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Team:RHIT/Project
From 2012.igem.org
Once the group agreed on a common purpose and principles, the team brainstormed ideas for the needs of the project. These ideas were then organized into categories based on similar themes. A few examples of the categories are team practices, mathematical modeling, presentation, laboratory, wiki, and gold criteria. Ideas were then flushed out by a group discussion, resulting in modifications to ideas’ category locations. This part served as the brainstorming and organization of Allen’s method.
Then, the team identified next actions for the project based off of the brainstormed ideas. Next actions are described as steps taken by the team to complete a specific portion of the project, and Allen’s method says that a project is only considered fully developed when all portions of the project have their potential next actions assigned. Once next actions were issued, all members of the team started to research how the task could be completed.
During the research phase, Devon found out that the team’s idea was previously attempted using a different method by the 2008 Johns Hopkins iGEM team. The team then switched gears into finding out what the Johns Hopkins team accomplished by examining their wiki, presentation, and poster. After learning helpful information about the project, the team decided to stick with their original idea.
Around the same time, research on possible pigment pathways, fluorescent colors, and promoters occurred. Robert then found the next big breakthrough on the project when he found a positive feedback loop in yeast cells form the work of Ajo-Franklin, Drubin, Eskin, et al. in their paper titled Rational Design of Memory in Eukaryotic Cells. Further research showed that many teams have used a very similar pathway in previous years of the iGEM competition, solidifying the part as a key component of the project design.
The next step in the process was finding DNA sequences that correlated to each component of the scheme. At this time, a modification from the initial layout occurred. All references and teams previously used a two-tiered system for the positive feedback loop, found in Figure 1. The team proposed to combine the two tiers into one, found in Figure 2.
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 Saccharomyces cerevisiae. 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 HIS3 plasmid.
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.
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.
As a secondary application of the project, two copies of this construct were implemented, each containing a different fluorescent protein. One of these constructs would eventually be integrated into the genome of one mating type of yeast. The purpose of this would be to allow for easy identification of the mating type of the unknown strain.
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.
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.
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.
