Overview
AstroYeast is the cellular component of the AstroYeast Microfarm system. The goal is to adapt a strain of Saccharomyces cerevisiae (baker's yeast) to be tolerant to microgravity induced stress, offering a reliable chassis for bioproduction in space. This is achieved by performing adaptive evolution experiments in a simulated microgravity environment.
The Problem
Our literature review brought us to the conclusion that gene expression in yeast was unpredictably affected in microgravity conditions, and that these changes were poorly understood (Sheehan, 2007). We also saw the lack of a standardized strain for microgravity research or chassis for bioproduction in microgravity conditions.
To create AstroYeast as a standardized chassis for space bioproduction, we needed to consider how yeast is affected in microgravity. The challenge was to overcome the unpredictable changes in phenotype when compared to control experiments here on Earth.
Yeast is affected by microgravity stress conditions and undergo cellular changes including:
- Abnormal, nonpolar or random budding directions. (Purevdorj-Gage, 2006)
- Cellular aggregates due to defects in cell separation processes (Purevdorj-Gage, 2006).
- Gene expression changes, including Stress Response Element (STRE) genes and Heat Shock proteins genes (Willeart, 2013).
- Activation of the Cell Wall Integrity and High Osmolarity Glycerol stress responsepathways (Willaert, 2013).
- Changes in growth rates and phases in some yeast strains (Willeart, 2013).
- Cytoskeleton alterations and changes in cell wall thickness (Willeart, 2013).
Figure 1: 1-5: Normal Budding, Earth control. 1g6-10: Abnormal Budding, Microgravity Conditions, 0g (Sheehan, 2006)
Figure 2: A: Microgravity, B: Normal Gravity Image Credit: NASA/JSC
We currently have many technologies for biomanufacturing applications here on Earth. A standardized microgravity-resistant AstroYeast strain, or chassis, would not only allow for these biological manufacturing advances to be easily transferred to a microgravity environment, they would also contribute to the progress of the research community, as experiments are performed on standardized and microgravity-specific Astroyeast strains.
Introduction
First, we inserted a fluorescent reporter into wild type BY4741 yeast, which served as a reporter that responded to stressors like microgravity. The functionality of the reporters was tested through exposure to various stressors. In these experiments, increased fluorescence was observed suggesting that the corresponding gene was upregulated. Second, we plan to evolve our yeast strain through adaptive laboratory evolution (ALE), which will mimic natural selection to adapt our yeast to a microgravity environment. To accomplish this, we are subjecting our yeast strains to simulated microgravity conditions with the use of simulators. For this, we built two simulators, a high aspect rotating vessel (HARV), and a 3D clinostat. The reporter consists of a promoter for a gene that has been seen to be highly misregulated in microgravity literature, GFP, and a high capacity terminator. These were inserted into the yeast using the CRISPR-Cas9 system. The insertion was assisted by the addition of homology arms to either end of the construct utilizing the yeast's endogenous homology directed repair mechanism. A total of eight strains, each with a unique promoter, were designed to monitor microgravity induced stress in different regulatory pathways. The change in the fluorescent signal of GFP will be observed over time. Once gene expression returns to its baseline expression, this suggests that the yeast has become tolerant to microgravity-induced stress. Once the yeast is tolerant, its efficiency as a chassis for bioproduction will be observed through the insertion of a gene that will produce a molecule of interest, such as limonene.
Methods
Reporter Design
Our reporter design follows a simple promoter, gene, terminator circuit. Our reporter is ENVY-GFP, a GFP variant that fluoresces at a higher intensity than the traditional GFP (Slubowski, 2015). The terminator chosen was that of the CPS1 gene as it is a high capacity terminator (Curran, 2013). To facilitate the addition of our construct with CRISPR, we added homology arms to either end of the construct. These come in the form of sequence 16 described in the paper (Flagfeldt, 2009). These two traits combined allow us to have a higher definition of reporter. These parts of the construct are the same for all of the reporters that we have tested. The reporter system described above was constructed using yeast homologous recombination.
