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The Disaster From Yeast

2019 SCU-China spared no efforts to complete and explore the project: CORegulaTIN. Finally, we synthesized cordycepin and pentostatin successfully in S. cerevisiae. Also, we verified our delay expression system step by step.

At the Very Beginning

Because this is the first year for SCU-China culturing yeast, there was no strain in our lab. Besides, only one or two professors major in the S. cerevisiae, so we turned to many people for a desirable yeast strain. At last, we found professor Ke Liu who offered us BY4742, and professor Xiaofeng Zhu applying us with AH109.

At first, we did not notice how big trouble these two strains might cause later.

Colony PCR

After transforming the plasmids, we constructed after a hard time into S. cerevisiae, we expected to confirm the transformants by colony PCR. Unfortunately, we always got negative results in this step. No band appeared on the gel after electrophoresis but DNA ladder. However, teammates of Tianjing University had a discussion with us and they asked us to test the moderate time for yeast lysis. 10 min for boiling water bath and 5 min for ice bath as 1 cycle, they advised us to set gradient cycle number for lysing the yeast. We then tried to extend the heating time in the first step of colony PCR, that is, we set 10 min for 98 degrees as the first step. In this way, we successfully confirmed our transformed yeasts by colony PCR.

3. AH109

We surprisingly found the transforming rate of AH109 was very high! Countless colonies appeared on plates without Ura. There were even biofilms! However, after the later confirmation steps we were no longer excited about it. "There are so many false positive colonies! OMG!" We then spread yeast solution of transformed AH109, which was diluted 20, 50 and 100 times into plates SD-Ura-. There were still close-set colonies appearing on all plates.

Is strain AH109 able to grow on SD plates without Ura？The information of this strain shows that it contain the gene mutation that prevent it grow on SD plates without Ura. We carried out negative control and extra control groups to verify that AH109 cannot grow on SD plates without Ura. But with some reasons still unknow, the control groups appeared no colony.

We consulted professor Liu about this strange phenomenon recently. Shocking reply comes, that strain AH109 does grow in SD medium even without Ura.We then replace strain AH109 with strain YM4271.

4. The green fluorescence

It is a common method that using green fluorescence as a reporter. But during the experiments this year, it seemed difficult to make the S. cerevisiae BY4741 express green fluorescence. We checked the design once and once again. It was strange why there was no green fluorescence. Later on, on a beautiful sunny day, we found a plate cultured seven days earlier with green fluorescence accidentally. It was a big surprise! But we were not so happy, because it was a simple reason for those transformants that did not shine: the culturing days were not enough!