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.

First, we construct three strains with Cre-EBD, 079 and 160 with ura tag ,085 with his tag through PCR, digestion and ligation. After proper induction and screening, we successfully obtained mutated 079, 085 and 160 strains hat have a manifest growing advantage in comparison with con-trol group 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, CRE-EBD and CYC1 terminator. We planned to use overlap to link this three parts and add them into our plasmids with URA3 and HIS nutrition labeling respectivly through digestion and ligation. Then, we use our forward primer of the promoter and the reverse primer of the terminator to sequence this part. The electrophoresis results showed that the length, about 2800bp, was correct. So we can start to screen strains.

OVERVIEW

After communication with professors, teachers and factory leaders, our HPers summarized that It is also difficult to monitor the copper concentration of the solution in real-time. Solving this problem by biosensor seems like a good idea.

The inspiration came from promoters found in nature, which can be induced by different metal ions. Ligating this kind of promoters with reporter gene like RFP is a common idea to monitor the concentration of metals. Take Cu ions for example, we first search for promoters induced by Cu in parts, and we found both promoters in E.coli and S.cerevisiae. Actually, The resistance to Cu ions in E.coli and S.cerevisiae differs from each other. The maximum resistance to Cu in E.coli is 9 mM, which is less than that in S.cerevisiae. The resistance in S.cerevisiae is over 15 mM. Considering the response rage, the budding yeast is a much better host for Cu detection.

We built a biosensor based on CUP1 promoter and yEmRFP to detect Cu ion’s concentration. The response range of this biosensor were characterized with fluorescent microplate reader. To improve the sensitivity of biosensor, and enlarge the response intensity when it is induced, we used error-prone PCR to obtain a large amount of promoter mutants and 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

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.