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 yeast strain with better tolerance to heavy metal ions .^[1].

We constructed three yeast strains namely 079, 160, and 085. They all have a plasmid containing the CRE-EBD sequence and different nutritional labels^[2]. 079 and 160 strains have a URA3 label, 085 strain has a HIS label. 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 PCLB2 promoter (BBa_K2407002), the Cre-EBD sequence (BBa_K2407005) and a CYC1 terminator(BBa_K2407003). 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 (BBa_K2407011）,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.

The experimental groups also have a survival advantage over control groups. From this picture, S5, S6, and S8 are able to develop a large single colony after diluted to 100000 times on SC solid media containing 4.8 mM copper ions. S7 is not as tough as the other three experimental groups, but it still shows higher resistance to copper ions compared with BY4741 when diluted to 100 times. BY4743 can hardly grow on this media, while synX cannot grow, which means that synX is unable to tolerate such high concentration of copper ions. The results clearly showcase that the mutated yeast strains have an improved 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 the seemingly best strain, S1, as the experimental strain to test its ability to survive high concentration of cadmium compared with the control strain, synX. Results are shown in the pictures and tables below.

The Fig.3-4 and Fig.3-5 showcase that the survival rate of S1 is higher than that of synX after yeast cells are immersed in cadmium ions solutions of identical concentration for the same amount of time. We painstakingly counted and recorded the number of the colonies on each individual growth media. The quantitative results are that compared with the control strain, the experimental strain S1's ability to tolerate cadmium ions has increased by 23.8% (30 minutes), 231.9% (1 hour), and 192.4% (2 hours). The longer the time of immersion is, the more obvious the difference of survival rates is. The results are consistent with the dilution assay, which is that the mutated strain has a better resistance level of cadmium ions .

As for copper, the seemingly best strain, S5, is chosen as the experimental strain to test its ability to survive high concentration of copper ions compared with synX. Results are shown in the pictures and tables below.

The Fig.3-6 and Fig.3-7 showcase that the survival rate of S5 is higher than that of synX after yeast cells are immersed in copper ions solutions of identical concentration for the same amount of time. The quantitative results are that compared with the control strain, the experimental strain S5's ability to tolerate copper ions has increased by 74% (1 hour), 72% (2 hours), and 698% (3 hours). It also can be extrapolated that the gap of survival rates between the mutated strain and synX strain will continue to widen as the immersion time increases. The results are consistent with the dilution assay too.

EXPECTATIONS

We are exhilarated to see that SCRaMbLE is really a feasible technology to enhance the yeast's ability to cope with adverse environmental conditions. Not just heavy metal ions, we are looking forward to seeing its future applications, be they, for example, alcohol tolerance or heat tolerance.

OVERVIEW

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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-induced 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-induced 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 1 mM, which is much less than that of S.cerevisiae's (over 15mM in YPD medium and 6.25 mM in SC medium). 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 yEmRFP to monitor the concentration of copper ions. The response range of this biosensor was characterized by a fluorescent spectrophotometer (Hitachi F-2700). To improve the sensitivity of the biosensor and enlarge the response intensity when it is induced, we used error-prone PCR to obtain lots of promoter mutants and then characterized them.

CONSTRUCTION

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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

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Based on the CUP1 promoter (BBa_K2165004) provided by iGEM16_Washington, we constructed this biosensor. 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 the fluorescent spectrophotometer (Hitachi F-2700) 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.

Figure 4-2 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_K2165004) & (BBa_K2407012) to build an effective Cu-induced biosensor in budding yeast. We noticed that this result is a little different with works done 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.

PARTS IMPROVEMENT

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The Cu-induced promoter CUP1 promoter is a previous BioBrick used by iGEM16_Washington, iGEM16_Waterloo, and other iGEM teams. However, the detailed characterization like what we did this year hasn't been shown on iGEM parts page. Moreover, this part hasn’t be improved by any means or in any ways. Under this situation, we plan to work on this promoter to improve its sensitivity and response peak, reduce the leakage expression, and create new parts for future work.

1) Redesign of CUP1 promoter

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First, based on the part (BBa_K2165004) provided by iGEM16_Washington, we tried to ensure the core sequence for transcription. Researches about this promoter mainly published in the 1990s, and the mechanism of induction has been researched thoroughly. There exist 5 ACE1 binding sites (UAS), 2 TATA boxes, and one initiation element in the promoter. The complex of ACE1 and copper ions will bind the promoter, which causes the activation of CUP1 promoter with TATA boxes’ help. ACE1 complex’s binding directly increases the possibility for TBP (TATA-Box Binding Protein) to bind the promoter, which can enhance the expression.

