Protocol for assembly of a serine integrase-based platform for functional validation of genetic switch controllers in eukaryotic cells-Plant

Grow Arabidopsis thaliana ecotype Columbia plants in a 150 mL plastic cup with aerated, moist, fertilized and autoclaved soil in an environmentally controlled chamber with a medium photoperiod (12 h light/12 h dark at 22°C) under low light (optimum light is approximately 150 µE.m-2.s-1) and 50-60% relative humidity.

Choose two to four healthy plants and take 20 leaves (3-4 cm long) by cutting each petiole vertically with the help of a scalpel.

(Optional) Sterilize the leaves by washing once in 70% ethanol, for 0h 5m 0s in a 1% sodium hypochlorite solution, and five times in sterile dH₂O.

Using a metal tweezer, transfer one leaf with the adaxial side facing upward to a glass cell culture dish (90 mm x 15 mm) containing 25 mL of the W5 solution. Using the scalpel, extract and discard the remaining petiole and make sequential cross-sections (1-2 mm thick) from the midrib to the leaf margin. It is not necessary to cut the leaves completely, only to make little “scratches” on the leaf surface.

Using a metal tweezer, carefully pass the chopped leaf on the cell culture dish´s edge to remove excess W5 solution and transfer the leaf with the adaxial side down to another glass cell culture dish (60 mm x 15 mm) containing 5 mL of the enzyme solution.

Transfer the cell culture dish with the enzyme solution and chopped leaves into the vacuum chamber and vacuum infiltrate 3 times for approximately 0h 0m 5s under 500 mm Hg pump pressure.

Cover the cell culture dish with aluminum foil and then incubate in the platform shaker with gentle swirling (up to 40 rpm) for 3h 0m 0s at Room temperature.

After 3 hours of incubation, release protoplasts by swirling the cell culture dish for 1 minute or until the solution turns green.

Filter the digested sample through a 74-µm cell mesh and carefully transfer it into a 30 mL glass round bottom centrifugation tube.

Wash the mesh with up to 10mL of ice-cold W5 solution to remove all remaining protoplasts.

Centrifuge the sample at 100x g,4°C.

Carefully remove supernatant, leaving enough solution to cover the protoplasts (green pellet).

Carefully resuspend the protoplasts in 20mLof ice-cold W5 solution and gently rock the tube until the protoplasts are resuspended.

Centrifuge 100x g,4°C. Remove the supernatant with a 5000 µl pipette, again leaving enough to cover the protoplasts.

Carefully resuspend protoplasts in 20mL of ice-cold W5 solution and then incubate protoplasts On ice for 0h 30m 0s.

Swirling the protoplast tube gently until the protoplast pellet is completely resuspended and centrifuge at 100x g,4°C

Carefully remove the supernatant with a 5000 µl micropipette, again leaving enough to cover the protoplasts.

Resuspend the protoplasts to bring the total volume in the 30 mL tube to 1mL with ice-cold MMg solution.

Count protoplasts and adjust the concentration to 4 x 10⁵ protoplasts/mL with MMg solution (see steps 20-22).

Counting the protoplasts:

In a 1.5 mL microcentrifuge tube, dilute 5µL of the protoplast solution obtained in step 18 above into 1mL of MMg solution, then pipet 2µL of the diluted protoplasts on a microscope slide and count the total number of protoplasts in that 2 µl drop.

To dilute to 4 x 105 protoplasts/mL, apply the formula:

Final volume of MMg solution (mL)= (D_(A)× V_S×100)/(4×〖10〗^5)

where DA is the average number of protoplasts from the five 2 µl drops and VS is the exact volume of resuspended protoplasts in microliters.

Add MMg solution to bring the original protoplast solution to the final volume calculated in the previous step.

In a 15 mL glass round bottom centrifugation tube, mix by gently swirling the protoplast solution, DNA solution, and 40% PEG solution, according to Table 5. Add the components in this order. Increase the number of reactions as needed. Use one tube for each reaction.

Table 5 - Reaction mix for protoplast transformation

A	B	C
Order	Component	Instructions
01	100 µL of protoplast solution (concentration 4 x 105 protoplasts/mL)	Swirl the tube with protoplast solution gently and thoroughly to make sure that no pellet is formed. Use a wide-bore pipette tip to prevent damage to the protoplasts. Do not make bubbles.
02	10 µL of each plasmid DNA (concentration 1 µg x mL-1)	Use a filter pipette tip to prevent contamination.
03	110 µL 40% PEG solution	The solution is very thick; pipette very carefully. Gently swirl the tube until the solution is well mixed and layers can no longer be seen in the solution. Do not introduce bubbles.
320 µL final volume		The final volume will vary according to the number of plasmids used in the transformation (up to 5).

Incubate the 15 mL tubes for 0h 15m 0s at Room temperature.

To stop the reaction, add 2 volumes of ice-cold W5 Solution. Gently swirl the tubes to mix.

Centrifuge at 100x g,4°C.

The supernatant was carefully removed, leaving enough solution to cover the protoplast.

Resuspend the protoplast in 500µL of ice-cold W1 Solution. Gently swirling the tube to mix. This volume corresponds to one single replica transformation reaction.

Transfer the protoplast solution to a 12-well cell culture plate. If you perform multiple transformation reactions, transfer each reaction to a single and labeled well of the plate.

Place the lid on the plate and seal with Parafilm. Set the plate in a wet chamber and incubate for 24 h in the dark under gentle shaking (up to 40 rpm) at 25°C-30°C. To make a dark wet chamber, put some paper towels in a plastic box/tray, wet with distilled water, place the plates and cover with aluminum foil.

After 24 h of incubation, put the sealed 12-well cell culture plate in an Axiovert 135 M fluorescence microscope under UV light with filter set 15 (Carl Zeiss). Excitation: BP 546; beam splitter: FT 580; emission: LP 590. Capture images of GFP emission with attached DS-Ri1 digital camera (Nikon).

Transfer all of the well contents to a labeled 1.5 mL microcentrifuge tube.

Centrifuge at 100x g,4°C in a swing-bucket rotor for plates with 1.5-2 mL microcentrifuge tubes block adapter.

Carefully remove the supernatant, leaving approximately 50µL of the solution.

Flick the tube gently to thoroughly resuspend the pellet.

Analyze the presence and intensity of GFP fluorescence in the Amnis® brand FlowSight® Imaging Flow Cytometer (see Equipment Setup).

Repeat steps 32-36 for each well/sample.