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The creation and transformation into a budding yeast vector of β-synuclein 1-60 and 1-74 truncation variants for Parkinson’s disease research
Kevin Duarte, Blakely Drake, and Ryan Oliver
Lake Forest College
Lake Forest, Illinois 60045
Neurodegenerative diseases are becoming more prevalent as people continue to live longer. Parkinson’s disease (PD) is one of the most common of these diseases and is characterized by both motor and nonmotor complications. It is known that one of the primary causes of Parkinson’s disease is the aggregation of the protein α-synuclein in the brain. However, there is much less known about the effects of β-synuclein in Parkinson’s disease. In order to gain a deeper understanding of Parkinson’s disease, we aimed to create truncated fragments 1-74 and 1-60 of β-synuclein which would then be purified and placed into a budding yeast model. We hypothesized that these truncations would be properly created and transformed into yeast for further experimentation. For our experimentation process, we aimed to (1) design and create two β-synuclein gene variants through the use of PCR, (2) isolate and purify the truncation variants by gel electrophoresis, (3) subclone the variants into a plasmid vector, (4) transform those vectors into bacteria, (5) check and confirm correct gene fragment orientation in vector within bacteria, and (6) transform the plasmid vectors into yeast. By completing each of our aims, we were able to successfully obtain yeast expressing our truncation variants. Due to this successful transformation, future studies will be conducted in order to determine β-synuclein’s role in Parkinson’s disease.
With life spans increasing over the ages, neurodegenerative diseases are becoming more and more prevalent due to age being a major risk factor (Holtzman et al., 2015). Currently, Parkinson’s disease (PD) is the second most common neurodegenerative disease in the world (Alshehri, 2017). It is a progressive degenerative disorder associated with motor and nonmotor complications such as motor fluctuations, dyskinesias, psychosis, falls, infections, and complications of immobility (Paul et al., 2019). Parkinsonism is an umbrella term used to describe the collection of signs and symptoms found in Parkinson’s disease (PD), but there are seven types: idiopathic Parkinson’s disease (most common), drug-induced parkinsonism, vascular parkinsonism, normal pressure hydrocephalus (NSA), corticobasal degeneration (CBD), progressive supranuclear palsy (PSP), and multiple system atrophy (MSA) (“Types of Parkinson’s Disease”).
The characteristic motor symptoms of PD are a result of the midbrain dopamine neurons in the substantia nigra being lost to degeneration, but PD is a disorder of more than just the motor system (Alexander, 2004). Since intraneuronal Lewy body inclusions and a reduced number of surviving neurons—a key part of PD—are similarly represented in each of the targeted neuron groups, this suggests that the neurodegenerative process is the same for each system affected by the disease (Alexander, 2004). The underlying cause of neuron loss differs based on the form of PD, but we want to focus on familial PD, which is caused by mutations in the following genes: LRRK2, SNCA, PARK7, PINK1, or PRKN (“Parkinson disease,” 2019). While these genes are important, there are other gene mutations associated with familial PD as well (Ross et al., 2008).
Since mutations in the α-synuclein gene have been linked to familial Parkinson’s disease, many studies have been conducted to see how the aggregation of α-synuclein may play a central role in the pathogenesis of PD (Sekigawa et al., 2015). Lewy bodies, which are primarily composed of α-synuclein, have been shown to accumulate heavily in PD patients, suggesting that they play a role in neuronal death (Leitao et al., 2018). Further studies have suggested a possible solution to this problem. β-synuclein, another protein in the synuclein family has been suggested to play a protective role in PD pathology by inhibiting the aggregation of α-synuclein (Brown et al., 2016). Despite this suggestion, the role of β-synuclein is still widely unknown by the Parkinson’s community. Although β-synuclein may be helpful, other studies have revealed that under the right conditions, it will also fibrillate into a potentially neurodegeneration-inducing protein like the others associated with PD (Taschenberger et al., 2013).
Since there is no definitive proof that β-synuclein is either helpful or harmful, it is important that its role be further analyzed within the context of PD. Like α-synuclein, β-synuclein is composed of three domains: an N-terminal amphipathic domain, a central hydrophobic core domain known as the non-amyloid component (NAC), and an acidic C-terminal domain (Landau, 2017). Studies have revealed that β-synuclein fibrillization is regulated by all three domains, but each in different ways (Landau, 2017). By analyzing the effects of truncated versions of this protein, we can determine the impact that each individual part has on the progression of PD.
