in+transgenic+crops:+challenges+and+opportunities.">Trait stacking in transgenic crops: challenges and opportunities.</a> GM crops. 1 (4), 220-229 (2010).</li><li>Reddy, A. S. N., Rogers, M. F., Richardson, D. N., Hamilton, M., Ben-Hur, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deciphering+the+plant+splicing+code:+experimental+and+computational+approaches+for+predicting+alternative+splicing+and+splicing+regulatory+elements.">Deciphering the plant splicing code: experimental and computational approaches for predicting alternative splicing and splicing regulatory elements.</a> Frontiers in Plant Science. 3 (18), (2012).</li><li>Hickey, S. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transgene+regulation+in+plants+by+alternative+splicing+of+a+suicide+exon.">Transgene regulation in plants by alternative splicing of a suicide exon.</a> Nucleic Acids Research. 40 (10), 4701-4710 (2012).</li><li>Syed, N. H., Kalyna, M., Marquez, Y., Barta, A., Brown, J. W. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alternative+splicing+in+plants+-+coming+of+age.">Alternative splicing in plants - coming of age.</a> Trends in Plant Science. 17 (10), 616-623 (2012).</li><li>Black, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+alternative+pre-messenger+RNA+splicing.">Mechanisms of alternative pre-messenger RNA splicing.</a> Annual Review of Biochemistry. 72, 291-336 (2003).</li><li>Dinkins, R. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changing+transcriptional+initiation+sites+and+alternative+5'-+and+3'-splice+site+selection+of+the+first+intron+deploys+Arabidopsis+protein+isoaspartyl+methyltransferase2+variants+to+different+subcellular+compartments.">Changing transcriptional initiation sites and alternative 5'- and 3'-splice site selection of the first intron deploys Arabidopsis protein isoaspartyl methyltransferase2 variants to different subcellular compartments.</a> The Plant Journal: for Cell and Molecular Biology. 55 (1), 1-13 (2008).</li><li>Kebeish, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chloroplastic+photorespiratory+bypass+increases+photosynthesis+and+biomass+production+in+Arabidopsis+thaliana.">Chloroplastic photorespiratory bypass increases photosynthesis and biomass production in Arabidopsis thaliana.</a> Nature Biotechnology. 25 (5), 593-599 (2007).</li><li>Maier, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transgenic+Introduction+of+a+Glycolate+Oxidative+Cycle+into+A.+thaliana+Chloroplasts+Leads+to+Growth+Improvement.">Transgenic Introduction of a Glycolate Oxidative Cycle into A. thaliana Chloroplasts Leads to Growth Improvement.</a> Frontiers in Plant Science. 3 (38), 1-12 (2012).</li><li>Kumar, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Remodeling+the+isoprenoid+pathway+in+tobacco+by+expressing+the+cytoplasmic+mevalonate+pathway+in+chloroplasts.">Remodeling the isoprenoid pathway in tobacco by expressing the cytoplasmic mevalonate pathway in chloroplasts.</a> Metabolic Engineering. 14 (1), (2012).</li><li>Sapir-Mir, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peroxisomal+localization+of+Arabidopsis+isopentenyl+diphosphate+isomerases+suggests+that+part+of+the+plant+isoprenoid+mevalonic+acid+pathway+is+compartmentalized+to+peroxisomes.">Peroxisomal localization of Arabidopsis isopentenyl diphosphate isomerases suggests that part of the plant isoprenoid mevalonic acid pathway is compartmentalized to peroxisomes.