TITLE: Protocol for assembly of a serine integrase-based platform for functional validation of genetic switch controllers in eukaryotic cells-Plant
AUTHORS: Marco A. de Oliveira, Lilian H. Florentino, Thais T. Sales, Rayane N. Lima, Luciana R. C. Barros, Cintia G. Limia, Mariana S. M. Almeida, Maria L. Robledo, Leila M. G. Barros, Eduardo O. Melo, Daniela M. Bittencourt, Stevens K. Rehen, Martín H. Bonamino, Elibio Rech

[DESCRIPTION]
This protocol details the  assembly of a serine integrase-based platform for functional validation of genetic switch controllers in eukaryotic cells in plant.

[GUIDELINES]
Troubleshooting:

TABLE 6. Troubleshooting for the Plant system stages

 
 
 

[STEPS]
SECTION: Plant growth ● Timing 4–6 weeks
1. 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.
 


SECTION: Protoplast isolation. ● Timing 4-6 hours
2. 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.
 

SECTION: Protoplast isolation. ● Timing 4-6 hours
3. (Optional) Sterilize the leaves by washing once in 70% ethanol, for 5 min in a 1% sodium hypochlorite solution, and five times in sterile dH2O.
 


SECTION: Protoplast isolation. ● Timing 4-6 hours
4. 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.
 

SECTION: Protoplast isolation. ● Timing 4-6 hours
5. 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.
 

SECTION: Protoplast isolation. ● Timing 4-6 hours
6. Transfer the cell culture dish with the enzyme solution and chopped leaves into the vacuum chamber and vacuum infiltrate 3 times for approximately 5 s under 500 mm Hg pump pressure.
 


SECTION: Protoplast isolation. ● Timing 4-6 hours
7. Cover the cell culture dish with aluminum foil and then incubate in the platform shaker with gentle swirling (up to 40 rpm) for 180 min at Room temperature.
 



SECTION: Protoplast isolation. ● Timing 4-6 hours
8. After 3 hours of incubation, release protoplasts by swirling the cell culture dish for 1 minute or until the solution turns green.
 

SECTION: Protoplast isolation. ● Timing 4-6 hours
9. Filter the digested sample through a 74-µm cell mesh and carefully transfer it into a 30 mL glass round bottom centrifugation tube.
 

SECTION: Protoplast isolation. ● Timing 4-6 hours
10. Wash the mesh with up to 10 mL of ice-cold W5 solution to remove all remaining protoplasts.

SECTION: Protoplast isolation. ● Timing 4-6 hours
11. Centrifuge the sample at 100 x g, 2 min, 4 °C.
 


SECTION: Protoplast isolation. ● Timing 4-6 hours
12. Carefully remove supernatant, leaving enough solution to cover the protoplasts (green pellet).
 

SECTION: Protoplast isolation. ● Timing 4-6 hours
13. Carefully resuspend the protoplasts in 20 mLof ice-cold W5 solution and gently rock the tube until the protoplasts are resuspended.
 


SECTION: Protoplast isolation. ● Timing 4-6 hours
14. Centrifuge 100 x g, 2 min, 4 °C. Remove the supernatant with a 5000 µl pipette, again leaving enough to cover the protoplasts.

SECTION: Protoplast isolation. ● Timing 4-6 hours
15. Carefully resuspend protoplasts in 20 mL of ice-cold W5 solution and then incubate protoplasts on ice for 30 min.
 




SECTION: Protoplast isolation. ● Timing 4-6 hours
16. Swirling the protoplast tube gently until the protoplast pellet is completely resuspended  and centrifuge at 100 x g, 2 min, 4 °C

SECTION: Protoplast isolation. ● Timing 4-6 hours
17. Carefully remove the supernatant with a 5000 µl micropipette, again leaving enough to cover the protoplasts.
 

SECTION: Protoplast isolation. ● Timing 4-6 hours
18. Resuspend the protoplasts to bring the total volume in the 30 mL tube to 1 mL with ice-cold MMg solution.
 


SECTION: Protoplast isolation. ● Timing 4-6 hours
19. Count protoplasts and adjust the concentration to 4 x 105 protoplasts/mL with MMg solution (see steps 20-22).
	Counting the protoplasts:
SECTION: Protoplast isolation. ● Timing 4-6 hours
20. In a 1.5 mL microcentrifuge tube, dilute 5 µL of the protoplast solution obtained in step 18 above into 1 mL of MMg solution, then pipet 2 µL of the diluted protoplasts on a microscope slide and count the total number of protoplasts in that 2 µl drop.
 

SECTION: Protoplast isolation. ● Timing 4-6 hours
21. 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.
 

SECTION: Protoplast isolation. ● Timing 4-6 hours
22. Add MMg solution to bring the original protoplast solution to the final volume calculated in the previous step.
SECTION: Protoplast transformation. ● Timing 1-2 hours
23. 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
 
 
 
SECTION: Protoplast transformation. ● Timing 1-2 hours
24. Incubate the 15 mL tubes for 15 min at Room temperature.

SECTION: Protoplast transformation. ● Timing 1-2 hours
25. To stop the reaction, add 2 volumes of ice-cold W5 Solution. Gently swirl the tubes to mix.
SECTION: Protoplast transformation. ● Timing 1-2 hours
26. Centrifuge at 100 x g, 2 min, 4 °C.
 


SECTION: Protoplast transformation. ● Timing 1-2 hours
27. The supernatant was carefully removed, leaving enough solution to cover the protoplast.
SECTION: Protoplast transformation. ● Timing 1-2 hours
28. Resuspend the protoplast in 500 µL of ice-cold W1 Solution. Gently swirling the tube to mix. This volume corresponds to one single replica transformation reaction.
 


SECTION: Protoplast transformation. ● Timing 1-2 hours
29. 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.
 

SECTION: Protoplast transformation. ● Timing 1-2 hours
30. 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.
 



SECTION: Protoplast transformation. ● Timing 1-2 hours
31. 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).
SECTION: Flow cytometry ● Timing 30 min by sample
32. Transfer all of the well contents to a labeled 1.5 mL microcentrifuge tube.
 

SECTION: Flow cytometry ● Timing 30 min by sample
33. Centrifuge at 100 x g, 1 min, 4 °C in a swing-bucket rotor for plates with 1.5-2 mL microcentrifuge tubes block adapter.
 


SECTION: Flow cytometry ● Timing 30 min by sample
34. Carefully remove the supernatant, leaving approximately 50 µL of the solution.

SECTION: Flow cytometry ● Timing 30 min by sample
35. Flick the tube gently to thoroughly resuspend the pellet.
 

SECTION: Flow cytometry ● Timing 30 min by sample
36. Analyze the presence and intensity of GFP fluorescence in the Amnis® brand FlowSight® Imaging Flow Cytometer (see Equipment Setup).
SECTION: Flow cytometry ● Timing 30 min by sample
37. Repeat steps 32-36 for each well/sample.