The work that has been done by iGEM in 2017 was centered on TRPV1 and its ability to import calcium into the cell in response to temperature changes, with the intention of one day using this as a method of In-Silico control of genetic circuits in synthetic biology. Our project explored the characterization of a calcium-sensitive reporter in Saccharomyces cerevisiae to establish the extracellular calcium concentration that would be optimal to regulate gene expression at the activation threshold temperature of TRPV1. Considering the native cytosolic calcium concentration ranges between 50 pM to 200 pM we worked on models that would predict our reporter gene expression under conditions near the native cytosolic concentration and far greater than the native calcium concentration. These models allowed us to decide on a low sensitivity promoter to activate our reporter genes known as PMC1 which we worked to construct in the wet lab.
We also built on work from previous iGEM teams to further develop a deterministic model for the calcium pathway in yeast that will activate our reporter genes. Additionally, we developed a stochastic model to predict the nature of ion channel gating in response to temperature change. These models will help guide future work with TRPV1 as a thermal actuator in yeast.
Several independent studies have shown that electromagnetic (EM) fields can non-invasively regulate blood glucose levels in mice and activate the stimulation of neuronal activity [8-10]. This stimulation has the potential to overcome the disadvantages of traditional input signals and offer a responsive and versatile In Silico control method for feedback regulation of cellular systems. The application of EM waves to stimulate gene expression, also known as magnetogenetics, currently relies on the tethering of a ferritin nanoparticle with an iron oxide core to a thermal sensitive cation selective channel of the TRP Family such as TRPV1 [11-14]. Although the known biophysical mechanism that allows for gating by the production of sufficient heating from ferritin nanoparticles in response to EM waves has received criticism [15, 16], there are efforts to explain this phenomenon . There is experimental evidence from independent studies [11-14] that provides empirical data, which demonstrates calcium influx from EM stimulation by using calcium fluorescence imaging and blood glucose regulation in mice via the activation of a calcium sensitive promoter that induces the expression of a synthetic insulin gene. Considering the possibility of TRPV1 as a thermal actuator for genetic circuit regulation, our team decided explore the optimal calcium influx conditions due to TRPV1 that could be used to regulate gene expression in S. cerevisiae.
Once TRPV opens and allows Calcium to enter the cell, the Calmodulin/Calcineurin pathway can be activated. The Calmodulin/Calcineurin pathway is a eukaryotic pathway dependent on calcium(II) influx. The pathway results in Crz1p acting as a transcription factor for various promoters. The promoter of interest is PMC1. The pathway begins in the cytosol, where calcium interacts with yeast calmodulin, which binds 3 calcium(II) ions before becoming active. Calmodulin then goes on to complex with calcineurin which activates its phosphatase activity. The newly formed complex dephosphorylates Crz1p, which then becomes active and is able to target the PMC1 promoter sequence. Transcription is then initiated.
It is important to note that PMC1 was chosen due to its relatively low affinity for the Crz1p transcription factor . This means that calcium influx must be enough to exceed homeostasis conditions in order to generate a response. The normal Calcium(II) concentration within yeast cells range from 50nm-200nm  which was taken into account during BioBrick and experimental designs.
The specific fluorescent proteins were chosen based on maturation time and brightness. We found in literature review that the maturation time for sfGFP is just shy of 6 minutes even in an environment with high calcium(II) content . mNeonGreen was chosen as a second reporter fluorescent protein due to its intensity upon maturation. The goal in picking these two proteins was to add parts to the registry that could be used with this pathway to provide rapid feedback to the synthetic biologist or a monitoring device. These reporter genes would be crucial in the characterization of parts that would be used to trigger this pathway. These two BioBricks were problematic during the build phase of the process. After optimization of the fluorescent gene sequence of the BioBrick using the IDT Codon Optimization Tool, we used the Genscript table to exchange codons contributing to illegal restriction sites for codons that were suboptimal. Since we were designing BioBricks with the PMC1 promoter, we could not fully optimize them as changing this sequence could have unforeseen consequences. This became a larger issue during gene synthesis since they contained sites that could not be modified where the GC content was too low.
â€” Experiments â€”
These characteristics were taken into account when we designed our experiments. The biggest implementation of the engineering design process was in this portion of our efforts.
Our primers for PCR amplification were designed using the added sequence for TspMI, but initially failed. The protocol we designed and tested used a suggested annealing temperature from the manufacturer, but it was to high. We went back to the drawing board and redesigned our protocol. We used a higher volume reaction so we could run more on our gel, and we created a program with a gradient to look for the best temperature for annealing. These modifications allowed us to pinpoint the proper annealing temperature on the next rounds of building and testing. We returned to the design phase to make the necessary modifications, build, and then test once more before upscaling and moving on to the next step of our project.
Our BioBricks are both nearly 2000 base pairs, and the shipping vector is 2070 base pairs. These sizes made the ligation step of cloning difficult for us. The original protocol we built for this used a roughly 1:1 ratio of vector to BioBrick. This proved ineffective after building and testing, so we returned to the design step. We decided on a higher ratio by increasing the concentration of our BioBrick, but halving the concentration of our vector. This new design was built and allowed us to obtain a higher ratio while maintaining the concentrations of our ligase and buffer. The test phase showed that the new protocol worked. We then took this and applied it to our expression vector, YEp352. After cloning, we attempted our yeast transformation. Our team did not have much experience with yeast so there was a steep learning curve. Our initial design of the protocol used one day old culture of BY4741 with a ccc1 knockout, but the transformation failed in the test phase. We examined the concentrations of our yeast using a Coulter Counter and found that the cultures were already in stationary phase, so we modified the protocol to only incubate for 12 hours in 5mL cultures. These were then split and given another two hours of incubation to ensure they were in the log phase of growth and that we had enough for the rest of the protocol.
All of these protocols that were designed, built, and tested can be found in the protocols section of the wiki.
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