</a> Plant Physiology. 148 (3), 1219-1228 (2008).</li><li>Voges, M. J., Silver, P. A., Way, J. C., Mattozzi, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+a+heterologous+protein+to+multiple+plant+organelles+via+rationally+designed+5'+mRNA+tags.">Targeting a heterologous protein to multiple plant organelles via rationally designed 5' mRNA tags.</a> Journal of Biological Engineering. 7 (20), (2013).</li><li>Gibson, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enzymatic+assembly+of+overlapping+DNA+fragments.">Enzymatic assembly of overlapping DNA fragments.</a> Methods in Enzymology. 498, 349-361 (2011).</li><li>Gibson, D. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enzymatic+assembly+of+DNA+molecules+up+to+several+hundred+kilobases.">Enzymatic assembly of DNA molecules up to several hundred kilobases.</a> Nature Methods. 6 (5), 12-16 (2009).</li><li>Gibson, D. G., Smith, H. O., Hutchison, C. A., Venter, J. C., Merryman, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+synthesis+of+the+mouse+mitochondrial+genome.">Chemical synthesis of the mouse mitochondrial genome.</a> Nature Methods. 7 (11), 901-903 (2010).</li><li>Lowenson, J. D., Clarke, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recognition+of+D-aspartyl+residues+in+polypeptides+by+the+erythrocyte+L-isoaspartyl/D-aspartyl+protein+methyltransferase.+Implications+for+the+repair+hypothesis.">Recognition of D-aspartyl residues in polypeptides by the erythrocyte L-isoaspartyl/D-aspartyl protein methyltransferase. Implications for the repair hypothesis.</a> The Journal of Biological Chemistry. 267 (9), 5985-5995 (1992).</li><li>Lanyon-Hogg, T., Warriner, S. L., Baker, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Getting+a+camel+through+the+eye+of+a+needle:+the+import+of+folded+proteins+by+peroxisomes.">Getting a camel through the eye of a needle: the import of folded proteins by peroxisomes.</a> Biology of the Cell / under the auspices of the European Cell Biology Organization. 102 (4), 245-263 (2010).</li><li>Reumann, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteome+analysis+of+Arabidopsis+leaf+peroxisomes+reveals+novel+targeting+peptides,+metabolic+pathways,+and+defense+mechanisms.">Proteome analysis of Arabidopsis leaf peroxisomes reveals novel targeting peptides, metabolic pathways, and defense mechanisms.</a> The Plant Cell. 19 (10), 3170-3193 (2007).</li><li>Woods, G., Zito, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+gene+gun+bullets+and+biolistic+transfection+of+neurons+in+slice+culture.">Preparation of gene gun bullets and biolistic transfection of neurons in slice culture.</a> J. Vis. Exp. (12), (2008).</li><li>Hollender, C. A., Liu, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bimolecular+Fluorescence+Complementation+(BiFC)+Assay+for+Protein-Protein+Interaction+in+Onion+Cells+Using+the+Helios+Gene.">Bimolecular Fluorescence Complementation (BiFC) Assay for Protein-Protein Interaction in Onion Cells Using the Helios Gene.</a> (40), (2010).</li><li>Li, M. Z., Elledge, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Harnessing+homologous+recombination+in+vitro+to+generate+recombinant+DNA+via+SLIC.">Harnessing homologous recombination in vitro to generate recombinant DNA via SLIC.</a> Nature Methods. 4 (3), 251-256 (2007).</li></ol>

