Obtaining the chassis

Aiming to achieve MTS for environmental use, it is essential to make sure that when the MAT locus has DSB (double strands break) cleaved by HO, our type-a (MATa) yeast can only become type-α (MATα). Therefore, we used a Ura-tag to replace the HMRa domain in chromosome Ⅲ. In this way the HMRa will no longer be the donor for the homologous recombination in the repairing process for MAT cleavage. Since the change of mating type may appear successively, there is a great possibility that the same type haploid mate with each other. To avoid the existence of meaningless mating, we built an vector to express MATα genes to produce a1-α2 stable corepressor so that the haploid will regard itself as a diploid and prevent mating unless the MATa locus changes to the other one. After selection, by homologous recombination, we deleted the Ura-tag for further usage. We selected the target colonies (SynⅩ-dUra) via 5-FOA plates.

Fig.1.We successfully acquired the target yeasts as our chassis (SynⅩ-dUra).

The result for constructing the Gal systems

In this pathway, we chose Gal1 as our inducible promoter for the expression of HO gene, CYC1 as the terminator, and PRS416(with Ura-tag) as our vector. As for segments ligation, we designed the cutting sites for Bsa1 enzyme in each part, hoping to achieve seamless ligation of these three parts.

We adopted the PCR method to amplify the Gal1-part and CYC1-part from a Gal1-Vika plasmid we had used in our former lab work with specially designed primers for this procedure. After PCR, the Gal1 has the cutting sites for SalⅠand BsaⅠon both ends, and CYC1 has that for BsaⅠand BamhⅠon both ends. Meanwhile, the HO gene was obtained by gene synthesis, flanked by specific hangtags for BsaⅠin order to be cohesive with Gal1 (upstream) and CYC1 (downstream). Thus, we have built our composite part (GHC).

After the ligation ofGHC and PRS416 Plasmid (GHC-416), we transformed the E. coli for the augment of our new plasmid——GHC-416. We examined the transformation result by PCR method to amplify the HO gene in the E. coli which we randomly selected in the plate.

Fig.2.The results of PCR of #6, #7, #16, #20, #27, #36, #37, #55 colonies. HO gene (length of 1770bp). As we can see, HO gene in all 8 colonies has been amplified, which indicated that we succeeded in constructing the device for HO gene expression.

The result of mating type switching(MTS)

We transformed the chassis yeasts for the new device——GHC-416, the new yeasts we selected in the Sc-Ura plate is named as SynⅩ-dUra-416.

In this section, we only got to test the Gal System due to time limit. And we figured that the result for Gal System is adequately enough to represent the feasibility of our designed strategy for MTS.

The whole test process can be divided into three steps.

1) Step one

Activate the Gal1 promoter. After that, the expression of HO gene in the SynⅩ-dUra-416 can be initiated.

2) Step two

Cultivate two groups of yeasts together. (one is SynⅩ-dUra-416, the other is normal BY4741 MATa) If the MTS has been accomplished (SynⅩ-dUra-416 can become MATα), the two groups of haploids can mate with each other and become diploids.

3) Step three

Test the results of mating by PCR method. We designed the primers for both MATa locus and MATα locus. The amplification of both MATa locus and MATα locus indicates that the yeasts has turned into diploids, the MTS has been achieved in other words.

According to our design, after activating the Gal1 promoter, the expression of HO gene in the SynⅩ-dUra-416 can be initiated.

Then we cultivated two groups of yeasts together. (one is SynⅩ-dUra-416, the other is normal BY4741 MATa) If the MTS has been accomplished (SynⅩ-dUra-416 can become MATα), the two groups of haploids can mate with each other and become diploids.

To test whether MTS has happened, we selected some colonies in the selective plates (Sc-Ura ) and adopted PCR method. With designed primers for both MATa locus and MATα locus, the amplification of both MATa locus and MATα locus can indicate that the yeasts has mated with each other, and turned into diploids, in other words, the MTS has been achieved.

Fig.3.(a) showed the PCR results for MATa locus. The MATa gene was amplified in all colonies except the first 24 colonies. (b)showed the PCR results for MATα locus. The MATα gene was amplified in all 96 colonies.