One model organism that is good for studying Parkinson’s disease is the yeast, Saccharomyces cerevisiae due to its short generation time, easy growth, and readily manipulated eukaryotic cell (Allendoerfer et al., 2008). Because its genome is relatively short and fully sequenced, it is relatively easy to analyze the different genes within it, and most of its genes are like those of human proteins with nearly half having clear human counterparts (Allendoerfer et al., 2008). Since there are many similarities in basic cellular mechanisms such as protein quality control, vesicle recycling, and mitochondrial function between yeast and other eukaryotes, yeast has been used as a model for learning about human disease (Amen et al., 2015).
So far, studies have shown that α-synuclein affects yeast biology in ways similar to how it affects neurons, suggesting that yeast is a good model for studying the effects of α-synuclein on Parkinson’s disease (Allendoerfer et al., 2008). It has revealed that overproduction of α‐synuclein causes stress on the endoplasmic reticulum (ER), lowers the capacity of cellular degradation, and affects vesicle trafficking from the ER to the Golgi (Amen et al., 2015). Because yeast has already revealed so much about α-synuclein, our hope is that it will also be a good model for the effects of β-synuclein.
The purpose of our project is to create truncation variants of the Parkinson’s disease related β-synuclein gene by making forward and reverse primers for 1-74 and 1-60, which will then be introduced (sub-cloned in DNA into vector) into a budding yeast vector. We hypothesize that these β-synuclein mutants will be properly created and transformed into yeast so that further experimentation can be carried out in the future. For our experimentation process, we aim to (1) design and create two β-synuclein gene variants through the use of PCR, (2) isolate and purify the truncation variants by gel electrophoresis, (3) subclone the variants into a plasmid vector, (4) transform those vectors into bacteria, (5) check and confirm correct gene fragment orientation in vector within bacteria, and (6) transform the plasmid vectors into yeast.
By carrying out these experiments, our goal is to create truncation variants of the Parkinson’s disease related β-synuclein gene and provide a research tool for the Parkinson’s disease community. We hope that by providing this tool, the community will receive a clearer understanding of Parkinson’s disease through the interactions of β-synuclein with yeast. If β-synuclein’s role in PD is more clearly understood, that means researchers are one step closer to finding a cure, or at least some way of improved treating of Parkinson’s disease.
Overall Project Design and Gene Fragment Amplification by PCR
We hypothesized that we would properly create and transform β-synuclein mutants into yeast so that further experimentation can occur in the future. To test our hypothesis, we first needed to obtain β-synuclein mutants. Therefore, we created two unique truncation variants of the Parkinson’s disease β-synuclein gene by performing gene amplification using PCR. When comparing the wild-type (WT) β-synuclein fragment to our fragments, we predicted that the resulting proteins from our fragments will have different folding and binding properties than its wild-type counterpart, resulting in either more fibrillation of β-synuclein or less fibrillation of α-synuclein in yeast (Figure 1A). To do these truncations, the following steps were followed as shown in our experimental design schematic (Figure 1B). First, we needed 3 PCR primers: a N-terminal GFP forward primer, a 1-74 β-synuclein reverse primer, and a 1-60 β-synuclein reverse primer (Figure 1C). After amplifying our gene fragments using PCR as described in the plasmid-based PCR method, we continued with the next steps of our project.
Gene Fragment Purification by Gel Electrophoresis
After amplifying our gene fragments using PCR, we then needed to separate, identify, and purify the fragments using gel electrophoresis as described in the PCR purification method. We had an idea of what an ideal gel would look like before starting the purification process, with a band around .934 kbp for our 1-74 fragments and a band around .892 kbp for our 1-60 fragments (Figure 2A). Our original gel with the PCR product was photographed for analysis; it displays multiple bands for our 1-74 fragment lanes and for our 1-60 fragment lanes. Bands are observed for the 1-74 fragment lanes at a little below 1 kbp and bands are observed for our 1-60 fragment lanes just a bit below the 1-74 bands (Figure 2B). After the original analysis, our gel was photographed again, but this time it was missing the part containing the DNA. Analysis of the cut gel confirmed that the gene fragment was properly cut out of the gel (Figure 2C). The DNA extracted from our cut gel was used by the peer teacher to make a primer check gel. Analysis of the primer check gel displays bands at .9 kbp. The band at .9 kbp is for fragment 1-74. No band appears for fragment 1-60 (Figure 2D). The rerun of the gene fragment purification gel resulted in the second lane showing a band at .8 kbp for fragment 1-60 (Figure 2E).