The authors have nothing to disclose.

In this study, simple strategies are described for localizing a single transgenic protein to multiple cellular compartments in plants. The goal was to design construct that would express a single gene in more than one organelle in Nicotiana benthamiana. Strategies include rational design of GFP-based DNA constructs, Gibson assembly, delivery of the plasmids to leaf cells with the Gene Gun, and observation of the results with confocal microscopy.
Three different short, N-terminal tags ...

This work is a metabolic engineering / synthetic biology project wherein plant cells are engineered to express a reporter protein in multiple organelles but with only a single DNA construct.
One approach to target proteins to more than one location involves cloning multiple genetic copies, each containing a different localization peptide. Each copy must be introduced by successive retransformation, or alternatively, by backcrossing single transforms1. This involves additional cloning, and is limited by one localization tag per terminus.
Another way to localize a protein to multiple locations is through alternative splicing2&#8211;5. RNA is transcribed from a single gene, but different copies of the transcript are processed differently, often in more than one way per cell. This can result in more than one messenger RNA in the cell transcribed from a single gene. These different messenger RNAs can encode for different isoforms of the same protein, or in the case of a frameshift, a different protein altogether. Although alternative splicing has been described in the literature for many years, the mechanisms of action and conserved donor and receptor splice sites are only being elucidated more recently6. As these sites are being better described, they open up opportunities for engineering.
Plant metabolic engineers are faced with a challenge when expressing a protein in multiple organelles. For a protein that is to be localized to more than one organelle, the engineer must clone the same gene multiple times, each with a separate signal sequence directing it to the organelle of interest. For a single gene in three organelles, this is simply three genes. But for a six-gene metabolic pathway, this expands to 18 genes, a significant cloning effort. Combining multiple localization sequences into a single, alternatively-spliced gene significantly reduces this effort. For example, re-engineering photorespiration7,8 and isoprenoid synthesis9,10 involves both the chloroplasts and peroxisomes. In our case we took advantage of splice sites as observed in a natural Arabidopsis thaliana system described previously6. We rationally redesigned the mRNA sequence leaving the natural splice sites alone, but placed sequences that would encode chloroplast- or peroxisome- targeting tags within the alternatively-spliced introns (Figure 1). The expressed protein may or may not have a tag, depending on whether the pre-mRNA that encoded it was excised as an intron (Figures 1g and 1h). For more information on the design of the constructs presented in this work, please see the companion article11.
Because this is still a significant cloning effort, Gibson assembly, a new method for cloning DNA constructs, is used in construction. The Gibson method may be used for any sequence, regardless of restriction sites (Figure 2)12&#8211;14. The specific mix of enzymes allows for a one-step, isothermal assembly. In this method, several double-stranded linear DNA parts are designed such that they have overlapping sequences of ~50 bp. The Gibson assembly enzyme mix partially digests the linear DNA parts, exposing single strands of homologous sequences. These partial single-stranded sequences are re-annealed in the reaction mixture, resulting in a rapid, one-step, sequence-independent, ligation-free subcloning reaction.
This work describes 1) rational design alternatively spliced constructs for expression in plant chloroplasts, peroxisomes, and cytosols, 2) their cloning using the new ligation-free method of Gibson assembly, 3) their delivery into tobacco leaf cells with the Gene Gun, and 4) results showing organelle targeting, as observed with GFP and confocal microscopy.

<table><tbody><tr><td>Oligonucleotide primers</td><td>IDT</td><td>(custom)</td><td>Design specifically for construct</td></tr><tr><td>GeneBlocks</td><td>IDT</td><td>(custom)</td><td>500 bp oligonucleotides</td></tr><tr><td>ApE software</td><td>U Utah</td><td>(download)</td><td>http://biologylabs.utah.edu/jorgensen/wayned/ape/</td></tr><tr><td>MinElute kit</td><td>Qiagen</td><td>28004</td><td>Used to purify PCR products</td></tr><tr><td>QIAprep spin miniprep kit</td><td>Qiagen</td><td>27104</td><td>Used to prepare cloning-appropriate amounts of plasmid</td></tr><tr><td>Phusion Master Mix with GC Buffer</td><td>NEB</td><td>M0532S</td><td>Used to PCR-amplify gene of interest</td></tr><tr><td>Gibson assembly reaction mix</td><td>NEB</td><td>E2611L</td><td>Master mix of the following 9 ingredients</td></tr><tr><td>1 M Tris-HCl pH7.5</td><td>Teknova</td><td>T1075</td><td>Gibson assembly mix</td></tr><tr><td>1 M MgCl2</td><td>G Biosiences</td><td>82023-086</td><td>Gibson assembly mix</td></tr><tr><td>dNTP mix</td><td>Fermentas</td><td>R0192</td><td>Gibson assembly mix</td></tr><tr><td>1 M DTT</td><td>Fermentas</td><td>R0861</td><td>Gibson assembly mix</td></tr><tr><td>PEG-8000</td><td>Affymetrix</td><td>19966</td><td>Gibson assembly mix</td></tr><tr><td>NAD</td><td>Applichem</td><td>A1124,0005</td><td>Gibson assembly mix</td></tr><tr><td>T5 exonuclease</td><td>Epicentre</td><td>T5E4111K</td><td>Gibson assembly mix</td></tr><tr><td>Phusion polymerase</td><td>NEB</td><td>F530S</td><td>Gibson assembly mix</td></tr><tr><td>Taq DNA ligase</td><td>NEB</td><td>M0208L</td><td>Gibson assembly mix</td></tr><tr><td>Gibthon.org</td><td></td><td></td><td>Website to simplify calculations</td></tr><tr><td>Plasmid PLUS Maxi kit</td><td>Qiagen</td><td>12963</td><td>Used to prepare DNA for gene gun bullets</td></tr><tr><td>Gene Gun system</td><td>BioRad</td><td>165-2451</td><td>Includes all parts necessary</td></tr><tr><td>Nicotania benthamiana</td><td>(n/a)</td><td>(n/a)</td><td>Gift of Jen Sheen, MGH</td></tr><tr><td>Confocal microscope</td><td>Leica</td><td>SP5 X MP</td><td>Imaging of resultant cells</td></tr><tr><td>Deep well slides</td><td>Electron Microscopy Sciences</td><td>71561-01</td><td>Used for confocal imaging</td></tr><tr><td>A Plasmid Editor (ApE) </td><td>University of Utah </td><td></td><td>http://biologylabs.utah.edu/jorgensen/wayned/ape</td></tr><tr><td>Gibthon:Ligation calculator</td><td></td><td></td><td>http://django.gibthon.org/tools/ligcalc/</td></tr></tbody></table>

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.