Figure 3: Homologous Recombination
To select genes of interest, for which our promoters were selected from promoterst, we used the AstroBio database, developed by our team last year (visit the wiki). This allowed us to see what genes were upregulated the most in microgravity. With this data, we then selected the most upregulated from several pathways such as heat shock, osmoregulation, ethanol stress, oxidative stress, and nutrient signalling. From this, we then searched the GEO2R database for visualization of the raw data, namely from the paper Yeast genomic expression patterns in response to low-shear modeled microgravity by Sheehan et al. to see which ones tended to be consistently upregulated. This gave us the following reporters to be tested in the lab. We tested each reporter, as we wanted to assure our reporter mimicked the expression of the native genes by expressing under the same conditions.
Figure 4: Reporter Design
Strain Improvement
Transformation
To develop our strain of microgravity tolerant yeast, a reporter gene was first inserted into yeast. The reporter gave us the ability to observe and quantify the yeast's response to the environmental change, with the goal of allowing us to see when the yeast is no longer stressed out by microgravity. The reporter's ability to track stress response was verified through experiments where the yeast was subjected to various stress inducers. The fluorescent reporter was designed to mimic the expression of key proteins related to microgravity stress responses selected using our AstroBio Database, our iGEM project from last year.
To insert the reporter gene, we used a CRISPR-Cas9 system. Our team transformed a plasmid encoding Cas9 and a single guide RNA (sgRNA) designed to target a chosen genomic locus with PCR amplified promoters, terminators, and GFP envy. The promoters used for the reporters were duplicates of promoters that already exist in the cell to allow for its function to continue. The insertion sequence was sequence 16 described by Flagfeldt et al (Flagfeldt, 2009). With CRISPR, a designed single guide RNA (sgRNA) traveled along the yeast DNA searching for a corresponding sequence to bind. When found, the sgRNA clamps onto the DNA and activates the Cas9 nuclease opening the DNA for our inserts.
To perform the experiment, we first cultured an E.coli stock which contained the Cas9 plasmid provided by Dr. Vincent Martin's Lab. The culture was then DNA mini prepped to isolate and purify the plasmid from E.coli. Following this, the plasmid was digested via a two hour restriction enzyme digest, the purpose was to prepare for our custom sgRNA sequence to be inserted. The next step was to perform the sgRNA insert assembly PCR reaction completed with Q5 polymerase, the primers used were the same flagfeld codons used to insert our reporter. Finally a transformation protocol was completed for our digested plasmid, sgRNA, and other components of our reporter system.
Confirming Transformation
The next step was to confirm that the promoters were transformed into the yeast cells successfully. This process first involved plating the transformed yeast onto an agar plate containing the antibiotic G418 (Geneticin). G418 resistance was conferred using the resistance gene on the plasmid which was used to insert the Cas9 gene. This means that any cells that had the plasmid inserted would be resistant to G418. However, just because a cell has the plasmid, it does not mean that it has our reporter system inserted. While the G418 serves to reduce the colony count on the plate, a colony on the plate cannot be said to carry our reporter. To screen for cells containing our reporter, colony PCR was used.
Colony PCR was used as a screening method to determine if a colony contains the construct that was inserted, by amplifying our construct directly from cells (Bergkessel, 2013). For colony PCR, cells were first lysed by boiling them in sodium hydroxide (NaOH) in order to remove the DNA from the cells, then put into a thermocycler for PCR to amplify the DNA sequence of interest, and results were confirmed by gel electrophoresis. If the colony was positive for the transformation, they were then restreaked onto a new agar plate containing no antibiotics. The colony PCR was repeated again in order to have a total of two restreaks in order to resolve for our transformed strains (overall process shown in the figure below).
