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.
Two nearly identical constructs were planned by the RHIT iGEM team. The differences between them were in the specific fluorescent domain; one coded for a red domain and the other coded for a blue domain. Both constructs were synthesized by GeneArt using sequence information provided by the RHIT iGEM team. Due to some miscommunication, the red construct sequence was optimized for synthesis, during which several additional EcoRI sites were introduced. The team was unaware of this when the construct was received, so numerous attempts at digestion, ligation and transformation were made, all of which were unsuccessful. Due to the added EcoRI sites, the red construct DNA is unusable both as a BioBrick and in the intended system without extensive changes being made, which time and cost prohibit. The blue construct was not similarly changed, however.
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 used to engineer useful machines. This approach is being applied to produce various things, including insulin from bacteria and biofuels from algae. And it can be used to address many of the world's grand challenges, 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. 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.
Yeast researchers must often determine the mating type of yeast haploids. This is a tedious and time-consuming task, which can take 2-3 days. 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.
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