The design effort was a result of significant planning. Novel to this project is the use of alternative splicing to create a pre-mRNA that is translated into differentially expressed proteins. These proteins are expressed in different organelles, in this case the chloroplast, peroxisome and/or cytosol. We adapted natural Arabidopsis gene that is alternatively spliced6, and placed known chloroplast6 and peroxisome17 targeting sequences in alternate exons (TriTag-1 and TriTag-2). T...

Watch this Scientific Journal Video about Transient Gene Expression in Tobacco using Gibson Assembly and the Gene Gun at JoVE.com

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

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 ...

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20.6K Views.  Harvard University.  Dit werk beschrijft een nieuwe werkwijze voor het selectief richten subcellulaire organellen in planten, bepaald met de BioRad Gene Gun. 

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

Generating Transgenic Plants with Single-copy Insertions Using BIBAC-GW Binary Vector

When generating transgenic plants, generally the objective is to have stable expression of a transgene. This requires a single, intact integration of the transgene, as multi-copy integrations are often subjected to gene silencing. The Gateway-compatible binary vector based on bacterial artificial chromosomes (pBIBAC-GW), like other pBIBAC derivatives, allows the insertion of single-copy transgenes with high efficiency. As an improvement to the original pBIBAC, a Gateway cassette has been cloned into pBIBAC-GW, so that the sequences of interest can now be easily incorporated into the vector transfer DNA (T-DNA) by Gateway cloning. Commonly, the transformation with pBIBAC-GW results in an efficiency of 0.2&#8211;0.5%, whereby half of the transgenics carry an intact single-copy integration of the T-DNA. The pBIBAC-GW vectors are available with resistance to Glufosinate-ammonium or DsRed fluorescence in seed coats for selection in plants, and with resistance to kanamycin as a selection in bacteria. Here, a series of protocols is presented that guide the reader through the process of generating transgenic plants using pBIBAC-GW: starting from recombining the sequences of interest into the pBIBAC-GW vector of choice, to plant transformation with Agrobacterium, selection of the transgenics, and testing the plants for intactness and copy number of the inserts using DNA blotting. Attention is given to designing a DNA blotting strategy to recognize single- and multi-copy integrations at single and multiple loci.

Using a pBIBAC-GW binary vector makes generating transgenic plants with intact single-copy insertions, an easy process. Here, a series of protocols is presented that guide the reader through the process of generating transgenic Arabidopsis plants, and testing the plants for intactness and copy number of the inserts.

When generating transgenic plants, generally the objective is to have stable expression of a transgene. This requires a single, intact integration of the ...

Genetics

Transient Expression in Nicotiana Benthamiana Leaves for Triterpene Production at a Preparative Scale

The triterpenes are one of the largest and most structurally diverse families of plant natural products. Many triterpene derivatives have been shown to possess medicinally relevant biological activity. However, thus far this potential has not translated into a plethora of triterpene-derived drugs in the clinic. This is arguably (at least partially) a consequence of limited practical synthetic access to this class of compound, a problem that can stifle the exploration of structure-activity relationships and development of lead candidates by traditional medicinal chemistry workflows. Despite their immense diversity, triterpenes are all derived from a single linear precursor, 2,3-oxidosqualene. Transient heterologous expression of biosynthetic enzymes in N. benthamiana can divert endogenous supplies of 2,3-oxidosqualene towards the production of new high-value triterpene products that are not naturally produced by this host. Agro-infiltration is an efficient and simple means of achieving transient expression in N. benthamiana. The process involves infiltration of plant leaves with a suspension of Agrobacterium tumefaciens carrying the expression construct(s) of interest. Co-infiltration of an additional A. tumefaciens strain carrying an expression construct encoding an enzyme that boosts precursor supply significantly increases yields. After a period of five days, the infiltrated leaf material can be harvested and processed to extract and isolate the resulting triterpene product(s). This is a process that is linearly and reliably scalable, simply by increasing the number of plants used in the experiment. Herein is described a protocol for rapid preparative-scale production of triterpenes utilizing this plant-based platform. The protocol utilizes an easily replicable vacuum infiltration apparatus, which allows the simultaneous infiltration of up to four plants, enabling batch-wise infiltration of hundreds of plants in a short period of time.

