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.