The purpose of restreaking was two fold. First, the plasmid containing Cas9 was removed in order to prevent further genome editing. Having an antibiotic free plate eliminated the need for the Cas9-carrying plasmid for growth. Second, restreaking allowed us to resolve mixed colonies through further dilutions on plates, ensuring that there were little to no cells without our reporter system.
At the end of this process, we obtained eight strains of yeast, all transformed with the correct constructs, without mixed colonies and with the Cas9 system removed.
Figure 5: The Overall Restreaking Process - which was used in order to resolve the final transformed strains.
Figure 6: Sample Pictures Of Our Agar Plates - showing that the transformed colonies are fluorescent. containing (1) transformed cells, (2) transformed cells after first restreak, and (3) after second restreak, which was used as the final strain.
Strain Selection
After successfully transforming eight strains of yeast, the next step was to determine which one would be the best candidate as a reporter strain. Ideally, we would want to see a strong and consistent change in GFP fluorescence intensity caused by microgravity induced stress. Before subjecting the yeast to simulated microgravity, we needed to evaluate the effect of well known stressors on fluorescence intensity under normal gravity conditions. By doing this, we will be able to see how responsive our yeast strains are to different stressors. With knowing how each strain responds, we can select the one with a strong and consistent change in fluorescence intensity as our reporter strain.
In order to evaluate the fluorescence intensity of each strain, we performed a series of experiments in which yeast was put under different stress conditions. The stressors included ethanol, salt, peroxide and heat. After subjecting each strain to each stressor for one hour, the mean population fluorescence was measured using flow cytometry (Xiong, 2018). These results are shown in the graphs below (fig. 1 and 2).
From these experiments, we were able to evaluate how each strain responded under each stressor. We observed that the strain containing the SOD1 promoter was the best candidate to use as a reporter strain, because in all four stress conditions, there was a significant change in fluorescence intensity as compared to the control. Since SOD1 was the most responsive to the tested stressors, it was the most likely to respond to microgravity induced stress. Therefore, SOD1 was selected as the reporter strain to be used in our evolution experiments.
Figure 7: The Fold Change Of The Mean Population Fluorescence Of The Stress Condition Compared To The Corresponding Control - all samples were exposed to each stressor for one hour At 30 oC (with the exception of heat stress, which was at 42 oC), and the mean population fluorescence was quantified using flow cytometry. Results are shown as mean ± standard deviation. *p<0.05, **p<0.01, ***p<0.001, all with an increase in fold change, #p<0.05, ##p<0.01, all with a decrease in fold change. All statistical tests were done using a t-test.
Figure 8: The Fold Change For HSP30 - all samples were exposed to each stressor for one hour at 30 oC (with the exception of heat stress, which was at 42 oC), and mean population fluorescence was quantified using flow cytometry. Results are shown as mean ± standard deviation. **p<0.01, ***p<0.001, all with an increase in fold change. All statistical tests were done using a t-test.
We performed stress experiments similar to the previous ones but using simulated microgravity provided by our 3D clinostat. The goal of this was to confirm our conclusions from the previous stress experiments, as well as evaluating each strain's ability to report microgravity induced stress. For this, we grew our strains in the 3D clinostat for 24 hours and then measured their mean population fluorescence using flow cytometry. From these experiments, we found that SOD1 may not be the best reporter for microgravity, seeing as it does not have any significant upregulation. However, we found that both HSP30 and SAF1 are upregulated under microgravity conditions, suggesting that they should be investigated further to see if they would make good microgravity reporters.
Figure 9: The Fold Change Of The Mean Population Fluorescence Of The Yeast Strains Grown In Simulated Microgravity (Clinostat) Compared To The Normal Gravity As A Control - all cells were grown for 24 h at 25 oC, and the mean population fluorescence was quantified using flow cytometry. Results are shown as mean ± standard deviation. *p<0.05, all statistical tests were done using a t-test.
Directed Evolution
We look forward to using adaptive laboratory evolution (ALE) to mimic the power of natural selection in laboratory settings to acquire an improved strain of yeast which thrives under microgravity conditions. In adaptive laboratory evolution, the microorganism is propagated for a certain period of time under the selective pressure of microgravity stressors to accumulate advantageous mutations that lead to increased fitness of the cultivated strain (Dragosits, 2013).
