Experiments

We used rapid yeast transformation for yHX0378, this type of transformation is simple and time-saving, therefore we were able to free up more time for our remaining experiments.

1.The yeast colonies were incubated in 5 mL of yeast peptone dextrose (YPD) overnight at 30℃ in the rocking device at 220 rpm.

2.The next day, we extracted 500 µL of the culture to 5 mL of YPD.

3. When OD600 reached 0.4-0.6, 1 mL of the yeast cells were harvested and washed twice with sterile water.

4.Then we mixed 240µL of 50% polyethylene glycol (PEG) 3350, 50 µL of ssDNA and 36 µL of 1 M LiOAc together, mixture served as transformation buffer. The ssDNA was preheated at 101 ℃ for 12 mins and then placed on ice for cooling down promptly.

5.Each kind of plasmid would be added 2 µL (pZJ-leu-30gRNA,pRS413-CBE) and about 20µL sterile water was added to the yeasts. The preconditioned yeast cells (about 34 µL) then were immersed into the transformation buffer and then mixed by being inverted up and down 7-10 times, afterwards we incubated the mixture at 42℃ for 40 min in water bath/metal bath.

6.The solution was centrifuged at 4000rpm for 1 min, after which we discarded the supernatant. Remained cells were plated onto a selective media.

We got a plasmid from the GENERAL BIOL which contained the information of Ode to joy, however, it is not reasonable to directly import the plasmids into Saccharomyces cerevisiae. The following operations are performed.

1.Firstly, we obtained the Ode to joy information fragments by restriction enzyme EcoRI and BamHI double digestions and gel extraction.

2.Meanwhile, KOD ONE PCR was performed on our original GIF plasmids and we also used gel extraction to obtain vectors. After the vectors and fragments were assembled by seamless cloning, we obtained the required information plasmids, pRS-hyg-joy.

3.Next, we transformed the plasmids into Escherichia coli for amplification. It also ensured the normally progress of our subsequent experiments.

4.After the plasmids were extracted from Escherichia coli, we added the designed primers and performed colony PCR and electrophoresis verify.

In order to make sure of the results' accuracy, we sent them to GENEWIZ for sequencing and obtained accurate plasmids sequences. The sequencing results will determine whether the plasmid-construction succeeded or not.

Although the rapid yeast transformation method is the same, it should be noted that there are some differences in the transformation of yHX0362.

1.We first integrated the CBE fragment on yHX0362 genome, used SC-U culture for incubation.

2.Then, we designed two sets of experiments of induced yeasts and constitutive yeasts to observe the differences between the two transformation methods. Induced yeasts contained pZJ-leu-30gRNA and information plasmids (pRS-hyg-joy and pRS-hyg-GIF). The constitutive yeasts possessed one more pRS413-CBE than the induced yeasts.

3.Because the plasmid has antibiotic tag, the yeasts need to be returned to its resistant growth state by overnight incubation after a heat shock. We used YPD to help them recover and gain the ability of growing in culture containing antibiotics (hygromycin).

4.We coated constitutive yeasts to SC-L-U-H+1/3 ADE + HYG and the other yeasts to SC-L-U+1/3 ADE + HYG.

1.The yeast colonies were incubated in 5 mL of SC-L-U + HYG about 1 day at 30 ℃ with shaking device at 220 rpm.

2.Take 1000µL yeast after culture, centrifuge(4000rpm×1min), clean the waste liquid, add 1mL sterile H2O and centrifuge again (4000rpm×1min). Repeat for two to three times, and 5mL SG-L-U+1/3 ADE + HYG (Galactose and Raffinose replaced the Glucose in SC) was put into the shaking tubes.

3.We then extracted 200µL SG-L-U+1/3 ADE + HYG into the centrifuge tubes, suspended yeast cells by suction, extracted 100 µL of which and transferred them back into the previous tubes, and induced them at 30℃ for 24h.

4.After inducing, we extracted 4µL yeast solution and add 200µL sterile water, put 102 µL of the mixture onto the appropriate selective SC media, waited for them to grow visible red colonies, then they were picked and sent for sequencing.

1.The yeast colonies were incubated in 5 mL of SC-L-U + HYG about 1 day at 30 ℃ with shaking device at 220 rpm.

2.Take 1000µL yeast after culture, centrifuge(4000rpm×1min), clean the waste liquid, add 1mL sterile H2O and centrifuge again (4000rpm×1min). Repeat for two to three times, and 5mL SG-L-U+1/3 ADE + HYG (Galactose and Raffinose replaced the Glucose in SC) was put into the shaking tubes.

3.We then extracted 200µL SG-L-U+1/3 ADE + HYG into the centrifuge tubes, suspended yeast cells by suction, extracted 100 µL of which and transferred them back into the previous tubes, and induced them at 30℃ for 24h.

4.After inducing, we extracted 4µL yeast solution and add 200µL sterile water, put 102 µL of the mixture onto the appropriate selective SC media, waited for them to grow visible red colonies, then they were picked and sent for sequencing.

Before sequencing, we need do some preprocessing. Select induced red colonies, boil and add primers for PCR amplification. They will then be sent to GENEWIZ for the results. The relevant experimental details of PCR can be referred to Protocol. (click to protocol)