Difference between revisions of "Part:BBa K5241009"

Revision as of 01:38, 29 September 2024

GAL1-Tyrosinase(BM)-GAL1

In brewing yeast, the GAL1 promoter, tyrosine as the target gene, and the CYC1 terminator are used to express the tyrosine synthesis pathway for the production of melanin in yeast.

Gene Circuit Diagram:

Description:

1. Turn on the ice maker and the water bath, set the temperatures to 42°C and 96°C respectively, adjust the shaker to 30°C (no shaking required), set the incubator to 29°C, turn on the laminar flow hood, and sterilize for 15 minutes.

2. Take out 50 µL of BY4741 competent cells, 1 µL of project plasmid (800 ng/µL), and 1 µL of control plasmid (800 ng/µL), place them on ice to thaw, and take out the corresponding single deficiency medium on the laminar flow hood. (For double transformation: the volume ratio of plasmid to competent cells should be 1:10, and the amount of transformation reagent should be increased or decreased synchronously according to the volume of competent cells.)

3. Take 5 µL of Carrier DNA and place it in the 96°C water bath for 3 minutes, then quickly place it in an ice bath for 3 minutes, and repeat the process once.

4. Take 250 µL of PEG/LiAc and centrifuge with the competent cells, plasmid, and Carrier DNA on a mini centrifuge.

5. Take two sterilized 1.5 mL centrifuge tubes.

- In one centrifuge tube, add 25 µL of competent cells, 1 µL of project plasmid, 2.5 µL of Carrier DNA, and 125 µL of PEG/LiAc in sequence, mix well by pipetting, label as Sample.

- In the other centrifuge tube, add 25 µL of competent cells, 1 µL of control plasmid, 2.5 µL of Carrier DNA, and 125 µL of PEG/LiAc in sequence, mix well by pipetting, label as Control.

6. Place in the shaker (no shaking required), 30°C, for 30 minutes (invert and mix 6-8 times at the 15-minute mark).

7. Transfer the mixture to the 42°C water bath and incubate for 15 minutes (invert and mix 6-8 times at the 7.5-minute mark).

8. Centrifuge at 5000 rpm for 40 seconds (if the centrifuge can only be set for 1 minute, time it manually), discard the supernatant, add 100 µL of ddH2O to each centrifuge tube, mix well by pipetting, centrifuge again for 30 seconds, and discard the supernatant.

9. Add 12.5 µL of ddH2O to each centrifuge tube, mix well by pipetting, spread on plates, and incubate in the incubator at 29°C for 48-96 hours, inverted.

Result:

We successfully expressed melanin in Saccharomyces cerevisiae and used it for ionizing radiation experiments.

Reference documentation

[1] https://2022.igem.wiki/sjtu-biox-shanghai/design

Sequence and Features