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Demonstrate

Mating-type Switch and Mating Switcher

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. 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 1-1 The PCR strategy for testing whether we deleted the HMRa in SynⅩ-dUra.

Fig 1-2 As we can see in the gel photo above, the UP and DOWN segments hasn’t been amplified in our SynⅩ-dUra comparing to the BY4741 as control. Which indicated that the HMRa gene has been successfully eliminated.

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 (BBa_K2407001) and CYC1-part (BBa_K2407107) 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 (BBa_K2407109) 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 (BBa_K2407100) 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 1-3 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 1-4 (a) showed the PCR results for MATa locus. The MATa gene was amplified in all colonies except the first 24 colonies.

Fig 1-4 (b) showed the PCR results for MATα locus. The MATα gene was amplified in all 96 colonies.

Discussion & Expectation

To our delight, we verified that our strategy, namely Gal System, is able to trigger the Mating Type Switching in yeasts when induced by galactose. Thereby, we succeeded in building a new kind of on-off device in our target yeast—— SynⅩ-dUra-416, where the whole system is ready to perform as a mating switcher to turn on/off other functional genes for further usage in our project——Romantic Switcher.

References

[1] Sara J. Hanson, and Kenneth H. Wolfe. An Evolutionary Perspective on Yeast Mating-Type Switching.

[2] James E. Haber, Mating-Type Genes and MAT Switching in Saccharomyces cerevisiae.

UP

Characterization of Mating Switcher

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 redid 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 (called PVUVC), BBa_K2407307 (called PVRVC). 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.

Fig.2-1.Red fluorescent protein expression vector construction flow chart.

Then we first inserted BBa_K2407306 to the Synthetic chromosome Ⅴ of Saccharomyces cerevisiae . 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 Saccharomyces cerevisiae BY4742 was used for modification. By digestion and ligation, we construct vika gene on plasmid pRS416 which contains a selective marker Ura3, and plasmid pRS413 which contains a selective marker His. Then we introduced those two different plasmids into BY4742 respectively.

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:

Fig.2-1.Three modified colonies and one resulting colony.

The upper left corner of the bacteria is synthetic Saccharomyces cerevisiae, we integrated modified fragment into its synthetic chromosome V. (PVUVC) The upper right corner is also synthetic Saccharomyces cerevisiae. (PVRVC) It is imported red fluorescent protein gene based on the upper left corner of the yeast. Both of them are single-celled organism called a. The lower right corner of the yeast is another mating type of haploid yeast called α. It has plasmid pRS416 with vika gene. The yeast in the lower left corner are diploid Saccharomyces cerevisiae, which is obtained by mating the two yeasts on the right side of the figure.

The yellow colony in the figure is mating successfully. After the induction of galactose, vika recombinase was expressed, and red fluorescent protein gene and terminator was deleted so that β-carotene expresses. The color of colony was changed from white to yellow. In addition to it, we also tried other methods to turn on the switch.

Fig.2-1.Bacteria after matingcultivated on the Sc-His plate.

There are 377 yellow colonies and 365 white colonies in the field of view.

We used another α-type yeast named BY4742, which has a plasmid called pRS413 with selective marker His. It could express vika recombinase before mating. It mated with a-type Saccharomyces cerevisiae PVRVC, and then yeast cultured on Sc-His plate. 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.

Fig.2-1.Bacteria after mating cultivated on the Sc-Ura plate.

There are 325 yellow colonies and 31 white colonies in the field of view.

Apart from mating, we also transformed plasmid pRS416 with vika gene into the PVRVC. The efficiency is up to 91.3 percent in this figure.

Compare above two methods, mating and transformation of plasmid, we find that mating is not as efficient as the transformation of the plasmid. After analysis, we came to the conclusions as follows. For the mating method, vika recombinase has stop expressing when BY4742 mated with PVRVC in YPD medium. The previously expressed vika recombinase may be degraded during the growth. In contrast to this, with another method that the plasmid was transformed into PVRVC directly, vika recombinase is continuously expressed during cell growth. So the efficiency of the second method is higher than the first method.

Fig.2-1.Four modified coloniesinserted with promotor-vox-RFP-terminators-vox-crt structure

We also used other a type Saccharomyces cerevisiae to achieve mating switcher. After changing TEF promotor to TDH3 promotor, we repeated the test according to the above two methods. The four strains are all a-type haploid synthetic Saccharomyces cerevisiae named TVRVC NO.2 (upper left), NO.4 (upper right), NO.11 (lower left) and NO.19 (lower right) respectively. The color appears to be white because β-carotene is not expressed.

Fig.2-1.Mating successfullycolonies

These are parts of successful result of mating mentioned above. Two figures one by one correspondence.

To sum up, mating switcher can be presented in kinds of yeast with different forms. This proves that our Mating switcher is fast, flexible and efficient.

3) Verification of RFP in the PVRVC

The main characteration method of verification of RFP in the PVRVC applied by us is observing the expression of red 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.

Fig.1.2.3 Microscope image of yeast cultured with SC-Leu with no yEmRFP gene transformed.

Fig.4.5.6 Microscope image of yeast cultured with SC-Leu with yEmRFP gene transformed.

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.

Normalized fluorescence value was calculated by dividing fluorescent value by cell concentration(OD₆₀₀600)

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, there was low red fluorescence was detected after mating, which was considered the influence of β-carotene.

DISCUSSION & FUTURE WORK

Our mating switch plays an important role in many respects, such as including heavy metal treatment and cell signal switching. And we created a novel method to prove the effectiveness of the switch in an intuitive and effective way. The terminator of the first part (PVUVC) terminates the expression of the downstream gene, proving the validity of the switcher, and the second part (PVRVC) creates an evident method of color conversion to determine the state of the switcher.

Aiming to increase the vika-vox system efficiency, we let vika enzyme saturate expression, but the efficiency was still relatively low. We hypothesized that this phenomenon was caused by degradation of the vika enzyme in the YPD culture medium. We’d better to change the composition or proportion of YPD ingredients to find out the best culture conditions. We are looking forward to more researches on this field so that we can make this system work better and even perfectly.

We use the RFP as the reporting protein. But there exists a drawback that it’s detected with expensive device. A more intuitive reporting strategy need to be developed, maybe it can be seen by bare eyes like E.coli in the near future.

Reference

Altamura E, Borgatti M, Finotti A, Gasparello J, Gambari R, Spinelli M, et al. (2016) Chemical-Induced Read-Through at Premature Termination Codons Determined by a Rapid Dual-Fluorescence System Based on S. cerevisiae. PLoS ONE11(4): e0154260. https://doi.org/10.1371/journal.pone.0154260

Leslie A. Mitchell, James Chuang, Neta Agmon, Chachrit Khunsriraksakul, Nick A. Phillips, Yizhi Cai, David M. Truong, Ashan Veerakumar, Yuxuan Wang, María Mayorga, Paul Blomquist, Praneeth Sadda, Joshua Trueheart, Jef D. Boeke; Versatile genetic assembly system (VEGAS) to assemble pathways for expression in S. cerevisiae, Nucleic Acids Research, Volume 43, Issue 13, 27 July 2015, Pages 6620–6630.https://doi.org/10.1093/nar/gkv466

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