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| | <h1>Description</h1> | | <h1>Description</h1> |
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| − | <h2>Background</h2>
| + | Saccharomyces cerevisiae is a single-celled organism with three types, called a, α, and a/α. In Saccharomyces cerevisiae, three cell types differ from each other in their DNA content at the MAT locus which specifies the cell types. In nature, the two haploid cell types (a and α) of this kind of budding yeast are able to interconvert in a reversible manner by DNA-rearrangement with a DSB at the MAT locus, and this process is called mating-type switching. |
| − | <p>Human existence on earth is almost impossible without the heavy metals. Even though important to mankind, exposure to them during production, usage and their uncontrolled discharge in to the environment has caused lots of hazards to man, other organisms and the environment itself. Heavy metals can enter human tissues and organs via inhalation, diet, and manual handling. As the process of urbanization and industrialization goes deeper and deeper, heavy metal pollution, a noticeable threaten to almost all the creatures, has become an essential problem to solve.</p>
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| − | <p>According to our human practice, the situation of heavy metal pollution (copper and cadmium ions) is marked on a world map, and the severity of heavy metal pollution has been increasing all over this map. Places with serious pollution includes middle Asia, eastern Asia, southern Europe, and Latin America. In addition, not only fresh water sources, but also soil and crops are seriously contaminated by heavy metals. On average, during three out of ten suppers we have, we absorb excess heavy metals over the standard concentration.</p>
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| − | <p>Considering the rigorous situation we face, our team decided to design an advanced system for typical toxic heavy metal disposal based on Saccharomyces cerevisiae.</p>
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| − | <h2>1) Mating-type switch and Mating Switcher</h2>
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| − | <p>Saccharomyces cerevisiae is a single-celled organism with three types, called a, α, and a/α. In Saccharomyces cerevisiae, three cell types differ from each other in their DNA content at the MAT locus which specifies the cell types. In nature, the two haploid cell types (a and α) of budding yeast are able to interconvert in a reversible manner by DNA-rearrangement with a DSB at the MAT locus, and this process is called mating-type switch. </p>
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| − | <p>The DSB at MAT locus is caused by HO endonuclease(a kind of site-specific endonuclease expressed by HO gene). DSBs in chromosomes can be repaired either by homologous recombination (HR) or by nonhomologous end-joining (NHEJ). In S.C haploids, the DSB caused by HO endonuclease mostly repaired by HR with HML(α) and HMR(a) as donors. If the donor is HML(α), the mating-type will become α, and vice versa. In this way, a haploid budding yeast is able to achieve mating-type switch.</p>
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| − | <p>In our design, we chose two a-type haploids as the starting strains. In one of them, we knocked out HMR(a) to ensure the mating type only switch from a to α. 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.</p>
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| − | <p>In laboratory, the species of budding yeast we usually used are BY4741 and BY4742, whose HO gene are knocked out. Therefore, we built another vector to make HO gene’s expression controllable. We used a modified GAL1 promoter with an artificial transcription factor (ATF) Z4EV (the Z4EV gene has been induced into the SynX chromosome of this group of haploids) to strictly control the expression of HO gene. Unlike common β-estradiol-induced or galactose-induced promoters, this modified promoter is designed to be activated only when it is specifically bound with activated Z4EV factor. </p>
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| − | <p>Z4EV is a kind of fusion protein with three domains – DNA binding domain (DBD), estrogen receptor (ER) and VP16 activation domain. In the absence of β-estradiol, the ER interacts with Hsp90 chaperone complex and keep the ATF out of the nucleus. This AFT will provide a strong transcriptional activator that is dependent on the presence of β-estradiol. By using a synthetic 4-time-repeated zinc-finger DBD array from the mouse TF Zif268, residual off-target effects have been completely eliminated.</p>
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| − | <p>We use this synthetic single-gene expression system to control the expression of HO gene, so that the mating-type switch will happen only under the presence of β-estradiol. After the switch of mating type, this group of haploids’ MATa becomes MATα, and are ready to mate with the other group of haploids whose MAT locus are α.</p>
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| − | <p>Next, we introduce Vika/vox recombination system into our design. We introduce Vika recombinase and vox sites into these two haploids respectively. The first kind of haploids whose mating type is designed to switch will express Vika recombinase, and the other ones contain functional genes whose expressions are controlled by vox-Terminator-vox structure. When these two haploids mate with each other, cell fusion happens, the components of Vika/vox system are all gathered in one cell and cutting off the terminator flanked by vox locus. Henceforth, the functional genes begin to express, enabling the application of our system. To sum up, the mating type switch and mating behavior in our system serve as a novel gene switcher, namely, Mating Swicher.</p>
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| − | <p>Mating Switcher is a flexible system which can easily incorporate with other proven techniques in Saccharomyces cerevisiae, like CRISPR/cas9 and other sit-specific recombination system. We intend to use this system to link different functions in the procedure of heavy metals’ enrichment for further disposal, and make each transition clear and controllable.</p>
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| − | <h2>2) Characterization of Mating Switcher (RFP to CRT)</h2>
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| − | <p>Mating Switcher is also a means of gene regulation. In our design, unlike the traditional ways, we regard the whole cell as a signal factor, which can carry components of different systems or transcription factors to influence the expression of downstream genes. The advantages of this switcher are its flexibility, feasibility and no leaky expression. </p>
