Engineering Success

Engineering Design Process

---

Throughout our project there were several instances where we underwent iterations of the engineering design process. The engineering design process includes designing, building, testing and then improving a design. This process is repeated until the goal of the project has been reached or the most ideal version of the design has been achieved.

Example: Protoplast Isolation Procedure

---

To test the expression of our genetic constructs in wheat our team decided to use wheat protoplasts. Protoplasts are plant cells where the cell walls have been enzymatically removed, making them easier to transform with foreign DNA. One challenge our team faced was optimizing our protoplast isolation procedure. This is one example of an instance where our team utilized the engineering design process.

The goal of the protoplast isolation procedure was to be able to successfully grow wheat plants and isolate enough protoplasts from clippings of their leaves to be able to perform proof of concept transformation experiments with our genetic constructs. Early on our team realized that this would be a challenging process. We knew that it could take a long time to optimize a new protocol, and there was limited time in the competition.

After going through five rounds of improvement of our procedure, we were successful in generating viable and sufficient amounts of protoplasts.

Initiation

---

We started by looking through the literature to find protoplast isolation protocols. We compared different protocols to find a consensus on reagents and steps. We also asked our advisors who had experience isolating protoplasts for their feedback on the protocols we had found. Eventually, the protocol we decided on was one used to make wheat and barley protoplasts successfully in a lab at UBC.

Protocol Iteration #1

---

This is the protoplast isolation procedure we used for our first trial run of making protoplasts

1. Grow about 50 wheat seedlings for 7 to 10 days.
2. Collect all leaves from the seedlings. Wash them and store them in 2x sterile dH₂O in 50 mL Falcon tubes.
3. Cut slits in leaves using a sterile scalpel (longitudinally, 0.5 mm strips).
4. Place cut leaves in 13 mL culture tubes and fill it with cell wall enzyme solution (~ 8-10 mL per tube) and leave them in the dark at room temperature for 4-5 hours with gentle shaking (50 rpm). Make sure temp is not > 25°C.
5. Very gently swirl the tubes during the incubation time in order to gently move the leaf tissue around in the enzyme solution to release the protoplasts.
6. Always use wide bore tips or 5 mL pipette (has wide bore) to pipette protoplasts.
7. After 4 hours, gently pipette the protoplasts into a 13 mL tube (culture tube) to a total vol of 10 mL (or fill to 8 mL and complete to 10 mL with washed leaf sucrose).
8. Overlay the solution with 1 mL of W5 media by adding gently to the top of the solution (do not mix). Spin the tubes at 250 g (~ 930 rpm), 10 min, no brake.
9. Remove the protoplasts from the boundary (middle layer) using a pipette and place them into a new 13 mL tube (it is ok if it takes a bit of other layers) (in this step 3 layers are formed, on top is W5, green protoplast in middle, and enzyme solution along with debris and dead cells on bottom).
10. Add gently W5 to a total volume of 5 mL (volume of protoplasts is >2-3 mL/tube). [Want to remove cellulase solution]. Invert the tube gently a couple of times and spin at 250 g, 5 min, no brake.
11. Remove the liquid on the top (top is W5 and protoplast will be in bottom) and gently suspend protoplasts in ~ 5 mL W5 by inverting tube gently (at this stage several tubes can be pooled).
12. Incubate them at 4°C (fridge) for at least 30 min.
13. Count the protoplasts. Want 4-5 x 106 per mL for transfections.

The first time we tried to make protoplasts we used the protocol given to us by a lab at UBC without any modifications or changes. The wheat plants used for the protocol grew successfully. 15 plant leaves were used for the protoplast isolation.

However, no protoplasts were successfully isolated from the clippings of the wheat leaves.

Protocol Iteration #2

---

Considering the results from the first trial of protoplast making, improvements were planned to the protocol:

1. We found that the wheat seeds planted at 4.5 cm depth sprouted more than those planted at 2 cm depth, so for future trials all seeds will be planted at 4.5 cm depth.
2. Only 15 leaves were used for this protoplast isolation. For future protoplast isolation trials more will be used. Plan to start the next trial with 30-40 leaves.
3. During cutting of the wheat leaves, try as hard not to cut through the entire leaf and just make very small slits, this may reduce debris and dead protoplasts.
4. When adding W5 for the first time, add directly to a falcon tube instead of a culture tube so that they don’t mix.
5. Will only use p1000 pipette/serological pipettes moving forward since protoplasts are so delicate and tear easily.
6. Will wash the leaf debris post incubation with 0.5 M sucrose. After each wash the 10 mL aliquots of the solution will be taken and added to culture tubes.

Using the modified protoplast isolation procedure for the second trial run of making protoplasts, again the wheat plants used for the protocol grew successfully. We had a higher percentage of seeds sprout when they were all planted at the 4.5 cm depth. 33 wheat leaves were used when making protoplasts.

Very few protoplasts were made from the clippings of the leaves of the plants, and those made were fractured and torn. The photo of the protoplasts can be seen below:

Protocol Iteration #3

---

Considering the results from the second trial of protoplast making improvements were planned to the protocol:

1. Start the protoplast isolation immediately after harvesting the leaves. Waiting overnight could dehydrate the leaves and cells could die, reducing protoplast yields. Keep the cut leaves in deionized water after harvesting if there is a delay before the next step of the protocol.
2. Resuspend the protoplast solution in only 1 mL of W5 solution instead of 5 mL to concentrate them more.
3. Be gentler with mixing and centrifuging since the protoplasts are very delicate and easily burst.
4. Do not store cut leaves in cell wall enzyme solution overnight at a temperature > 25°C. Higher temperatures can cause the cells to die.

In the third trial run of making protoplasts, no protoplasts were obtained using the modified protoplast isolation procedure. What can be seen is torn apart plant cells.

Protocol Iteration #4

---

Considering the results from the third trial of protoplast making improvements were planned to the protocol:

1. Plant about 60 seedlings per protoplast trial as only about 50 will germinate and sprout. 50 seedling leaves are ideal for the protoplast isolation.
2. Cutting the wheat leaves latitudinally in 0.5 mm strips rather than longitudinally. The slits are made perpendicular to the midrib of the leaf. Dicing the leaves as much as possible increases surface area to allow thee enzyme solution to work better.
3. Let leaves soak in mannitol for 5-10 minutes after cutting. Mannitol helps plant cells to tolerate salt stress, also can be used as food so protoplasts can metabolize mannitol during the digestion phase.
4. For step 11 do 1 mL of W5 instead since it is better to have a more concentrated solution than a less concentrated solution of protoplasts). Place on ice to cool (this allows faster cooling to 4°C).

In the fourth run of making protoplasts very few protoplasts were obtained, however these protoplasts were not burst or damaged. This improvement indicated that the changes made to the protocol were beneficial in bringing us closer to our end goal.

Protocl Iteration #5: Success

---

After the fourth trial of protoplast making with little success previously. We went back to the literature and incorporated changes we found in other protoplast procedures. The following improvements were planned to the protocol:

1. Make the reagents on the same day as making protoplasts do avoid degradation of enzymes in the reagents such as the cell wall enzyme solution.
2. Reduce enzyme content in cell wall enzyme solution by 50% as many protoplasts were degraded in previous trials.
3. Remove the sucrose wash step.
4. Perform 3 W5 washes and concentrate steps with a 30 minute incubation on ice after each resuspension of the protoplast pellet.

In the fifth run of making protoplasts, we successfully made many protoplasts! We made protoplasts of good quality and in sufficient amounts that we could proceed to use them in transfection experiments.