March - Ideation, team sellection, fundraising April - Problem selection, fundraising, team application May - Research/experiment planing, DNA design, fundraising June - First DNA construction, GGA, the yeast arrives, discussion with Makerere_Uganda 2022, start of the collaboration with Uppsala 2022 team July - Summer camps, InterLab study 2022, growth media optimisation, collaboration with ESTONIA-TUIT 2022 August - InterLab study 2022, Baltic Jamboree 2022, yeast transformation, growth media optimisation, collaboration with Technion-Israel 2022 September - Carotenoid content analysis, yeast transformation, composit GGA, ELTE 2022 Mock Jamboree, Researcher's Night 2022 October - yeast transformation, wiki building, the GRAND Jamboree
GGA for TUs
1. Take 10μL of promoter, terminator and gene of choice (10ng/uL). 2. Add 2μL of 10x ligase buffer. 3. Add 1μL of T4 ligase. 4. Add 1μL of BsaI restrictase. 5. Add 6μL of nuclease free water. 6. Incubate 5min at 37°C. 7. Incubate 5min at 10°C. 8. Cycle 10-20 times. 9. Incubate 5min at 60°C.
PCR for TUs1. Add 5μL 10x reaction buffer. 2. Add 1μL dNTP mix. 3. Take 10μL of each assembled TU. 4. Add 1μl of the respective forward and reverse primers for each TU. Each TU needs the respective primer pair which will extend the fragments and add SapI sites with unique fusion sites. 5. Add 1μL Taq polymerase. 6. Add 31 μL water. 7. Anneal at 58°C for 5min, 20 cycles.
R. toruloides transformation1.Inoculate yeast in 10 mL of YPD medium and incubate overnight with a stirring speed of 160 rpm at 30°C. 2. Dilute the culture to an OD600 of ~0.2 in 10 mL of YPD and incubate again until an OD600 of ~0.8. 3. Harvest the culture in a sterile 50 mL centrifuge tube at 3000 g for 10 min. 4. Resuspend the cell pellet in 25 mL of sterile water and centrifuge again. 5. Resuspend the cells in 1 mL 100 mM lithium acetate (LiAc) (pH 7.5) and transfer 500 μl of the suspension to a 1.5 mL microfuge tube. 6. Centrifuge the cells at 3000 g for 30s and remove LiAc. 7. In the following order, add 240 μL of polyethylene glycol (PEG) 4000 (50% w/v), 36 μL of 1M LiAc (pH 7.5), 24 μL of sterile water. 8. Transfer 10 μl of pre boiled salmon sperm DNA (10 mg/mL, at 100°C for 10 min) to the tube. 9. Add 50 μL of transforming DNA (0.1–10 μg, removed from the bacterial backbone by either restriction enzymes or by PCR amplification). 10. Mix the cell pellet and the added reagents by vigorous vortexing. 11. Incubate at 30°C for 30 min. 12. Add 34 μL of dimethyl sulfoxide (DMSO) 13. Heat shock at 42°C for 15 min. 14. Centrifuge the tube at 3000 g for 30 s and remove the supernatant. 15. Resuspend the pellet in 200 uL of water and spread on an agar YPD plate, grow at 30°C overnight. 16. The next day replica plate onto multiple solid YPD plates containing hygromycin/G418 and leave to grow at 30°C for two days.
Ura3 mutant selection1. Grow R. toruloides aerobically grown at 30°C in SD medium supplemented with uracil for 18h. 2. Prepare 5FOA solution in DMSO (100mg/mL). 3. Spread 150uL of 5FOA solution on a 15mL agar SD plate supplemented with uracil. 4. Spread 200uL of the culture (OD=0.05) on SD+5FOA uracil containing agar plates. 5. Incubate at 30°C for 2 days. 6. Replica plate onto SD plates with and without uracil to isolate potential ura3 mutants.
Carotenoid extraction1. Weigh harvested and washed cells were weighed (wet cell biomass). 2. Add 5 mL of DMSO to each sample. 3. Vortex the samples carefully to make sure that the cell pellet has been resuspended completely. 4. Add 5 mL of acetone, 5 mL of hexane and 5 mL of 20% NaCl solution in deionized water and vortex after each addition. 5. Centrifuge (2500 g, 5 min). 6. Collect the upper layer of the extract.
UV-Vis measurementsAll measurements were carried out with the PerkinElmer Lambda 35 UV-Vis spectrometer. 1. Pour 3 mL of each extract in a 10 mm quartz cuvette. 2. Fill the reference cuvette with hexane. 3. Record the spectra from 350 to 600 nm. 4. Dissolve the beta-carotene standard in hexane at 1.3·10-5 M.
HPLC analysis1. Dissolve the commercial beta-carotene standard in a mixture of tetrahydrofuran (THF) and acetonitrile (50:50, v/v). 2. Prepare calibration samples in concentrations ranging from 16 to 250 mg/mL. 3. Prepare a C18 column with the UV detector operating at 450 nm. 4. Maintain the column temperature at 25 °C. Use a mixture of acetonitrile-THF (60:40, v/v) as the mobile phase with a flowrate of 1 mL/min. 5. Create a calibration curve based on the area of beta-carotene peaks at different sample concentrations. 6. Remove the solvent from the yeast beta-carotene extracts using a rotary evaporator. 7. Redissolve the extract powder in 1 mL of acetonitrile-THF mixture (50:50, v/v). 8. Perform the analysis as described in steps 3 – 4. 9. Using the standard calibration curve, determine the beta-carotene concentrations in the extracts.