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| − | <p>To characterize our Mating Switcher, we built a gene route to switch the expression from RFP to β-carotene. In this route, we combined RFP with TEF-1 promoter. To prevent leaky expression, we choose two kinds of terminators——ADH1 and Ura3‘s. So β-carotene’s expression is controlled by promoter-vox-RFP-Terminators-vox structure. Before the mating-type switch, our yeast presents reddish color due to RFP’s expression. After the Mating Switcher, with the deletion of RFP and terminators flanked by vox locus, β-carotene expresses and the strains take on an orange color. In Saccharomyces cerevisiae, these two colors are easy to distinguish. In this way, we can easily visualize the function of our switcher, as well as measure its efficiency and error rate.</p>
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| − | <h2>3) Resistance to heavy metals (SCRaMbLE)</h2>
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| − | <p>We want to exploit the genetic and biomedical capacities of Saccharomyces cerevisiae to remediate contaminated water by heavy metals, which means that the yeast strain we use should have an inherent high resistance to heavy metal. Statistics reveal that in the natural environment, Saccharomyces cerevisiae can barely survive in the medium that contains copper or cadmium ions of which the concentrations is higher than 1.5×10-3 and 0.55×10-3 mole per litre, respectively. Since the concentration of heavy metals in industrial effluents is usually higher than these, we decide to increase the heavy metal resistance of the yeast strain, conferring a survival advantage on it in terms of coping with adverse environmental conditions. Because our laboratory spearheads efforts in the de novo synthesis of the yeast chromosomes and the cutting-edge technology— SCRaMbLE (Synthetic Chromosome Rearrangement and Modification by loxPsym-mediated Evolution) in China, we want to apply this novel technology to obtain a geneticallyengineered yeast strain with the highest heavy metal resistance.</p>
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| − | <p>Cre/Loxp system is a site-specific DNA recombination technology, used to conduct deletions, inversions, insertions, and translocations of DNA fragments in cells. This system consists of one enzyme, Cre recombinase, which recognizes and recombines a pair of short sequences called loxp sites. Results of the recombination events are dependent on the directionality of the loxp sites. Because of the asymmetry of the loxp sites, when a pair of loxp sites is present in the same DNA strand and they are in opposite orientations, the Cre recombinase will catalyze the inversion of the gene in between the loxp sites; when the loxp sites are in same orientation, this gene will be deleted.</p>
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| − | <p>In order to bestow more genetic capacities on yeast strain containing synthetic chromosomes, our laboratory has done many genetic alterations to the original yeast chromosome V and chromosome X before embarking on their de novo synthesis, one of which being to introduce loxpsym (sym: symmetric) sites to synthetic chromosomes. To be more specific, virtually every genes present in the synthetic chromosome V and X are flanked by the loxpsym sites. Unlike the naturally-occurring loxp sites, the loxpsym sites are deprived of directionality, which means the results of recombination events are no longer contingent upon the relative orientation of loxp sites (which they do not have). Under the catalysis of Cre recombinases, these loxpsym-flanked genes in synthetic chromosomes are stochastically deleted, inverted, inserted, and translocated. To put it simply, these genes are “shuffled”. Considering the intricacies of interreactions between genes as well as metabolic pathways in cells, these recombination events of chromosome fragments will somehow exert influences on the complicated metabolic network in yeast cells, engendering a dramatic shift in their biochemical properties and thus a great diversity of phenotypes amid a viable increase in heavy-metal resistance. SCRaMbLE provides us with a new method of rapid evolution of species harboring synthetic chromosomes. With this powerful genetic tool, we are able to not only select strains with highest heavy metal resistance but also other desired traits, be they alcohol tolerance, heat tolerance et cetera.</p>
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| − | <h2>4) Detection and enrichment of copper ions (parts’ improvement)</h2>
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| − | <p>In Saccharomyces cerevisiae S288C, there is a natural copper-induced promoter – CUP1p. The CUP1 promoter enables binding of RNA Polymerase II and the subsequent transcription of downstream DNA to mRNA. It is activated by ACE1, a transcription factor which binds to copper ions. It is previously available as a standalone part as BBa_K945002, produced by Tec-Monterrery’s 2012 iGEM team, and team iGEM16_Washington modified this part with illegal restriction sites removed to make this part (BBa_K2165004) easier to control and operate.</p>
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| − | <p>Based on the part provided by iGEM16_Washington, we built a vector for detection of copper ions which combines CUP1 promoter with RFP. In fact, this promoter is leaky in the absence of inducer. To make it more sensitive and lower the threshold of expression, we decide to transform the promoter with error-prone PCR. After finishing the library, the sensitivity of engineered promoter will be characterized by the fluorescence intensity.</p>
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| − | <p>After the detection, we will use Mating Switcher to open following gene’s expression rapidly. We can either overexpress CUP1 to concentrate copper in the yeast cell or display the Metallothionein on the surface of budding yeast.</p>
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| − | <h2>5) Separation of different irons (copper and cadmium ions) </h2>
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| − | <p>Our HP shows that the most challenging point in dealing with heavy metals is the separation of different metal ions. To tackle this problem, we designed a gene route based on Mating Switcher to enrich two different heavy metal respectively.</p>
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| − | <p>Aiming to separate two different ions effectively, we redesign two kinds of metallothionein. In addition to CUP1, another metallothionein was found that can specifically combine with cadmium ions. Semi-rational design and error PCR help us improve the specificity of each proteins, which make sure the separation of two kinds of metal ions can be accomplished accurately. </p>
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| | </p> | | </p> |
| − | | + | <p>This year, we are planning to utilize the natural phenomenon of mating-type switching to create a new concept called mating switcher for functional transformation and safeguards in Saccharomyces cerevisiae with gene-editing technique’s help. We will take this new kind of switcher into some very interesting applications, including heavy metal treatment and cell signal swiching, to improve the maneuverability of this yeast. Moreover, we will discuss the possibility of this concept’s utilization in other eukarya.</p> |
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