OVERVIEW

After realizing that we need more intuitive characterization of Mating Switcher, we thought of two kinds of gene expression products in different colors, red fluorescent protein and β-carotene. We carried out a reasonable experimental design, and decided to realize functional conversion from red fluorescent protein to β-carotene by Mating Switcher.

At first, we built expression vector with TEF promoter, which was a strong promoter in Saccharomyces cerevisiae. Although we obtained results as we expected, it is not so perfect that we decided to change a stronger promoter. Then, we constructed another expression vector with TDH3 promoter. We re-did the same qualitative and quantitative experiments to characterize our results.

CONSTRUCTION

In the early stage of the project, we constructed two composite parts with TEF promoter: BBa_K2407306, BBa_K2407307. At the end of our project, we also constructed one composite part with TDH3 promoter called TVRVC. Among them, yEmRFP is modified from a mCherry mRFP to adapt to the transcription environment in yeast. We did overlap PCR to combine them together. After that, we sequenced these parts, and sequencing result showed that this construction was successful.

Then we first transformed BBa_K2407306 into Saccharomyces cerevisiae with Synthetic chromosome Ⅴ. Through the screening of SC-Ura solid medium and PCR experiments, we obtained the required strains called PVUVC. Second, we integrated the second composite part into this chromosome through homologous recombination, allowing the RFP gene to replace the Ura3 gene. The 5-FOA solid medium and PCR experiments were used to screen correct colony PVRVC. The conversion of the last fragment refers to the previous method. This process is graphically displayed on the above figure.

To achieve mating, another mating type of wild type haploid yeast BY4742 was used for modification.

Results of Characterization of Mating Switcher

1) Proof of Existence of Composite Parts

We built three composite parts in total. They are integrated into the chromosomes of Saccharomyces cerevisiae by transformation. We use colony PCR to proof the existence of these three parts in our strain. The result is showed as below.

The PCR’s results confirmed that the target genes were ligated into chromosome correctly.

2) Result of Mating

After we got the strain that introduced the red fluorescent protein gene, we let it mate with another mating type haploid yeast, which had plasmid with vika gene. The result is showed as follows:

The yellow colony in the figure is mating successfully. After the induction of galactose, vika enzymes was expressed, and red fluorescent protein gene and terminator was deleted so that β-carotene expresses.As can be seen from the figure above, the reorganization efficiency is high, which reaches 50.8 percent.This proves that our Mating switcher is fast and efficient.

3) Verification of RFP in the TVRVC

The main characteration method of verification of RFP in the TVRVC applied by us is observing the expression of fluorescent protein under the fluorescence microscope. By this way, it will be much more intuitive so that we can directly get the results. We took pictures under different visions and the results are as follows.

From these images we can clearly see the expression of yEmRFP. These images undoubtedly verify the yEmRFP gene has been transformed succeessfully.

Meantime, We cultured the transformed yeast in several 5mL liquid SC-Leu at 30℃ and 220 rpm for 12 hours ( Take three samples at a time). We used one sample for centrifugation to precipitate the bacterial and the remaining two remained unchanged. The difference is the fluorescence value we need, then we calculated the value of average them. The excitation wavelength is set at 540nm and the emission wavelength is set at 635nm. Hereafters, we measured the bacterial concentration at OD600. At last, we divided the fluorescence value by OD600 to normalize the value and the result data are as follows.

From the data we can find that the successful transformation of the bacteria's fluorescence intensity is more than twice that of the wild type.

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 label, 085 strain has 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 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.

Figure 1. This is a simplified version of this vector expressing the Cre recombinase enzyme. CRE-EBD is the coding sequence of Cre recombinase; PCLB2 is a constitutive promoter in yeast; CYC1 is a terminator.

Figure 1. This is a simplified version of this vector expressing the Cre recombinase enzyme. CRE-EBD is the coding sequence of Cre recombinase; PCLB2 is a constitutive promoter in yeast; CYC1 is a terminator.

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.

Figure 2. Experimental groups: S1, S2, S3, and S4; control groups: synX, BY474, and BY4743. Yeast cells were picked up from single colonies on solid growth media and diluted by 10, 100, 1000, 10000, and 100000 times.