Subcloning into Plasmid Vector and Bacterial Transformation
After purifying the fragments using gel electrophoresis, we then subcloned the fragments into a plasmid vector and transformed the plasmid into bacteria as described in the vector subcloning and bacterial transformation method. After bacterial transformation, growth was observed in all the plates; however, more growth was observed in the plates with 200 µL of the fragments. The plates with 200 µL of the fragments showed more colonization than the plates with 30 µL as well (Figure 3A). The positive control shows some cell growth and colonization while the negative control does not display any cell growth or colonization (Figure 3B).
Orientation Check: Bacterial & Plasmid PCR Gel Electrophoresis
After bacterial transformation, we ran our PCR products through gel electrophoresis; half were in purified plasmid as described in the plasmid purification method and the other half were whole cell PCR as described in the bacterial whole cell PCR method. A peer teacher verification gel of the purified plasmids was first analyzed, showing bands between 4 and 3 kb (Figure 4A). Before running the gel electrophoresis, we had an idea of what the ideal gel result would look like for fragment 1, 1-74 (Figure 4B) and fragment 2, 1-60 (Figure 4D). Our image from the gel electrophoresis was missing bands for fragment 1-74 (Figure 4C). Our gel electrophoresis was also missing bands for fragment 1-60 (Figure 4E). Due to the lack of bands, new reverse primers were used to redo the experimentation process. The redo gel from this process displayed bands for fragments 1-74 and 1-60, which were observed at around 1 kbp, the expected size (Figure 4F).
The DNA sequencing was done by the University of Chicago for fragment 1-60 and fragment 1-74. For fragment 1-74 mini prep 3 and 4 were used, while for fragment 1-60 only mini prep 2 was used. Fragment 1-60 showed the expected sequence while fragment 1-74 displayed a changed sequence (Figure S1).
Plasmid Vector Transformation into Yeast
After isolating the plasmid vector with our DNA fragments from E. coli, we wanted to transform the plasmid into yeast cells as the final step of our project. After about five days, images were taken to display the growth of the colonies for the positive control (Figure 5A) and negative control (Figure 5B) YPD broth. There was also growth for the 1-60 fragment (Figure 5C) and 1-74 fragment (Figure 5D) in the YPD broth. In the SC-URA broth there is one plate that had the positive control, which displayed cell growth (Figure 5E) and one plate with the negative control, which did not display any cell growth (Figure 5F). Two plates had cell with the 1-60 fragment transformed into them. One plate had less of the 1-60 fragment (Figure 5G) and the other had more of the cells with the 1-74 fragment (Figure 5H); both plates displayed growth. The last two plates contained cells with the 1-60 fragments. One had less of the 1-74 fragment, and it displayed growth (Figure 5I) and the other had a larger amount of the 1-74 fragment, and it didn’t display growth (Figure 5J). Major growth was only observed in the plates with the YPD broth. Plates with the SC-URA medium either did not show much or any growth.
The goal of project was to create and transform 1-74 and 1-60 β-synuclein gene truncations into yeast for future Parkinson’s disease research. The first aim of our project was to isolate and purify two different β-synuclein gene fragments from the original plasmid vector. When isolating and purifying the selected fragments by gel electrophoresis, we expected to see bands for the 1-74 fragment at 934 base pairs and we expected to see bands for the 1-60 fragment at 892 base pairs. Strong and clear bands appeared at these expected sizes, which indicates that these truncations were successfully formed. Because the 1-60 fragment appeared lower on the gel than the 1-74 fragment, it was confirmed that the 1-60 fragment was shorter than the 1-74 fragment. Also, since our gel only yielded one band in the 1-74 fragment lane, it can be inferred that our 1-74 gene fragment was purified successfully, especially since it was located at the expected location.
After isolating and purifying our β-synuclein gene fragments, we next aimed to insert these fragments into an open plasmid vector and transform them into E. coli. After inserting them into E. coli, large colonies appeared in the LB+AMP plates. This indicates that the DNA was properly inserted into the YES2 vector. This vector contains Ampr which is a gene that gives E. coli the ability to grow in these plates. The bacterial colonies that survived must have the vector and the fragment.