Based on this mechanism, we redesigned the part sequence provided by iGEM16_Washington. We deleted irrelevant bases on the two ends of this promoter and retained the core sequence. In this way, this promoter played its key role with fewer bases. Strains of S. cerevisiae BY4742 containing either BBa_K2165004-yEmRFP and BBa_K2407000-yEmRFP 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 0.1 mM Cu²⁺. Samples were tested with fluorescent spectrophotometer (Hitachi F-2700) after 1, 3, 6, 12, and 24 hours.

Figure 4-4 shows the expression of yEmRFP with both the two promoters were very similar, so we can tell the deletion didn’t influence the core function of CUP1 promoter. Based on the new part, we carried out a further improvement.

2) Error-Prone PCR

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In our experiment, we noticed that CUP1 promoter still has a certain degree of leakage expression. To make a better biosensor, we planned to reduce the leakage expression and increase the sensitivity. To reach this goal, we took the fluorescence intensity at both inductions or not into evaluation indexes.

The technology of error-prone PCR was taken into our experiment. Although there are many methods to introduce genetic diversity into a parent sequence, error-prone PCR is the most common way of creating a combinatorial library based on a single sequence. By adding some heavy metal ions into the PCR buffer and preparing dNTPs with different composition, new mutants were introduced into the CUP1 promoter. The whole procedure is shown in the following figure.

The library of promoter mutants obtained from error-prone PCR were ligated into plasmid pRS416 with two restriction sites (BamHI and XbaI). After that, we enriched different plasmids from E.coli and established the plasmid library with 132 samples. Then, different plasmids were transferred into S.cerevisiae BY4742 to test the fluorescence intensity under different conditions.

First, we tested and selected mutants with less leakage. Compared with control group, we test the fluorescence intensity with no induction and selected two mutants with lower fluorescence intensity. Actually, we test a lot of mutants, but most of them were not positive result. We picked EP-3, EP-5, EP-9, and EP-28 whose fluorescence intensity was less or close to the control group, and sequenced them. The sequencing results can be found in the parts' information: BBa_K2407013 (EP-3), BBa_K2407014 (EP-5), BBa_K2407015 (EP-9), BBa_K2407016 (EP-28).

Second, we worked on the sensitivity of the biosensor. Leakage expression was not the only thing needed to be solved, and we also needed to increase the response range when it was induced. A good biosensor needs less leakage and more sensitivity.

We tested the 4 selected biosensors and control group with 0, 10, 100, 500, and 1000 μM Cu²⁺ induced for 20 min, and the result is shown below with logarithmic coordinates.

The figure shows the response ranges of biosensors with different promoters within 20 min. For most biosensors, the fluorescence intensity increases as copper ion’s concentration increases from 0 to 100 μM. However, when the concentration exceeds 100 μM, the responses of most biosensor become slow, and the fluorescence intensity decreases. A reasonable explanation is that high concentrations of copper can inhibit the biosensor's response within a short time.

Fortunately, we still found a biosensor who met the requirements of an excellent biosensor. EP-5 has a less leakage and a higher sensitivity. Its fluorescence intensity is lower than the control group by 17 units with no induction and is higher by 21 units with 100-μM-Cu induction. By aligning the sequence with the CUP1 promoter, we found altered bases mainly located at the both sides of UASs and a deletion of one base even occurred between two UASs. We suspected that the change of sensitivity and leakage expression mainly due to the change of spatial distribution and the increase of A/T concentration, which both could influence the binding procedure of transcription factors.

DISCUSSION & FUTURE WORK

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In our characterization of both primary and improved promoters, we found the effect of induction is not as obvious as expected (Previous iGEM team’s results). After reading some references, we found the activation process is related to the acetylation of H3 and H4 located at CUP1 promoter, which showed nucleosome reposition and transcription factors binding might be the main reason for the activation. However, our biosensors were ligated on plasmid pRS416, which usually exists in the nucleus in a supercoiled state. There is only little possibility for a plasmid to binds to histones, so the transcription process shows less activation than that on a chromosome.

In the future, we plan to construct this biosensor on chromosomes to see whether the result will be more positive. Meanwhile, we will continue enlarging the response peak and range to improve this biosensor.

overview

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Our HPers have conducteded research in Shanxi Province. They found some factories directly choose to only deal with one main kind of the heavy metals, then abandon others. The convenient treatment obviously can't satisfy the environmental requirements. Hence, we demand a type of bacterium to handle two types of heavy metal ions together.