Transient Expression in Nicotiana Benthamiana Leaves for Triterpene Production at a Preparative Scale

Transient heterologous expression of biosynthetic enzymes in Nicotiana benthamiana leaves can divert endogenous supplies of 2,3-oxidosqualene towards the production of new high-value triterpene products. Herein is described a detailed protocol for rapid (5 days) preparative-scale production of triterpenes and analogs utilizing this powerful plant-based platform.

The triterpenes are one of the largest and most structurally diverse families of plant natural products. Many triterpene derivatives have been shown to ...

Biochemistry

Characterization of Complex Systems Using the Design of Experiments Approach: Transient Protein Expression in Tobacco as a Case Study

Plants provide multiple benefits for the production of biopharmaceuticals including low costs, scalability, and safety. Transient expression offers the additional advantage of short development and production times, but expression levels can vary significantly between batches thus giving rise to regulatory concerns in the context of good manufacturing practice. We used a design of experiments (DoE) approach to determine the impact of major factors such as regulatory elements in the expression construct, plant growth and development parameters, and the incubation conditions during expression, on the variability of expression between batches. We tested plants expressing a model anti-HIV monoclonal antibody (2G12) and a fluorescent marker protein (DsRed). We discuss the rationale for selecting certain properties of the model and identify its potential limitations. The general approach can easily be transferred to other problems because the principles of the model are broadly applicable: knowledge-based parameter selection, complexity reduction by splitting the initial problem into smaller modules, software-guided setup of optimal experiment combinations and step-wise design augmentation. Therefore, the methodology is not only useful for characterizing protein expression in plants but also for the investigation of other complex systems lacking a mechanistic description. The predictive equations describing the interconnectivity between parameters can be used to establish mechanistic models for other complex systems.

We describe a design of experiments approach that can be used to determine and model the influence of transgene regulatory elements, plant growth and development parameters, and incubation conditions on the transient expression of monoclonal antibodies and reporter proteins in plants.

Plants provide multiple benefits for the production of biopharmaceuticals including low costs, scalability, and safety. Transient expression offers the ...

Bioengineering

Virus-induced Gene Silencing (VIGS) in Nicotiana benthamiana and Tomato

RNA interference (RNAi) is a highly specific gene-silencing phenomenon triggered by dsRNA1. This silencing mechanism uses two major classes of RNA regulators: microRNAs, which are produced from non-protein coding genes and short interfering RNAs (siRNAs). Plants use RNAi to control transposons and to exert tight control over developmental processes such as flower organ formation and leaf development2,3,4. Plants also use RNAi to defend themselves against infection by viruses. Consequently, many viruses have evolved suppressors of gene silencing to allow their successful colonization of their host5.
Virus-induced gene silencing (VIGS) is a method that takes advantage of the plant RNAi-mediated antiviral defense mechanism. In plants infected with unmodified viruses the mechanism is specifically targeted against the viral genome. However, with virus vectors carrying sequences derived from host genes, the process can be additionally targeted against the corresponding host mRNAs. VIGS has been adapted for high-throughput functional genomics in plants by using the plant pathogen Agrobacterium tumefaciens to deliver, via its Ti plasmid, a recombinant virus carrying the entire or part of the gene sequence targeted for silencing. Systemic virus spread and the endogenous plant RNAi machinery take care of the rest. dsRNAs corresponding to the target gene are produced and then cleaved by the ribonuclease Dicer into siRNAs of 21 to 24 nucleotides in length. These siRNAs ultimately guide the RNA-induced silencing complex (RISC) to degrade the target transcript2.
Different vectors have been employed in VIGS and one of the most frequently used is based on tobacco rattle virus (TRV). TRV is a bipartite virus and, as such, two different A. tumefaciens strains are used for VIGS. One carries pTRV1, which encodes the replication and movement viral functions while the other, pTRV2, harbors the coat protein and the sequence used for VIGS6,7. Inoculation of Nicotiana benthamiana and tomato seedlings with a mixture of both strains results in gene silencing. Silencing of the endogenous phytoene desaturase (PDS) gene, which causes photobleaching, is used as a control for VIGS efficiency. It should be noted, however, that silencing in tomato is usually less efficient than in N. benthamiana. RNA transcript abundance of the gene of interest should always be measured to ensure that the target gene has efficiently been down-regulated. Nevertheless, heterologous gene sequences from N. benthamiana can be used to silence their respective orthologs in tomato and vice versa8.