Even with the observed growth of the bacterial cells, we were still not be able to be sure if they were correctly oriented because you cannot observe orientation by looking at the colonies alone. So, in order to confirm the orientation of the cells, we had to rely on GAL 1-FP. This is the promoter of the vector used in the transformation. If our DNA was oriented properly, then the eGFP reverse primer and GAL 1-FP would end up binding in opposite directions, and the PCR would end up amplifying the DNA inside the bacterial cells. If the DNA was not oriented properly, then gene amplification would not occur because the primers would be going in the same 5’ to 3’ direction which in turn makes amplification impossible. By running the gel electrophoresis of the whole-cell and plasmid PCRs, we expected to observe bands at specific locations to confirm properly oriented vectors. However, in our gels no bands seemed to show up. This would indicate that none of the vectors were properly oriented.
We were not the only group to obtain no bands; none of the groups in the class observed bands. After further analysis, we came up with possible reasons for why no bands appeared on the gels. These possible reasons are that the primer may have been wrong which would then lead to it not being able to amplify itself, or there may not have been enough DNA initially which would lead to it not showing up on the gel. Since we did not observe any bands, Yoan, Dr. Bottero, and Niam went back and redid this process with new reverse primers. After redoing the process, the new gel displayed bands for 1-74 and 1-60 of the PCR orientation check at the expected lengths.
After checking that our PCR products were correctly oriented, our DNA was isolated and sent off for sequencing at the University of Chicago DNA Sequencing & Genotyping Facility. Our sequencing results show that fragment 1-60 was properly sequenced meaning we properly truncated the 1-60 fragment from β-synuclein. However, we had to borrow mini prep 3 and 4 from group 3 for our fragment 1-74 sequencing to be done. The sequencing came back for this truncation showing that there was a portion of the sequence that did not belong to this truncation. This means that along the way our DNA was contaminated, but that doesn’t mean the whole fragment is messed up.
The final aim of our project was to transform the plasmid including our desired β-synuclein into yeast. When we inserted our DNA fragments into yeast cells, we found that there was ample growth for all the cells within the YPD medium while there was little or no growth for the cells within the SC-URA medium. This makes sense because all yeast grow on YPD but only yeast with the plasmid correctly transformed into it can grow on SC-URA. This is due to the yeast needing uracil to survive. Because the SC-URA plates don’t contain uracil, the yeast need to obtain uracil from the plasmid. The only plate that did not have any growth was the SC-URA plate in which we added 200uL of the cells containing the 1-74 fragment. This could be from either the cells dying from a type of contamination, the cells in the broth not successfully having the 1-74 fragment transformed into them, or there not being yeast cells in the solution which was added to the plate. The fact that the positive control for SC-URA showed growth and the negative control for SC-URA did not suggests that we correctly followed clean aseptic technique. However, because the growth for the SC-URA plates was minimal and doesn’t look like the YPD plates, we suspect that our β-synuclein truncation variants were not successfully transformed into yeast and the visible growth is due to contamination. This means that the process will have to be redone in order to obtain truncation variants that can be studied in yeast.
As mentioned previously, transforming these β-synuclein truncation variants into yeast is only the first step in a larger scale research project that will be carried out over the summer. The goal of this project was to create β-synuclein truncation variants that can then be analyzed in yeast to determine the role it plays in Parkinson’s disease. While we will not be involved in the larger research project, we do have a few ideas involving analysis of these gene fragments in the future. Since β-synuclein has been linked to PD, we suggest a series of assays be performed on these mutated yeast cultures. First, we suggest using a protein assay to help determine the specific concentrations of the β-synuclein protein in the yeast cells. This would be done as a preliminary analysis on the yeast (Martinez, 2019). Another assay we suggest is using serial dilution spotting to assess toxicity by comparing the growth levels of each truncation (Duennwald, 2012). Fluorescence microscopy can be used to track the localization of the β-synuclein protein. This can be done by taking advantage of the eGFP tag attached to the truncations (Rizzo & Davidson, 1970). Western blotting is a technique that can measure relative expression levels for each of the truncations. This could be used to figure out which amino acids correlate to observed phenotypes. The lost properties would be attributed to the amino acids lost in the truncations. We believe that it may be interesting to even look into what would happen if you were to co-express β-synuclein with α-synuclein. There have been studies that suggest that β-synuclein can moderate the effects of the aggregation of α-synuclein.
