Name: Transient Gene Expression in Tobacco using Gibson Assembly and the Gene Gun (Video) | JoVE Journal
Uploaded: 2014-04-18T18:30:00.000+00:00
Duration: 12 min 2 s
Description: Learn transient gene expression in tobacco leaf cells using Gibson assembly and gene gun delivery. This method enables visualization of GFP in chloroplasts and peroxisomes without restriction enzymes.

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5 minutes

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/pubmed?term=((Trait+stacking+in+transgenic+crops%3A+challenges+and+opportunities%5BTitle%5D)+AND+%22GM+crops%22%5BJournal%5D)">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/pubmed?term=((Deciphering+the+plant+splicing+code%3A+experimental+and+computational+approaches+for+predicting+alternative+splicing+and+splicing+regulatory+elements%5BTitle%5D)+AND+%22Frontiers+in+Plant+Science%22%5BJournal%5D)">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/pubmed?term=((Transgene+regulation+in+plants+by+alternative+splicing+of+a+suicide+exon%5BTitle%5D)+AND+%22Nucleic+Acids+Research%22%5BJournal%5D)">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/pubmed?term=((Alternative+splicing+in+plants+-+coming+of+age%5BTitle%5D)+AND+%22Trends+in+Plant+Science%22%5BJournal%5D)">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/pubmed?term=((Mechanisms+of+alternative+pre-messenger+RNA+splicing%5BTitle%5D)+AND+%22Annual+Review+of+Biochemistry%22%5BJournal%5D)">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/pubmed?term=((Changing+transcriptional+initiation+sites+and+alternative+5%27-+and+3%27-splice+site+selection+of+the+first+intron+deploys+Arabidopsis+protein+isoaspartyl+methyltransferase2+variants+to+different+subcellular+compartments%5BTitle%5D)+AND+%22The+Plant+Journal%3A+for+Cell+and+Molecular+Biology%22%5BJournal%5D)">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/pubmed?term=((Chloroplastic+photorespiratory+bypass+increases+photosynthesis+and+biomass+production+in+Arabidopsis+thaliana%5BTitle%5D)+AND+%22Nature+Biotechnology%22%5BJournal%5D)">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/pubmed?term=((Transgenic+Introduction+of+a+Glycolate+Oxidative+Cycle+into+A.+thaliana+Chloroplasts+Leads+to+Growth+Improvement%5BTitle%5D)+AND+%22Frontiers+in+Plant+Science%22%5BJournal%5D)">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/pubmed?term=((Remodeling+the+isoprenoid+pathway+in+tobacco+by+expressing+the+cytoplasmic+mevalonate+pathway+in+chloroplasts%5BTitle%5D)+AND+%22Metabolic+Engineering%22%5BJournal%5D)">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/pubmed?term=((Peroxisomal+localization+of+Arabidopsis+isopentenyl+diphosphate+isomerases+suggests+that+part+of+the+plant+isoprenoid+mevalonic+acid+pathway+is+compartmentalized+to+peroxisomes%5BTitle%5D)+AND+%22Plant+Physiology%22%5BJournal%5D)">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/pubmed?term=((Targeting+a+heterologous+protein+to+multiple+plant+organelles+via+rationally+designed+5%27+mRNA+tags%5BTitle%5D)+AND+%22Journal+of+Biological+Engineering%22%5BJournal%5D)">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/pubmed?term=((Enzymatic+assembly+of+overlapping+DNA+fragments%5BTitle%5D)+AND+%22Methods+in+Enzymology%22%5BJournal%5D)">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/pubmed?term=((Enzymatic+assembly+of+DNA+molecules+up+to+several+hundred+kilobases%5BTitle%5D)+AND+%22Nature+Methods%22%5BJournal%5D)">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/pubmed?term=((Chemical+synthesis+of+the+mouse+mitochondrial+genome%5BTitle%5D)+AND+%22Nature+Methods%22%5BJournal%5D)">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/pubmed?term=((Recognition+of+D-aspartyl+residues+in+polypeptides+by+the+erythrocyte+L-isoaspartyl%2FD-aspartyl+protein+methyltransferase.+Implications+for+the+repair+hypothesis%5BTitle%5D)+AND+%22The+Journal+of+Biological+Chemistry%22%5BJournal%5D)">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/pubmed?term=((Getting+a+camel+through+the+eye+of+a+needle%3A+the+import+of+folded+proteins+by+peroxisomes%5BTitle%5D)+AND+%22Biology+of+the+Cell+%2F+under+the+auspices+of+the+European+Cell+Biology+Organization%22%5BJournal%5D)">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/pubmed?term=((Proteome+analysis+of+Arabidopsis+leaf+peroxisomes+reveals+novel+targeting+peptides%2C+metabolic+pathways%2C+and+defense+mechanisms%5BTitle%5D)+AND+%22The+Plant+Cell%22%5BJournal%5D)">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/pubmed?term=((Preparation+of+gene+gun+bullets+and+biolistic+transfection+of+neurons+in+slice+culture%5BTitle%5D)+AND+%22J.+Vis.+Exp%22%5BJournal%5D)">Preparation of gene gun bullets and biolistic transfection of neurons in slice culture.</a> J. Vis. Exp. (12), (2008).</li>
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</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