Virus-induced Gene Silencing VIGS in Nicotiana benthamiana and Tomato

Description of a virus-induced gene silencing (VIGS) method for knock-down of gene expression in Nicotiana benthamiana and tomato.

RNA interference (RNAi) is a highly specific gene-silencing phenomenon triggered by dsRNA1. This silencing mechanism uses two major classes of RNA ...

Biology

Agrobacterium-Mediated Virus-Induced Gene Silencing Assay In Cotton

Cotton (Gossypium hirsutum) is one of the most important crops worldwide. Considerable efforts have been made on molecular breeding of new varieties. The large-scale gene functional analysis in cotton has been lagged behind most of the modern plant species, likely due to its large size of genome, gene duplication and polyploidy, long growth cycle and recalcitrance to genetic transformation1. To facilitate high throughput functional genetic/genomic study in cotton, we attempt to develop rapid and efficient transient assays to assess cotton gene functions.
Virus-Induced Gene Silencing (VIGS) is a powerful technique that was developed based on the host Post-Transcriptional Gene Silencing (PTGS) to repress viral proliferation2,3. Agrobacterium-mediated VIGS has been successfully applied in a wide range of dicots species such as Solanaceae, Arabidopsis and legume species, and monocots species including barley, wheat and maize, for various functional genomic studies3,4. As this rapid and efficient approach avoids plant transformation and overcomes functional redundancy, it is particularly attractive and suitable for functional genomic study in crop species like cotton not amenable for transformation.
In this study, we report the detailed protocol of Agrobacterium-mediated VIGS system in cotton. Among the several viral VIGS vectors, the tobacco rattle virus (TRV) invades a wide range of hosts and is able to spread vigorously throughout the entire plant yet produce mild symptoms on the hosts5. To monitor the silencing efficiency, GrCLA1, a homolog gene of Arabidopsis Cloroplastos alterados 1 gene (AtCLA1) in cotton, has been cloned and inserted into the VIGS binary vector pYL156. CLA1 gene is involved in chloroplast development6, and previous studies have shown that loss-of-function of AtCLA1 resulted in an albino phenotype on true leaves7, providing an excellent visual marker for silencing efficiency. At approximately two weeks post Agrobacterium infiltration, the albino phenotype started to appear on the true leaves, with 100% silencing efficiency in all replicated experiments. The silencing of endogenous gene expression was also confirmed by RT-PCR analysis. Significantly, silencing could potently occur in all the cultivars we tested, including various commercially grown varieties in Texas. This rapid and efficient Agrobacterium-mediated VIGS assay provides a very powerful tool for rapid large-scale analysis of gene functions at genome-wide level in cotton.

We present the detailed protocol for Agrobacterium-mediated virus-induced gene silencing (VIGS) assay in cotton. The tobacco rattle virus (TRV)-derived VIGS vectors were deployed to induce RNA silencing of cotton GrCLA1, Cloroplastos alterados 1 gene. The albino phenotype caused by silencing GrCLA1 was observed at the seedling stage within 2 weeks after inoculation.

Cotton (Gossypium hirsutum) is one of the most important crops worldwide. Considerable efforts have been made on molecular breeding of new varieties.  The ...

Efficient Agroinfiltration of Plants for High-level Transient Expression of Recombinant Proteins