Parkinson’s disease is one of the most common neurodegenerative diseases. This disease is caused by the misfolding of proteins and the aggregation of these proteins in the brain. In order to gain more insight on how the protein misfolding occurs, we wanted to successfully create β-synuclein truncations and transform them into yeast. Specifically, we focused on fragments 1-74 and 1-60 of β-synuclein. We wanted to do this in order to better understand the properties of β-synuclein, but our yeast transformation failed. Hopefully future experiments will be done to obtain modified yeast that can be studied.
Materials & Methods
Primer Design and Synthesis
Primer design and synthesis was done as described (DebBurman, 2013). In order to design the forward primers, we used wild type β-synuclein as the template. For the 1-74 primer, we began counting at the 1st amino acid of the WT β-synuclein and counted out nucleotides from there. For the 1-60 primer, we also began counting nucleotides from the WT β-synuclein; however, this time we counted out less nucleotides. The forward primers then will bind to the template strand (3’-5’) so that the sequence would be the same as the (5’-3’) strand for WT β-synuclein. The WT DNA had no stop codon making it possible to fuse with the eGFP. The reverse primer was done by counting out nucleotides in the reverse direction from the 5’-3’ strand of the eGFP. The stop codon was excluded because the reverse primer will bind to the coding strand. We then took the complements for each of the nucleotides.
Template DNA, Subcloning Plasmids, and Bacterial and Yeast Cells Used
The template DNA used was wild-type-β-synuclein-eGFP which was isolated from E. coli. This was done as described (DebBurman, 2013). We used this template for both of the truncations. For subcloning, the truncated DNA samples were put into TOPO pYES2 vectors which will work for E. coli. This vector contained the Ampr gene and the Ura3 gene. These genes allow for E. coli to grow in presence of ampicillin and for yeast to grow without uracil. The E. coli cells used for bacterial transformation were TOP10 OneShot E. coli cells. The cells used for the yeast transformation were S. cerevisiae cells of the BY4741 strain (DebBurman, 2013). Yeast were plated on YPD and SC-URA plates, while bacteria were only plated on LB+AMP plates.
Amplification of the gene fragments was performed by plasmid-based PCR as described (DebBurman, 2013). Due to frigid weather, the lab session had been cancelled. The PCR reactions were performed by Dr. Bottero and the peer teacher, Niam. Each PCR reaction will contain the forward and reverse primers, the template DNA, the mastermix (Taq, MgCl2, dNTPs) and dH2O. The PCR was performed in 3 main steps which include preparation for the reaction, preparing positive and negative controls, and finally running the PCR reaction. In preparation for the reaction, 0.2 mL PCR tubes were labeled appropriately and placed in a benchtop ice bucket. Then, a series of ingredients were pipetted into the PCR tubes to a total of 100 μL of product.
For the positive control, the template and primers used were:
For the positive control, the control is beta-synuclein template along with beta synuclein forward primer and beta synuclein reverse primer It will amplify the full-length beta synuclein.
Finally, the PCR reaction was run by Dr. Bottero and Niam, the peer teacher. In order to run it, the PCR tubes were placed into the PCR machine and then was programmed to run the following: 95°C for 30 seconds. Then at 55°C for 30 seconds, followed by 72°C for 30 seconds. Finally after the previous conditions were ran 29 times, the machine was ran at 72°C for 30 minutes before being stored in a freezer at -4°C until ready to be purified by gel electrophoresis.
Gene fragment purification was performed by gel electrophoresis as described (DebBurman, 2013). The gel was prepared by combining 0.3 g of Agar powder and 40 mL of 1X TAE Buffer solution into a 250 mL Erlenmeyer flask. The flask was then swirled until the agar dissolved before being placed into a microwave for 1 minute of heating (brought solution to a boil). Flask was then moved to hood for the addition of 1 μL of Ethidium Bromide. With the agarose solution complete, it was then poured into a properly aligned gel casting tray. Gel was then left to solidify for 20 minutes before being lifted and aligned to face the nodes of apparatus. Enough IX TAE solution was then added into apparatus to touch top of gel but not go over it. Finally, the comb was lifted to expose wells, and gel was ready for sample loading.