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...

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 ...

The overall goal of the following experiment is to observe transient gene expression of A GFP tagged reporter in tobacco leaf epidermal cells. This is achieved by first designing a DNA construct that will be expressed in the chloroplast and peroxisomes. The construct is then built using PCR amplification and Gibson assembly.Next, a gene gun is used to deliver the construct to tobacco leaf epidermal cells. Ultimately, imaging of the tobacco leaf by confocal microscopy reveals GFP expression in the chloroplast and peroxisome. The main advantage of this technique over existing methods, such as classical cloning, is that it does not require restriction enzymes, and thus, any sequence that can be amplified by PCR may be cloned.Because of the flexibility of the genetic design, the applications of these techniques extend towards a larger scale. Genetic engineering of agricultural plants Begin this protocol by determining the sites within the plant cell for final protein expression. For this work, the aim is to target expression to the chloroplast, peroxisome, and cytosol.Next, design the expression construct. The construct must contain a promoter, a detectable gene, and a terminator within a single plasmid. In this case, the constitutive promoter P end cup two drives the expression of GFP, which is followed by the Nolene Synthe, terminator or nos t.Note that in this experiment, we will be testing a series of tags, trit tags, one, two, and three expressed end terminal to GFP for expression in chloroplast and peroxisomes. The Gibson assembly method depends on the action of several enzymes in a pre-made mix to partially digest the ends of double stranded DNA and facilitate the annealing of complimentary base pairs of neighboring DNA sequences. The final construct has three parts that must be amplified in four PCR reactions prior to Gibson assembly.The first and second reactions amplify an 8, 000 base pair plasmid vector, PORE, which contains GFP NOS T, and a cany resistance marker. The resistance marker is a good spot to divide this large DNA into two smaller templates that are more easily PCR amplified. The third reaction amplifies the promoter P end cup two, and the fourth reaction amplifies triag one using in silico design software such as ape design, the construct base for base, such that there is a 50 base pair overlap between each piece.When ordering primers for PCR, it is possible to use the 50 base pair overlap as the primers 25 base pairs each direction After performing PCR to amplify each component, purify the PCR product separately and dilute them in distilled deionized water. If the PCR template was a mini prep from e coli treatment with restriction enzyme DPN, one will remove the contaminating template DNA. Next, measure the concentrations of each of the DNA pieces.Adjust the vector concentration to approximately 150 nanograms per microliter for each construct to be assembled, including a control, take a 15 microliter aliquot of the Gibson Assembly mix out of the freezer and thaw it on ice. Using the online open source tool@gibbon. org, determine the volumes for equimolar amounts of each of the fragments.Add the calculated volume of each of the fragments to the 15 microliter Gibson mix. Also prepare a control of the vector DNA alone. The total volume of each reaction should be 20 microliters.Use water to adjust the volume if needed. Incubate the tubes at 50 to 55 degrees Celsius for 30 to 60 minutes. After the incubation, place the tubes back on ice.Remove a micro fuge tube containing a commercial preparation of 200 microliters of calcium chloride competent e coli from a minus 80 degrees Celsius freezer, and warm it slightly by hand. Then add all 20 microliters of the reaction to the e coli. Incubate the cultures on ice for 30 minutes, then transfer them to a water bath at 42 degrees Celsius and heat shock for 60 seconds.Do not use electro competent cells. The relatively high salt concentration and low DNA concentration may cause the cells to arc Following transformation. Return the cultures to ICE for two minutes, then apply 750 microliters of SOC or LB medium and allow them to recover for 30 minutes at 37 degrees Celsius.Next plate, the mixture on LB plates with cany or other appropriate antibiotic incubate at 37 degrees