The aim of this class is to transform yeast with four linear DNA fragments which will recombine in the cell into a new circular DNA molecule which is an expression vector for the S. cerevisiae ATF1 gene. The resulting plasmid will have the systematic name pTA1_TDH3_ScATF1_PGI1.

The class has a 40 min incubation.

Transfer 1 mL ultra pure water to an Eppendorf tube and put the tube on ice. Cold water is needed later in the protocol.

1. Put one empty and clean 1.5 mL Eppendorf tubes on ice.
2. Add 15 µL digested pTA1 vector
3. add 15 µL promoter PCR product
4. add 15 µL ddH2O
5. add 15 µL terminator PCR product

1. Put one empty and clean 1.5 mL Eppendorf tubes on ice.
2. Add 15 µL digested pTA1 vector
3. add 15 µL promoter PCR product
4. add 15 µL gene PCR product
5. add 15 µL terminator PCR product

The DNA mix should be marked with "➖" or "➕" and kept on ice until needed.

The yeast strain to be transformed is called CENPK111-32D.

1. Transfer 100 µL of the cell suspension to each of two empty Eppendorf tubes (Step 1). Keep the tubes with cells on ice when you are not pipetting.
2. Spin the cells for 20-30 s at the highest speed in the microcentrifuge.
3. Remove supernatants with a P200 pipette. Leave the cell pellet at the bottom of the tube, do not resuspend.
4. Add 300 µL PLS to each of the tubes. Put this tube back on ice.
5. Add all of the DNA mix + to one tube and DNA mix - to the other. Put this tube back on ice.
6. Mark the tubes "➕" and "➖".
7. Vortex the tube until the cells are well resuspended, but not longer.
8. Put the tubes in a floating tube rack in a water bath at 42°C. Be sure you can identify your tube.
9. Incubate for 40 min.

10. Remove tubes with cells from the water bath and put on ice for 2 min.
11. Add about 1/2 mL glass spheres (~10-15 spheres) each to two empty Petri dishes with the appropriate solid medium for selection.
12. Spin tubes for 30 s to 1 min at highest speed.
13. Remove supernatant with a P1000 pipette. Leave the cell pellet at the bottom of the tube.
14. Add 200 µL ice cold water (from the tube with sterile water on ice) and resuspend with the pipette by slowly pipetting up and down using a blue tip, try not make foam.
15. Transfer 180 µL of the cell suspension to the Petri dish with the solid medium (90% of the cells).
16. Transfer 200 µL cold water and 20 µL of the cell suspension to another Petri dish (10% of the cells).
17. Spread the cells by shaking (using the samba method).
18. Mark you plates with "➖" or "➕", group number, date and either "90%" or "10%".
19. Incubate the plates upside down for 2-3 days at 30°C.
20. Give the tubes with the rest of the cells to the instructor.

The transformation protocol is based on the Gietz High-efficiency yeast transformation using the LiAc/SS carrier DNA/PEG method.

Finish the DNA assembly on your computer.

If the transformation is successful, we would expect fewer colonies on the "➖" control plates than on the "➕" plates. See results from 2023-10-02 and 2023-10-03.

Colonies will be picked and resuspended in 1 mL YPD medium containing 250 ppm (mg/mL) isoamyl alcohol (Miller 2020).

Miller, R. A., Lee, S., Fridmanski, E. J., Barron, E., Pence, J., Lieberman, M., & Goodson, H. V. (2020). “Scentsor”: A Whole-Cell Yeast Biosensor with an Olfactory Reporter for Low-Cost and Equipment-Free Detection of Pharmaceuticals. ACS Sensors, 5(10), 3025–3030. link

bioshock_plasmid.webp

Lista de materiais para a preparação de aula

Meio YPD líquido 100 mL
Proveta estéril vazio 100 mL
Água ultrapura estéril 50 mL
Palitos estéreis
Micropipetas P1000, P100, P20 e P10
Pontas amarelas, azuis e brancas estéreis, uma de cada cor para cada grupo.
Copo com tubos Eppendorf 1.5 mL **Novos**, um por grupo.
Suporte para tubos Eppendorf, um por grupo.
40 placas de meio SD sólido.
50 Tubos vazios de vidro estéreis com tampa (tubos de cultura)
Suportes para tubos de vidro.
Isoamyl alcohol 0.1 - 1 mL
Um tubo FALCON 50 mL com ~25 mL de esferas de vidro (~ 4-5 mm) estéreis (para espalhar células)

Banho maria 42°C
Microcentrífuga
Caixas de esferovite para gelo, um por grupo.
Marcadores
Lamparinas, um por grupo.

Meio YPD:
- 20 g/L peptone or tryptone
- 10 g/L yeast extract
- 20 g/L glucose

Meio sólido SD:
- 6.7 g/L YNB (Yeast Nitrogen Base w/o amino acids)
- 20 g/L Glicose
- 20 g/L Agar