The genetic circuit based on the Vika-Vox system enables stepwise treatment, owing to a switch from the expression of Cup1 (copper accumulation) to LIMT (cadmium accumulation). We grow our yeasts and measure the concentration of heavy metal ions in the supernatant at equal intervals to test the efficiency of our system.

Construction

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The TEF promoter, the Cup1 gene, and the Ura3 terminator are ligated together, integrated into vox-ura3-vox system by homologous recombination. 5-FOA plate helps us to screen the correct cell. Similarly, the LIMT gene and the Ura3 nutritional label are integrated into the synthetic chromosome V, too.

PCR is used to check if we successfully completed the molecular biology construction.

Fig.5-1 the results of PCR. We use 2k plus Ⅱ as the marker. On four parallel lanes of the gel (number 1,2,3,4), run were four set of DNA molecules of known size ( 319bp for number 1, the LIMT; 186bp for number 2 and 3, the Cup1; 3114bp for number 4,the whole sequence contained Cup1). From the DNA band of number 1, we could analyze that vika has been expressed to delete the Cup1 and its terminor, so we can get the LIMT. From the DNA band of number 2, 3 and 4, we could delightedly prove that the fragments (TEF promoter, Cup1 and ura3 terminator) have successfully transformed to synthetic chromosome V.

Accumulation

Firstly, S.C-Cu, S8 (screened by SCRaMbLE) and BY4741 are cultured in YPD liquid media for 24 hours. Then add the 430 mg/L copper ions solution. Cells are cultured for another 45 hours (30℃). Atomic absorption spectroscopy is used to measure the concentration of copper ions in the supernatant every 5 hours. We depict the adsorption curve of copper ions.

In the same way, we culture Cd Yeast, S1 (screened by SCRaMbLE) and BY4741 in YPD liquid medium for 24 hours and then add xx mg/L cadmium ions solution to the media for another xx hours (30℃). the concentration of cadmium ions in the supernatant is measured every x hours.

In terms of the respective ability to adsorb copper and cadmium, we compare genetically-engineered yeast, SCRaMbLE yeast and original one.

As is illustrated in Fig.5-1 and Fig.5-2, engineered yeast significantly absorbs more ion than control group without any improvement. Furthermore, SCRaMbLE yeast also shows excellent adsorption capacity, comparable to genetically-engineered one. Fig.X1 reveals the adorption of copper ion, which relatively faster than cadmium, showed in Fig.X2.

Afterwards, we check if the vika enzyme could work well. The Cu yeast with a plasmid expressing vika enzyme is grew in the medium with raffinose, then transferred to heavy metal solution.

Figure 5-4. S.C-Cu is cultivated in medium with raffinose including 320mg/L copper ions and 6mg/L Cadmium.galactose is added at 12 hours to turn on "switch".

Fig.5-4 clearly shows the change of the concentration of heavy metal ions in the supernatant. Firstly, the Cu yeast works smoothly. The concentration of copper ions declines over time while that of cadmium ions barely changes. 12 hours later, we add galactose to the solution. Situation changes. Galactose induces the enzyme, changing Cu yeast to Cd yeast. It leads to faster adsorption of cadmium but slower for copper..

DISCUSSION & FUTURE WORK

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In the experiment, we find the LIMT doesn’t appear as ideal as we thought. In the future work, we plan to read involved references in order to complete protein modification by rational design.

Moreover, it’s a pity that we have no time to combine our genetic circuit and SCRaMbLE yeast. In subsequent experiments, it deserves a try to transform fragments into SCRaMbLE yeast, checking if it will double the absorption capacity or even better.

In the nutshell, we will be dedicated to improve the absorption efficiency and better our genetic circuit.

Reference

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Wang J, Chen C. Biosorption of heavy metals by Saccharomyces cerevisiae: A review[J]. Biotechnology Advances, 2006, 24(5):427.

C. Baumann, A. Beil, S. Jurt, M. Niederwanger, O. Palacios, M. Capdevila, S. Atrian, R. Dallinger, O. Zerbe, Angew. Chem. Int. Ed. 2017, 56, 4617.

Dönmez G, Aksu Z. The effect of copper(II) ions on the growth and bioaccumulation properties of some yeasts[J]. Process Biochemistry, 1999, 35(1–2):135-142.

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