Mammalian cell culture is the major platform for commercial production of human vaccines and therapeutic proteins. However, it cannot meet the increasing worldwide demand for pharmaceuticals due to its limited scalability and high cost. Plants have shown to be one of the most promising alternative pharmaceutical production platforms that are robust, scalable, low-cost and safe. The recent development of virus-based vectors has allowed rapid and high-level transient expression of recombinant proteins in plants. To further optimize the utility of the transient expression system, we demonstrate a simple, efficient and scalable methodology to introduce target-gene containing Agrobacterium into plant tissue in this study. Our results indicate that agroinfiltration with both syringe and vacuum methods have resulted in the efficient introduction of Agrobacterium into leaves and robust production of two fluorescent proteins; GFP and DsRed. Furthermore, we demonstrate the unique advantages offered by both methods. Syringe infiltration is simple and does not need expensive equipment. It also allows the flexibility to either infiltrate the entire leave with one target gene, or to introduce genes of multiple targets on one leaf. Thus, it can be used for laboratory scale expression of recombinant proteins as well as for comparing different proteins or vectors for yield or expression kinetics. The simplicity of syringe infiltration also suggests its utility in high school and college education for the subject of biotechnology. In contrast, vacuum infiltration is more robust and can be scaled-up for commercial manufacture of pharmaceutical proteins. It also offers the advantage of being able to agroinfiltrate plant species that are not amenable for syringe infiltration such as lettuce and Arabidopsis. Overall, the combination of syringe and vacuum agroinfiltration provides researchers and educators a simple, efficient, and robust methodology for transient protein expression. It will greatly facilitate the development of pharmaceutical proteins and promote science education.

Plants offer a novel system for the production of pharmaceutical proteins on a commercial scale that is more scalable, cost-efficient and safe than current expression paradigms. In this study, we report a simple and convenient, yet scalable approach to introduce target-gene containing Agrobacterium tumefaciens into plants for protein transient expression.

Mammalian cell culture is the major platform for commercial production of human vaccines and therapeutic proteins. However, it cannot meet the increasing ...

Optimization and Utilization of Agrobacterium-mediated Transient Protein Production in Nicotiana

Agrobacterium-mediated transient protein production in plants is a promising approach to produce vaccine antigens and therapeutic proteins within a short period of time. However, this technology is only just beginning to be applied to large-scale production as many technological obstacles to scale up are now being overcome. Here, we demonstrate a simple and reproducible method for industrial-scale transient protein production based on vacuum infiltration of Nicotiana plants with Agrobacteria carrying launch vectors. Optimization of Agrobacterium cultivation in AB medium allows direct dilution of the bacterial culture in Milli-Q water, simplifying the infiltration process. Among three tested species of Nicotiana, N. excelsiana (N. benthamiana &#215; N. excelsior) was selected as the most promising host due to the ease of infiltration, high level of reporter protein production, and about two-fold higher biomass production under controlled environmental conditions. Induction of Agrobacterium harboring pBID4-GFP (Tobacco mosaic virus-based) using chemicals such as acetosyringone and monosaccharide had no effect on the protein production level. Infiltrating plant under 50 to 100 mbar for 30 or 60 sec resulted in about 95% infiltration of plant leaf tissues. Infiltration with Agrobacterium laboratory strain GV3101 showed the highest protein production compared to Agrobacteria laboratory strains LBA4404 and C58C1 and wild-type Agrobacteria strains at6, at10, at77 and A4. Co-expression of a viral RNA silencing suppressor, p23 or p19, in N. benthamiana resulted in earlier accumulation and increased production (15-25%) of target protein (influenza virus hemagglutinin).

Optimization and Utilization of Agrobacterium-mediated Transient Protein Production in Nicotiana

Transient protein production in Nicotiana plants based on vacuum infiltration with Agrobacteria carrying launch vectors (Tobacco mosaic virus-based) is a rapid and economic approach to produce vaccine antigens and therapeutic proteins. We simplified the procedure and improved target accumulation by optimizing conditions of bacteria cultivation, selecting host species, and co-introducing RNA silencing suppressors.

Agrobacterium-mediated transient protein production in plants is a promising approach to produce vaccine antigens and therapeutic proteins within a short ...

Generation of Plasmid Vectors Expressing FLAG-tagged Proteins Under the Regulation of Human Elongation Factor-1&#945; Promoter Using Gibson Assembly

