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 media 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 S1, S2, S3, and S4, and as for copper, S5, S6, S7, and S8.
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 S1, S2, S3, and S4; control groups are synX (the yeast strain containing a synthetic chromosome X), BY4741 (wild-type haploid yeast), and BY4743 (wild-type diploid yeast). Results are shown in the picture below.
Apparently, the experimental groups have a survival advantage over control groups. In this picture, S1 is able to develop a large single colony even after it is diluted to 100000 times on SC solid media containing 0.14 mM cadmium ions; S3 and S4 are able to grow when diluted to 100000 times but the colonies are much smaller than that of S1. Although S2 is not as good as the other three, it still shows higher resistance to cadmium ions than the control groups do. Wild-type yeast strains BY4741 and BY4743 can barely grow on this growth media, while synX cannot grow, which means that synX is unable to survive such high concentration of cadmium ions. The results clearly demonstrate that these mutated yeast strains have an improved phenotype-increased resistance to cadmium ions.
Another assay was conducted on SC solid media containing 4.8 mM copper ions. Results are shown in the picture below.
Apparently, the experimental groups also have a survival advantage over control groups.From this picture, S5, S6 and S8 can still get a large single colony when it is diluted to 100000 times on SC solid media containing 4.8 mM copper ions. Although S7 is not as good as the other three, it still shows higher resistance to copper ions compared BY4741 when diluted to 100 times . In the same time, BY4743 just grow slightly in the highest concenatration. And synX cannot grow on SC solid media containing 4.8 mM copper ions which means that synX is unable to tolerate such high concentration of copper ions. These results intuitively indicate that our screened strains have a better phenotype, increased resistance to copper ions.
Survival Rate Experiments
This experiment aims to quantify mutated yeast strains’ ability to survive in 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, namely 10min, 30min, 1h, and 2h, then diluted and plated on YPD solid media. After yeast colonies emerge from the growth media, the number of the colonies are counted and recorded to calculate the survival rate of this strain in this solution.
We choose one optimal strain, S1, to test it's ability to survive compared with synX. Results are shown in the figures and tables below.
From figure 5, the survival rate of S1 is higher than synX after immersing for the same time. We can get the following conclusion that SCRaMbLE really makes sense. What’s more, through these datas, we can also get the percentage of resistance improvement compared with synX which are 23.8%, 231.9% and 192.4% respectively. With the prolonging of soaking time, the difference of survival rates are more and more obvious, which means that S5 has better tolerance to cadmium ions. The results of the three experiments are consistent show that the strain is stable and has good reproducibility.
For copper, We choose one optimal strain, S5, to test it's ability to survive compared with synX. Results are shown in the figures and tables below.
From figure 7, the survival rate of S5 is higher than synX after immersing for the same time. What’s more, through these datas, we can also get the percentage of resistance improvement compared with synX which are 74%, 72% and 698% respectively. With the prolonging of soaking time, the difference of survival rates are also more and more obvious, which means that S5 has better tolerance to copper ions. We don’t have enough time to do three times. But many times dilution assay have the coincident results which shows that SCRaMbLE really makes sense.
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