After preparing the gel, it was loaded using a 200 μL pipet as the following describes. 10.5 μL of the 10X loading dye was added to the 100 μL PCR sample, and 50 μL of this mixture was loaded on the desired gel lane. 4 μL of the 2000 bp DNA ladder plus 9.5 μL of water and 1.5 μL of 10X loading dye were mixed to create a 15 μL sample of the ladder that was then loaded on the first lane of gel.
After loading the gel, it was then running for around 10 minutes before more 1X TAE buffer was added to cover the gel. The gel electrophoresis was then running for about another 30 minutes so that the dye was at least one inch from the end of the gel. Apparatus was then turned off and the gel was removed for imaging by the molecular imaging system (Image Lab 4.0.1). After the initial imaging, the DNA was extracted from the gel by using a sharp scalpel, and the gel slab with missing DNA was imaged again.
The PCR product was then purified as described (DebBurman, 2013). 100 μL of turbo salt solution was added per .1 g of gel slice to a tube containing the gel slice. The tube was then incubated at 55°C for 10 minutes (gel slice was completely dissolved). We then transferred 600 μL or less of the gel and turbo salt solution to a turbo cartridge placed within a 2 mL catch tube for 30 seconds of centrifugation at 13,000x g. Catch tube was then emptied of liquid. Afterwards, 500 μL of turbo wash/ethanol solution was added and centrifugation at 13,000x g occurred for another 30 seconds. The catch tube was emptied again, and centrifugation at 13,000x g occurred for 4 minutes. Detachable cap was then removed from a new turbo catch tube and set aside. The turbo cartridge containing the bound DNA was inserted into the new catch tube, and 30 μL of turbo elution solution was added directly onto the membrane. Then incubated for 5 minutes at room temperature before centrifuging at 13,000x g for another minute. Catch tube was capped, and purified PCR product was ready for cloning.
Vector Subcloning and Bacterial Transformation
Vector subcloning was performed by creating reactions in 1.5 mL microcentrifuge tubes as described (DebBurman, 2013). Each tube contained 4uL (microliters) of their respective purified PCR product, 1uL of salt solution and 1uL of TOPO Vector. The negative control used the same formula except with 1uL of water instead of TOPO Vector. Each reaction was gently mixed and incubated for 5 minutes at room temperature. After the reaction was done 2uL of the cloning reaction solution and Chemically competent E. coli into a different TOP10 One Shot. A negative and positive control were done by the peer teacher. Negative control used 2uL of water instead of the TOPO cloning reaction, and the positive control has 2uL of control plasmid given by the peer teacher. The reaction tubes were then placed in ice for 5 minutes before being heat shocked for 30 seconds in water that’s heated to 42° Celsius, then immediately returned to ice. 250uL of SOC medium was then added to each tube, the tubes were then capped and shaken horizontally for 60 minutes. After being shaken, 30 and 200uL of each transformation was spread on pre-warmed LB ampicillin plates (one plate had 30uL and another had 200uL, for each transformation totaling 4 plates with transformation spread onto them). Plates were then incubated overnight at 37° C.
Bacterial Whole Cell PCR
Bacterial whole cell PCR was performed by using cell colonies from our growth plates as described (DebBurman, 2013). They were labeled 5-8 to know which sample was used where in the following gel electrophoresis. The PCR preparation required LABELED 0.2mL PCR tubes. Each of the PCR tubes contained 25uL of Master Mix, 22uL of Sterile RNase-free H2O, 1.5uL of forward primer, 1.5uL of reverse primer, and a very small amount of each cell colony dabbed from the growth plates. Each tube only had 1 labeled colony in the reaction, and the cell colony samples were acquired using a sterile toothpick to transfer the culture. A Negative control was made by simply not adding a cell colony sample to one PCR tube. By the end there were 9 PCR tubes, 4 to be used with the 1-60 fragment, 4 to be used for the 1-74 fragment, and the control. The 9 tubes were then loaded onto the PCR machine just like it was done for the purified plasmid PCR. The program ran 95°C for 30 seconds. Then at 55°C for 30 seconds, followed by 72°C for 30 seconds. Finally, after the previous conditions were ran 29 times, the machine was ran at 72°C for 30 minutes before being stored in a freezer at -4°C until ready for analysis.
