OVERVIEW
After doing relevant literature reading, we found that yeast’s tolerance level of ambient copper and cadmium ions has a threshold concentration, approximately 3mM and 0.5mM in SC culture medium respectively.
In order to increase yeast strains’ inherent tolerance of copper or/and cadmium ions in their growing environment, we used this cutting-edge biological technology—SCRaMbLE, which stands for Synthetic Chromosome Rearrangement and Modification by Loxpsym-mediated Evolution, to obtain mutated yeast strains.
We constructed three yeast strains namely 079, 160, and 085. They all have a plasmid containing the CRE-EBD sequence and different nutritional labels. 079 and 160 strains have URA3 tag, 085 strain has HIS tag. After proper induction and screening, we successfully obtained mutated 079, 085 and 160 strains that have a manifest growing advantage over control groups when cultured in SC solid media which contain 0.14 mM cadmium ions or 4.8 mM copper ions. We named those mutated strains with increased tolerance capacity of cadmium ions 1s, 2s, 3s, and 4s, and as for copper ion, 5s, 6s, 7s, and 8s.
In order to characterize their increased tolerance of copper or/and cadmium ions, we designed and conducted two different sets of experiments, in both visible and quantitative manner, to test their ability to cope with adverse environmental conditions.
CONSTRUCTION
This vector consists of three parts, an estrogen-inducible promoter, the Cre-EBD sequence and a CYC1 terminator. We used overlap PCR to ligate these three parts and then the plasmids with URA3 and HIS nutritional label respectively through enzymatic digestion and ligation. Then this composite part was sequenced and proved to be accurate by using the promoter's forward primer and the terminator's reverse primer. The electrophoresis results below also showcased that the sequence length (about 2800bp) was correct.
CHARACTERIZATION
Dilution Assay
We conducted dilution assay on SC solid media containing 0.14 mM cadmium ions. Experimental groups are 1s, 2s, 3s, and 4s; control groups are synX (the yeast strain containing a synthetic chromosome X), BY4741 (wild type yeast), 160, and 085. Results are shown in the picture below. Apparently, the experimental groups have a survival advantage over control groups.
Another assay was conducted on SC solid media containing 4.8 mM copper ions. Experimental groups are 5s, 6s, 7s, and 8s; control groups are synX(the yeast strain containing a synthetic chromosome X), BY4741(wild type yeast), 160, and 085. Results are shown in the picture below. Apparently, the experimental groups also have a survival advantage over control groups.
Survival Rate Experiments
This experiment aims to quantify mutated yeast strains’ ability to survive copper or cadmium ions solution. Same amount of yeast cells are added to the copper or cadmium ions solution at the beginning; after that, a certain amount of this solution is taken out at regular intervals, diluted and plated on YPD solid media, namely 30 minutes, 1hour, 2hours, and 3hours. After yeast colonies emerge from the growth media, we count and record the number of the colonies as the survival rate of this strain in this solution.
For cadmium ions, we use 5mM cadmium ions solution and the survival rates of 1s, 2s, 3s, 4s and synX were compared. Results are shown in the pictures and tables below. These datas indicate the degree of improvement of our optimal strains directly.
For copper ions, we use 0.5M copper ions solution and the survival rates of 5s, 6s, 7s, 8s and synX were compared. Results are shown in the pictures and tables below. These datas indicate the degree of improvement of our optimal strains directly.
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