Team:RHIT/Project
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<p>The goal of the Rose-Hulman iGEM team’s project was to devise a cellular circuit that would allow for the determination of the mating type of <i>Saccharomyces cerevisiae</i>. This mating type sensor was created by introducing a self-perpetuating fluorescent heteroprotein by means of a plasmid vector. The heteroprotein contained several distinct segments including an Ste12 responsive element, a LexA-reg element, two separate fluorescent domains, a LexA binding domain, a VP64 activator domain, a nuclear localization sequence, and a terminator, as illustrated below. This protein was contained on a <i>HIS3</i> plasmid.</p><br /> | <p>The goal of the Rose-Hulman iGEM team’s project was to devise a cellular circuit that would allow for the determination of the mating type of <i>Saccharomyces cerevisiae</i>. This mating type sensor was created by introducing a self-perpetuating fluorescent heteroprotein by means of a plasmid vector. The heteroprotein contained several distinct segments including an Ste12 responsive element, a LexA-reg element, two separate fluorescent domains, a LexA binding domain, a VP64 activator domain, a nuclear localization sequence, and a terminator, as illustrated below. This protein was contained on a <i>HIS3</i> plasmid.</p><br /> | ||
<div align="center"><img src="https://static.igem.org/mediawiki/igem.org/9/94/Designed_construct.png" /><br /> | <div align="center"><img src="https://static.igem.org/mediawiki/igem.org/9/94/Designed_construct.png" /><br /> | ||
- | < | + | <h4>Figure 1: Designed construct.</h4></div> |
<p>Ste12 is a transcription factor that is activated as part of the pheromone response pathway. The initial production of the heteroprotein is controlled by the Ste12 responsive element, taken from the Fus1 gene. Once the protein is produced, the LexA binding domain binds to the LexA-regulatory element, and in conjunction with the VP64 activator domain, facilitates the further production of the heteroprotein in the form of a positive feedback loop.</p><br /><br /> | <p>Ste12 is a transcription factor that is activated as part of the pheromone response pathway. The initial production of the heteroprotein is controlled by the Ste12 responsive element, taken from the Fus1 gene. Once the protein is produced, the LexA binding domain binds to the LexA-regulatory element, and in conjunction with the VP64 activator domain, facilitates the further production of the heteroprotein in the form of a positive feedback loop.</p><br /><br /> | ||
<p>In addition to the planning described in the project planning section, this circuit was rationally designed with several potential problems in mind. The first problem that often arises in similar circuits is the “leaky” nature of some promoters. Extensive research was performed on the various proteins involved in the yeast pheromone response pathway in order to choose one that is tightly regulated, and is only activated in presence of mating factor. Furthermore, the use of a non-native activator as the predominant control mechanism reduces the probability of interactions between other similar transcription factors.</p><br /><br /> | <p>In addition to the planning described in the project planning section, this circuit was rationally designed with several potential problems in mind. The first problem that often arises in similar circuits is the “leaky” nature of some promoters. Extensive research was performed on the various proteins involved in the yeast pheromone response pathway in order to choose one that is tightly regulated, and is only activated in presence of mating factor. Furthermore, the use of a non-native activator as the predominant control mechanism reduces the probability of interactions between other similar transcription factors.</p><br /><br /> | ||
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<p>Synthetic biology combines DNA sequences discovered in nature and synthetic DNA sequences designed in the laboratory (parts) to produce new functions in living cells (machines). Different types of regulatory and protein-encoding parts are used to engineer useful machines. This approach is being applied to produce various things, including insulin from bacteria and biofuels from algae. It can also be used to address many of the world's large problems, from hunger to disease epidemics and alternative energy.</p><br /> | <p>Synthetic biology combines DNA sequences discovered in nature and synthetic DNA sequences designed in the laboratory (parts) to produce new functions in living cells (machines). Different types of regulatory and protein-encoding parts are used to engineer useful machines. This approach is being applied to produce various things, including insulin from bacteria and biofuels from algae. It can also be used to address many of the world's large problems, from hunger to disease epidemics and alternative energy.</p><br /> | ||
<h2>Yeast background</h2><br /> | <h2>Yeast background</h2><br /> | ||
- | <p>Yeast is a single-celled eukaryote, which means that it shares many properties with cells of multi-cellular organisms, including humans. It is commonly used for laboratory research and commercial applications. Yeast can exist as diploid cells, which have two copies of each chromosome, like most animal cells, or as haploid cells, which have only one copy of each chromosome. <br /><div align=center><img src="https://static.igem.org/mediawiki/igem.org/ | + | <p>Yeast is a single-celled eukaryote, which means that it shares many properties with cells of multi-cellular organisms, including humans. It is commonly used for laboratory research and commercial applications. Yeast can exist as diploid cells, which have two copies of each chromosome, like most animal cells, or as haploid cells, which have only one copy of each chromosome. <br /><div align=center><img src="https://static.igem.org/mediawiki/igem.org/8/88/Diploiddd.png" width=38% /><br /><br />This cell is diploid, as it has two copies of each chromosome.<br /><br /><img src="https://static.igem.org/mediawiki/igem.org/3/3c/Haploiddd.png" width=38% /><br /><br />This cell is haploid, as it has one copy of each chromosome.</div><br /><br />Haploid cells also are one of two mating types, MATa or MATalpha. They are, therefore, similar in these respects to animal eggs and sperm. Diploid yeast cells are produced when haploid cells of opposite mating type sense one another and fuse together. What each haploid senses is mating pheromone produced by cells of the opposite mating type. MATa cells produce a-pheromone that binds a-receptors on MATalpha cells, while MATalpha cells produce alpha-pheromone, which binds alpha-receptors on MATa cells. This "cross-signalling" activates the mating pheromone response pathway (MPRP), leading to fusion of the two haploids and formation of a diploid cell.</p><br /> |
<h2>The Checkmate Project</h2><br /> | <h2>The Checkmate Project</h2><br /> | ||
<p>Yeast researchers must often determine the mating type of yeast haploids. This is a tedious and time-consuming task, which can take at least 40 hours. To streamline the process, we designed a cellular system called Checkmate, which produces a colorful protein in response to pheromone secreted by cells of the opposite mating type. We hope it will simplify the process and reduce the time necessary to identify a haploid's mating type. The system uses a genetic circuit that is turned on when the MPRP of a cell is activated. Once turned on, a positive feedback mechanism will maintain production of the colorful protein, even after the MPRP shuts off. Typical mating type testing will be done by mixing Checkmate mating type detector cells with unknown haploids and examining them for a colorful response. Such a response indicates that the mating type of the unknown is opposite that of the Checkmate detector used in the test.</p><br /><br /> | <p>Yeast researchers must often determine the mating type of yeast haploids. This is a tedious and time-consuming task, which can take at least 40 hours. To streamline the process, we designed a cellular system called Checkmate, which produces a colorful protein in response to pheromone secreted by cells of the opposite mating type. We hope it will simplify the process and reduce the time necessary to identify a haploid's mating type. The system uses a genetic circuit that is turned on when the MPRP of a cell is activated. Once turned on, a positive feedback mechanism will maintain production of the colorful protein, even after the MPRP shuts off. Typical mating type testing will be done by mixing Checkmate mating type detector cells with unknown haploids and examining them for a colorful response. Such a response indicates that the mating type of the unknown is opposite that of the Checkmate detector used in the test.</p><br /><br /> |
Latest revision as of 03:53, 4 October 2012