Gibson assembly (GA) cloning offers a rapid, reliable, and flexible alternative to conventional DNA cloning methods. We used GA to create customized plasmids for expression of exogenous genes in mouse embryonic stem cells (mESCs). Expression of exogenous genes under the control of the SV40 or human cytomegalovirus promoters diminishes quickly after transfection into mESCs. A remedy for this diminished expression is to use the human elongation factor-1 alpha (hEF1&#945;) promoter to drive gene expression. Plasmid vectors containing hEF1&#945; are not as widely available as SV40- or CMV-containing plasmids, especially those also containing N-terminal 3xFLAG-tags. The protocol described here is a rapid method to create plasmids expressing FLAG-tagged CstF-64 and CstF-64 mutant under the expressional regulation of the hEF1&#945; promoter. GA uses a blend of DNA exonuclease, DNA polymerase and DNA ligase to make cloning of overlapping ends of DNA fragments possible. Based on the template DNAs we had available, we designed our constructs to be assembled into a single sequence. Our design used four DNA fragments: pcDNA 3.1 vector backbone, hEF1&#945; promoter part 1, hEF1&#945; promoter part 2 (which contained 3xFLAG-tag purchased as a double-stranded synthetic DNA fragment), and either CstF-64 or specific CstF-64 mutant. The sequences of these fragments were uploaded to a primer generation tool to design appropriate PCR primers for generating the DNA fragments. After PCR, DNA fragments were mixed with the vector containing the selective marker and the GA cloning reaction was assembled. Plasmids from individual transformed bacterial colonies were isolated. Initial screen of the plasmids was done by restriction digestion, followed by sequencing. In conclusion, GA allowed us to create customized plasmids for gene expression in 5 days, including construct screens and verification.

Synthesis of custom plasmids is labor and time consuming. This protocol describes the use of Gibson assembly cloning to reduce the work and duration of custom DNA cloning procedure. The protocol described also produces reliable tagged protein constructs for mammalian expression at similar cost to the traditional cut-and-paste DNA cloning.

Gibson assembly (GA) cloning offers a rapid, reliable, and flexible alternative to conventional DNA cloning methods. We used GA to create customized ...

A Comparative Analysis of Recombinant Protein Expression in Different Biofactories: Bacteria, Insect Cells and Plant Systems

Plant-based systems are considered a valuable platform for the production of recombinant proteins as a result of their well-documented potential for the flexible, low-cost production of high-quality, bioactive products.
In this study, we compared the expression of a target human recombinant protein in traditional fermenter-based cell cultures (bacterial and insect) with plant-based expression systems, both transient and stable.
For each platform, we described the set-up, optimization and length of the production process, the final product quality and the yields and we evaluated provisional production costs, specific for the selected target recombinant protein.
Overall, our results indicate that bacteria are unsuitable for the production of the target protein due to its accumulation within insoluble inclusion bodies. On the other hand, plant-based systems are versatile platforms that allow the production of the selected protein at lower-costs than Baculovirus/insect cell system. In particular, stable transgenic lines displayed the highest-yield of the final product and transient expressing plants the fastest process development. However, not all recombinant proteins may benefit from plant-based systems but the best production platform should be determined empirically with a case-by-case approach, as described here.

In this study the expression of a target human recombinant protein in different production platforms was compared. We focused on traditional fermenter-based cultures and on plants, describing the set-up of each system and highlighting, on the basis of the reported results, the inherent limits and advantages for each platform.

Plant-based systems are considered a valuable platform for the production of recombinant proteins as a result of their well-documented potential for the ...

Imaging Spatial Reorganization of a MAPK Signaling Pathway Using the Tobacco Transient Expression System

Visualization of dynamic signaling events in live cells has been a challenge. We expanded an established transient expression system, the biomolecular fluorescent complementation (BiFC) assay in tobacco epidermal cells, from testing protein-protein interaction to monitoring spatial distribution of signal transduction in plant cells. In this protocol, we used the BiFC assay to show that the interaction and the signaling between the Arabidopsis MAPKKK YODA and MAPK6 occur at the plasma membrane. When the scaffold protein BASL was co-expressed, the YODA-MAPK interaction redistributed and spatially co-polarized with CFP-BASL. This modified tobacco expression system allows for quick examination of signaling localization and dynamic changes (less than 4 days) and can accommodate multiple of fluorescent protein colors (at least three). We also presented detailed methods to quantify protein distribution (asymmetric spatial localization, or &#34;polarization&#34;) in tobacco cells. This advanced tobacco expression system has a potential to be used widely for quick testing of dynamic signaling events in live plant cells.

At the subcellular level, signaling events are dynamically modulated by developmental and environmental cues. Here we describe a protocol that employs the tobacco transient expression system to monitor dynamic protein-protein interaction and to disclose spatial organization of signal transduction in plant cells.

Visualization of dynamic signaling events in live cells has been a challenge. We expanded an established transient expression system, the biomolecular ...

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

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