We will be assessing strain improvement by looking at the expression of our GFP reporter under microgravity conditions. The assumption is that initially, microgravity will be a stress condition for yeast and it will lead to upregulation of GFP. We are aiming for a viable population of yeast (through acquired and accumulated mutations) that have adapted to microgravity, and no longer exhibit upregulation of GFP. We will also introduce mutagenesis, either chemically or via UV, in order to increase the genetic diversity of the population in order to speed up the process of adaptive evolution.
Figure 10: Directed Evolution
We have developed and constructed two microgravity simulators for these experiments- the 3D Clinostat and the High Aspect Ratio Vessel (HARV). Each microgravity simulator has its own advantages and we are excited to compare experimental results from each machine. In both cases, dilutions of the culture are necessary throughout the simulation to assure the yeast cultures remain in the exponential, or log, phase. This is important as we want to assure they do not attain the stationary phase where undesirable adaptations can occur (Dragosits, 2013).
Figure 11: 3D Clinostat
The 3D-Clinostat, shown above, has the advantage of holding a 96-well plate. This allows us to perform experiments on many samples at the same time, as well as to measure their fluorescence quickly. The microgravity simulation is temporarily stopped to take fluorescence measurements and for dilutions to occur.
Figure 12: HARV Bioreactor
The HARV Bioreactor (High Aspect Ratio Vessel Bioreactor) simulates Low Shear Simulated Microgravity (LSSMG) as means to introduce microgravity on cell cultures (Huang et al., 2018). Performing evolutionary experiments in this microgravity simulator allows for continuous, non-stop evolutionary experiments. The HARV was designed with syringes attached to the rotating bioreactor which allow for dilutions to be performed without stopping the microgravity simulation. The advantage is that we can run experiments up to two weeks, including many generations of adaptation to the microgravity stress. In addition, the HARV is fitted with a fluorometer, which allows us to observe real-time fluorescence, or genetic expression, changes in our AstroYeast strains in the future.
Proof of Concept
To prove that our AstroYeast strain works, a proof of concept was developed. The end goal of the project is to engineer a strain of yeast which produces nutrients such as vitamins or flavor molecules. The yeast strain engineered would be less affected by microgravity induced stress than the wild type, allowing it to thrive in microgravity. Our project tried to simultaneously address the issue of space food being less palatable, or bland as described by the Canadian Space Agency though their Deep Space Food Challenge, by producing the lemon flavor molecule, L-limonene, in our AstroYeast Microfarm. As a proof of concept, L-limonene will be inserted into yeast and grown to optimal amounts in our bioreactor. We used restriction enzyme cloning to created the system used by our yeast to produce l-limonene.Primers were designed to amplify the gene of interest, L-limonene synthase, which will be inserted into our yeast vector, PYES2 plasmid, as it has all the characteristics to work in our yeast strain such as native promoter (GAL1) and terminator (CYC1). Yeast has intrinsic production of Geranylpyrophosphate (GPP) which can then be converted to Limonene by L-limonene synthase (Jongedijk). Due to time constraints, this was not able to be done by our team this year, but if given the chance to take the project further, it will be a priority.
Figure 13: Mechanism of Geranylpyrophosphate (GPP) Becoming L-limonene
Further Investigations
With continued improvements, AstroYeast will be able to biomanufacture an abundance of different nutrients in microgravity. This technology will be useful for astronauts and long term space travel as there is currently no way to overcome the nutritional needs once reserve supplies are depleted. With AstroYeast, we will be able to overcome the extreme environments of outer space, as the AstroYeast Microfarm system biomanufactures not only nutrients for survival but also fuels, materials, and even medications that are needed for the future of space exploration. With AstroYeast, human travel to Mars and beyond becomes one step closer to reality.
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