Figure 2. Experimental groups: S1, S2, S3, and S4; control groups: synX, BY474, and BY4743. Yeast cells were picked up from single colonies on solid growth media and diluted by 10, 100, 1000, 10000, and 100000 times.

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.

Figure 3. Experimental groups: S5, S6, S7, and S8; control groups: synX, BY4741, and BY4743. Yeast cells were picked up from single colonies on solid growth media and diluted by 10, 100, 1000, 10000, and 100000 times.

Figure 3. Experimental groups: S5, S6, S7, and S8; control groups: synX, BY4741, and BY4743. Yeast cells were picked up from single colonies on solid growth media and diluted by 10, 100, 1000, 10000, and 100000 times.

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.

Table 1. Colony numbers of synX and S1. Experiments were repeated for three times. Cadmium concentration: 5mM for the first and the third experiments; 10 mM for the second experiment.

Figure 4. Picture of the third survival rate experiment results. Growth media: YPD. Experimental strain: S1; control strain: synX. Cells were plated onto the media after immersed in 5 mM cadmium solution for 10 minutes, 30minutes, 1 hour, and 2hours.

Table 1. Colony numbers of synX and S1. Experiments were repeated for three times. Cadmium concentration: 5mM for the first and the third experiments; 10 mM for the second experiment.

Figure 4. Picture of the third survival rate experiment results. Growth media: YPD. Experimental strain: S1; control strain: synX. Cells were plated onto the media after immersed in 5 mM cadmium solution for 10 minutes, 30minutes, 1 hour, and 2hours.

The figure 5 showcases 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.

Table 2. Colony numbers of synX and S5. Due to the time limit, we conducted this experiment for only one time. Yeast cells were immersed in 50mM copper solutions for 10 minutes, 30 minuets, 1 hour, and 2 hours.

Figure 6. Picture of survival rate experiment results. Growth media: YPD. Experimental strain: S5; control strain: synX. Cells were plated onto the media after immersed in 50 mM copper solution for 10 minutes, 30minutes, 1 hour, and 2hours.

Table 2. Colony numbers of synX and S5. Due to the time limit, we conducted this experiment for only one time. Yeast cells were immersed in 50mM copper solutions for 10 minutes, 30 minuets, 1 hour, and 2 hours.

Figure 6. Picture of survival rate experiment results. Growth media: YPD. Experimental strain: S5; control strain: synX. Cells were plated onto the media after immersed in 50 mM copper solution for 10 minutes, 30minutes, 1 hour, and 2hours.

The figure 7 showcases 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

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 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.

Fig. 3-1. Sequencing result of CUP1p and yEmRFP (alignment support by SnapGene®)

Fig. 3-1. Sequencing result of CUP1p and yEmRFP (alignment support by SnapGene®)

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.

PARTS IMPROVEMENT

The Cu-induced promoter CUP1p is a previous BioBrick used by iGEM16_Washington, iGEM16_Waterloo, and other iGEM teams. However, the detailed characterization like what we did this year haven’t be showed 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

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. The promoter is activated by the complex of ACE1 and copper ions. There exist 5 ACE1 binding sites, 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.

Fig. 3-3. structure of redesigned CUP1 promoter used in our project, based on BBa_K2165004

Fig. 3-3. structure of redesigned CUP1 promoter used in our project, based on BBa_K2165004

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 less bases.

2) Error-Prone PCR

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 induction 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 CUP1 promoter. The whole procedure is shown as the following figure.

Fig. 3-4. The procedure for error-prone PCR experiment

Fig. 3-4. The procedure for error-prone PCR experiment

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 result can be found in the part information: BBa_K2407013 (EP-3), BBa_K2407014 (EP-5), BBa_K2407015 (EP-9), BBa_K2407016 (EP-28).

Fig. 3-5. The leakage expression of different promoters

Fig. 3-5. The leakage expression of different promoters

Second, we worked on the sensitivity of 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 Cu for 20 min, and the result is shown below with logarithmic coordinates.

Fig. 3-5. The leakage expression of different promoters

Fig. 3-5. The leakage expression of different promoters

The figure shows the response rages 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 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 space distribution and the increase of A/T concentration, which both could influence the binding procedure of transcription factors.

DISCUSSION & FUTURE WORK

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 rage to improve this biosensor.