Celsius overnight screen 10 colonies by sequencing. Note that this method may result in a higher background and higher number of non-target recombination events than traditional ligation based cloning. The biotic transfection of tobacco with a gene gun has been previously described in JoVE.Here, key steps and differences are reviewed from previous protocols. After amplifying and isolating the constructs, prepare gene gun bullets as described in the JoVE article by woods and zeto and load them into the gene gun for transfection of Nico Tania Benham Miana tobacco leaf epidermal tissue. Choose a large leaf close to the base of a three to five month old plant.Carefully place it bottom side up on wet paper towels in a 15 centimeter Petri dish wearing appropriate eye and ear protection. Set the helium pressure for the gene gun to 200 to 250 PS.I carefully aim the gene gun between the ribs on the bottom side of a leaf, about three to five centimeters above it. Release the safety and deliver the particles to the leaf.Use each bullet twice in the same location on the leaf. Move to another location and repeat the process if the leaf explodes, discard, and start a new leaf. There is some variance in leaf toughness due to growth conditions such as age, moisture, et cetera.After all shots have been delivered, cover the leaf and store it two to three days in low light on the bench. Examine the leaf in a dissecting microscope equipped with UV fluorescence and search for individual cells expressing GFP. When cells expressing GFP are found, use dissecting scissors to carefully extract five to 10 millimeter sections of leaf.Immediately submerge each individual leaf fragment in the well of a deep well microscope slide containing about 200 microliters of 0.1%Triton X 100. The detergent helps prevent air bubbles from forming on the surface and the deep well microscope slide allows for larger tissue imaging area image cells using a confocal microscope equipped with the appropriate filters and a 40 x water immersion objective. In this study, the three different GFP organelle targeting constructs were compared to untag GFP and a rubis.Coag.GFP confocal microscopy was used to detect GFP fluorescence in the transiently transformed single cells in control experiments in which untagged GFP was expressed. Fluorescence is observed in the nucleus and cytosol only, but not the chloroplast, which are identified by the red autofluorescence of chlorophyll as seen in this example of a cell transfected with a chloroplast only Rubis Oag. GFP construct GFP was expressed in the large organelles.The cell is outlined in white. The yellow indicates areas of colocalization of the construct with chlorophyll indicating that the construct is targeted to the chloroplast as shown in this image of a triag one transfected cell GFP expression is seen in the chloroplast, peripheries, cytosol and small punctate organelles believed to be peroxisomes. Likewise, triag two transfected cells also express GFP in the chloroplast cytosol and peroxisomes triag three, which consists of a peroxisome signal embedded within a low complexity region of the chloroplast sequence, localized only to the chloroplast and the peroxisome.Note that this cell is in a different leaf layer. This figure summarizes expression observed in the compartments of a typical tobacco leaf epidermal cell. The relative sizes and locations within the cell are shown and the relative expression levels observed via gene gun transfection and confocal microscopy.Note that the untag GFP localizes to the cytosol and nucleus, the PIMT two GFP control localizes to the chloroplast and triag one GFP, triag two GFP, and Triag 3G FP localized to the chloroplast and peroxisome as predicted, After watching this video, you should have a good understanding of how to construct a plasma with Gibson assembly, deliver it bally to tobacco leaves, and observe GFP expression within three days. While attempting this procedure, it's important to remember to use 150 nanograms per microliter of vector DNA for the Gibson assembly, and 1500 nanograms per microliter of your final DNA construct to make your gene gun bullets Following this procedure, other methods like agrobacterium transfection can be performed to obtain stable gene expression rather than transient.

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

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