Samples of the grown bacteria were removed, and the plasmid purification procedure was done on a total of 8 samples as described (DebBurman, 2013). The samples, which are in tubes, were mixed well using a vortex unit, and 1.5mL of cells were transferred to a 1.5mL microfuge tubes. The tubes were then centrifuged at 14,000xg for a minute, and afterwards the supernatant was removed using a pipette, leaving only cells. The cells were then washed with 300uL of 0.5 M NaCl solution, and vortexed and centrifuged again. Supernatant was again removed using a pipette. Next 250uL of Buffer P1, containing PNase, was added to the remaining cell pellet and tubes were vortexed again, but not centrifuged. Then 250uL of Buffer P2 was added and tubes were inverted multiple times to mix. Next 350uL of Buffer N3 was added and again repeatedly inverted until the solution in the tubes were no longer blue of color. The tubes were then centrifuged for 10 minutes at maximum speed in a tabletop microcentrifuge. The supernatant was then pipetted into QIAprep column and the tubes were centrifuged again for 30-60 seconds. The flow through was discarded. The QIAprep spin column was washed by adding .75mL of Buffer PE and centrifuging for 30-60 seconds. Flow through was discarded and column was centrifuged again for 1 minute. The QIAprep column was then placed into a clean micro-centrifuging tube. Then to elute DNA, 30uL of Buffer EB was added to the center of each QIAprep column. We let it stand for 1 minute before centrifuging for 1 minute. The flow through was purified plasmid, which was labeled and later used for PCR.
Our gene fragments underwent DNA sequencing at the University of Chicago DNA Sequencing & Genotyping Facility as described (DebBurman, 2013). We sent them some of our purified plasmid vector and a sequencing primer.
To transform our plasmid vector into yeast, we used LiAc transformation as described (DebBurman, 2013). 10 mL of liquid YPD was inoculated and incubated with shaking overnight (18-24 hours) at 30 °C. Then a 100X dilution (990μL water and 10μL culture) of the culture was made in a 1.5 mL microcentrifuge tube. Afterwards, a 10μL slide was prepared and the overnight culture was counted. Cell density was calculated by the following formula: Cell Density = (# cells/box) x (104) X dilution factor (i.e. 100 for 1:100 dilution). 50 mL of YPD was then inoculated to a cell density of 5.0x106/mL of culture. The culture was then incubated at 30 °C and 200 rpm until it was at 2x107 cells/mL (typically takes 3-5 hours and gives enough cells for 10 transformations). Individual transformation aliquots were then prepared.
Cells within transformation aliquot were centrifuged and supernatant was discarded. Cells were then resuspended by adding 0.5 mL of 100mM Lithium Acetate (LiAc) and mixed by vortexing. The cells were then spun in a centrifuge at 14,000xg for 30 seconds and LiAc was removed with a micropipette. The cell pellet was then resuspended by the addition of 200μL of 100 mM LiAc and vortexing. Cells were then centrifuged again and LiAc was again removed with a micropipette. Cells were then ready for transformation.
For transformation to occur, a transformation mix was created by adding the following to the transformation cell pellet: 240 μL of PEG (50% w/v), 36 μL of 1.0 M LiAc, 25μL of single-stranded carrier DNA (2.0 mg/mL), 46 μL of H20, and plasmid DNA (0.5-10μL; usually 5μL is fine). A negative control was prepared by adding all of the previous materials with yeast but without plasmid. Then each tube was vortexed vigorously until the cell pellet was completely mixed. Tubes were incubated for 30 minutes at 30 °C in the incubator. Then tubes were heat shocked for 20-25 minutes in a water bath at 42 °C. Then tubes were microfuged at 6000-8000 rpm for up to 30 seconds and transformation mix was removed with a micropipette. 1.0 mL of sterile H2O was then pipetted into each tube to re-suspend the pellet. Plates were dried in incubator for 30 minutes prior to plating. The samples were then plated as follows with 3 plates per sample: 30 μL on one Sc-Ura glucose, 200μL on one Sc-Ura glucose, and 30 μL on YPD. Plates were then placed in 30 °C incubator for yeast transformation to complete (colony growth can take up to 5 days).
We would like to thank our lab professor, Dr. Bottero, as well as our peer teacher, Niam A. for all the help that they provided throughout this experimental process. We would also like to thank Dr. DebBurman for his involvement in explaining the overall goal of this project, and Yoan Ganev for graciously taking the time to redo any failed experiments.
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