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.

From this picture, S1 can still get a large single colony when it is diluted to 100000 times on SC solid media containing 0.14 mM cadmium ions. In the same time, S3 and S4 can grow when they are diluted to 100000 times. Although S2 is not as good as the other three, it still shows higher resistance to cadmium ions than the control groups. Because BY4741 and BY4743 just grow slightly in the highest concenatration. And synX cannot grow on SC solid media containing 0.14 mM cadmium ions which means that synX is unable to tolerate such high concentration of cadmium ions. These results intuitively indicate that our screened strains have a better phenotype, increased resistance to heavy metal ions.

Another assay was conducted on SC solid media containing 4.8 mM copper ions. Experimental groups are S5, S6, S7, and S8; control groups are synX, BY4741, 160, and 085. Results are shown in the picture below. 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, namely 30 minutes, 1hour, 2hours, and 3hours, 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 as the survival rate of this strain in this solution.

The 5mM cadmium ions solution was used to test the experimental groups' ability to survive, and the survival rates of the S1, S2, S3, S4 (experimental groups) and synX (control group) strains were compared by the number of the colonies on the growth media. Results are shown in the pictures and tables below. These data are able to directly indicate to what extent these mutated yeast strains' ability to survive cadmium ions solution has improved.

For copper, the 50mM copper ions solution was used. Experimental groups are S5, S6, S7, S8; control group is synX. Results are shown in the pictures and tables below.

OVERVIEW

After communicating with professors, teachers, and factory superintendents, our HPers found that it was difficult to monitor the concentration of the copper ions in solution in real-time. Using a biosensor seems to be a good solution to this problem.

This idea was inspired by the naturally-occurring metal-ion-inducible promoters. Ligating this kind of promoters with a reporter gene such as RFP is a common idea to visibly monitor the concentration of metal ions. Take copper as an example: after browsing through parts(?), we found copper-ion-inducible promoters in both E.coli and S.cerevisiae. Actually, the ability of E.coli and S.cerevisiae to tolerate copper ions differs from each other. E.coli's maximum tolerance level to copper ions is 9 mM, which is less than that of S.cerevisiae (over 15mM). (这里和scramble数据貌似不一样) Considering the response range, the budding yeast is a much better host for copper detection.

We built a biosensor based on the CUP1 promoter and the yEmRFP to monitor the concentration of copper ions. The response range of this biosensor was characterized by a fluorescent microplate reader. To improve the sensitivity of the biosensor and enlarge the response intensity when it is induced, we used error-prone PCR to obtain plenty of promoters mutants and then characterized them.

CONSTRUCTION

The biosensor consists of two main parts. One is the Cu-induced promoter CUP1p, the other is yEmRFP, which is modified from a mCherry mRFP to adapt to the transcription environment in yeast. The promoter was synthesized without RFC sites (XbaI) and the RFP was amplified by PCR. We used overlap PCR to combine the two parts and added two restriction sites on the ends. By digestion and ligation, we constructed this biosensor on the plasmid pRS416 which contains a selective marker URA3. After that, we sequenced this part with M13F and M13R as primers. The sequencing result showed that this construction was successful, so we can take the next step – characterization.

CHARACTERIZATION

To characterize this biosensor, strains of S.cerevisiae BY4742 containing the plasmid with an initial OD₆₀₀ of 0.1 were grown for 24 hours in SC-URA medium at 30 degrees Celsius, and then were induced with copper sulfate. Samples in different copper concentration were tested with fluorescent microplate reader after 1, 6, 12, and 24 hours. This protocol was based on the experience used by Waterloo and Washington iGEM teams and amended by our team.

Fig.3-2. The fluorescence intensity of CUP1p-yEmRFP biosensor with different Cu concentration induced

Fig.3-2. The fluorescence intensity of CUP1p-yEmRFP biosensor with different Cu concentration induced

Figure 3-2 showed the relationship between fluorescence intensity with induction time and Cu concentration. With 0.1 mM CuSO₄ induced, the fluorescence intensity is 2 times over a control with no induction at 1 hour. As time went on, the fluorescence intensity slightly reduced. Moreover, as the Cu concentration increased, the fluorescence intensity decreased, and when the concentration reached 1 mM, the intensity was close to the control group. This might be due to the higher copper ion concentration influences the transcription, expression and even growth of yeast.

This result will be useful for teams who will use the parts BBa_K2407000 & BBa_K2407012 to build an effective Cu-induced biosensor in budding yeast. We noticed that this result is a little different with works down by Waterloo team. It may be due to the differences between Cu ion’s concentration and yeast species. However, we both verified the possibility of building a biosensor based on CUP1 promoter in yeast.

This result was provided for modeling of this biosensor and try to find a proper function to accurately describe the response procedure. Click here to see more information.