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

Podsumowanie

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

Streszczenie

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’ 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.

Wprowadzenie

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 splicing^2–5. 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 photorespiration^7,8 and isoprenoid synthesis^9,10 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)^12–14. 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.

Protokół

1. Design of Alternatively-spliced Sequences for Multiple Organelle Targeting

1. Determine the sites for final protein expression. For this work, the interest is in targeting the chloroplast, peroxisome, and cytosol.
2. 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 methyltransferase^6,15, and a peroxisome targeting sequence from Arabidopsis thaliana transthyretin-like S-allantoin synthase^16,17 were determined.
3. 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).

2. 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’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.

1. 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, (P_ENTCUP2, 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.
2. 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.
   1. 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.
      1. 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 <500 bp. In this case, the TriTag itself is 437 bp so it was advantageous to order it commercially.
      2. 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.
      3. Restriction Restriction enzyme DpnI cuts methylated DNA.  If the PCR template was a miniprep from E. coli, DpnI treatment will remove the contaminating template DNA.
3. Purify all the DNA sequences separately and elute in water, TE or Buffer EB.
   1. pORE vector part 1, ~3,000 bp (green arrows). PCR amplified and DpnI treated.
   2. pORE vector part 2, ~3,000 bp (black arrows). PCR amplified and DpnI treated.
   3. TriTag insert, 437 bp (blue arrows). Ordered commercially. Resuspend in ddH₂O.
   4. P_ENTCUP promoter, ~750 bp (orange arrows). PCR-amplified and DpnI treated.
4. Measure the concentrations of each of the DNA pieces. Aim for 150 ng/μl of the vector pieces.
5. 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 lab^12–14.
   1. There are two steps to this recipe: a viscous 5x master mix and 15 μl reaction-size 1.33x aliquots. The 5x master mix contains: 3 ml 1 M Tris-HCl pH 7.5, 300 μl 1 M MgCl₂, 600 μl 10 mM of each dNTP, 300 μl 1 M DTT, 1.5 g PEG-8000, 20 mg NAD, and ddH₂O to 6 ml. Prepare 320 μl aliquots (18) and freeze all but one at -20 °C. The solution will be quite viscous. Label these “5X isotherm buffer”
   2. To the one remaining (320 μl), add 1.2 μl T5 Exonuclease, 20 μl Phusion High-Fidelity DNA Polymerase, 160 μl Taq DNA Ligase and 700 μl ddH₂O. Prepare 15 μl aliquots (~80) on ice in PCR tubes and store at -20 °C.
6. 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 μl of all the DNA pieces. Add them to the 15 μl Gibson mix aliquot. If this requires volumes too small to pipette, dilute the DNA fragments and/or use more than one aliquot.
   1. 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.
7. Incubate the tubes in a thermal cycler at 50-55 °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.
   1. Apply all 20 μl to a commercial preparation of 200 μl calcium-chloride competent E. coli.
      Incubate on ice 30 min and heat shock 60 sec at 42 °C
   2. Return to ice 2 min, apply 750 μl SOC or LB medium and recover shaking in a microcentrifuge tube 30 min at 37 °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.
8. Confirm clone via sequence and store an aliquot at -80 °C

3. Biolistic Transfection of Tobacco with the Gene Gun

This is a technique that is established in JoVE^18,19. Key steps and differences are described below.

1. Grow 50-100 ml E. coli or 200 ml Agrobacterium. Maxi-prep the culture. A concentration of about 1,500 ng/μl is required to continue.
2. Prepare gene gun bullets as previously described. Load them into the Gene Gun.
3. 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.
4. Set the He pressure for the Gene Gun to 200-250 psi.
5. 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—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.
6. Store the leaf, covered with the top of a Petri dish for 2-3 days in low light on the bench.
7. 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.
8. Submerge the leaf in a deep well microscope slide under ~200 μl 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.

4. Confocal Microscopy of Transfected Tissue

These instructions vary for every instrument, so it is essential to get properly trained.

1. 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.

Wyniki

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...

Dyskusje

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 ...

Materiały

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