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Creating ∆61 and ∆80 β-synuclein N-Terminal Truncations to Further Explore β-synuclein Involvement in Parkinson’s Disease
Lake Forest College
Lake Forest, Illinois 60045
Creating ∆61 and ∆80 β-synuclein N-Terminal Truncations to Further Explore β-synuclein Involvement in Parkinson’s Disease
1 Department of Biology, Lake Forest College, Lake Forest, IL 60045
Parkinson’s Disease is a prevalent neurodegenerative disease. Strong evidence shows the involvement of β-synuclein in the development of Parkinson’s Disease, although the specific role of the protein is not fully understood. In this experiment, we sought to create two N-terminal truncations of the protein, at amino acid 61 and 80, and then subclone these truncated versions of the protein into a TOPO pYES2.1 vector, and transform it into E. coli and Saccharomyces cerevisiae. This was done to see what the effects of this truncation would have on its function. We predicted that these truncations would be possible and that S. cerevisiae would be capable of accepting the modified plasmid containing the truncated DNA. Although both ∆61 and ∆80 were successfully subcloned into the TOPO pYES2.1 vector, only the ∆61 truncation was successfully transformed into S. cerevisiae. Our project provides the basis for future studies of the successfully transformed yeast. We predict that the truncations would affect β-synucleins ability to bind to the cell membrane, potentially causing it to aggregate in the cytoplasm. Observing the affinity of the modified β-synuclein to aggregate could provide more insight into the protein’s role in Parkinson’s Disease.
Parkinson’s Disease (PD) is the most common kind of parkinsonism that is characterized by a combination of six specific motoric features: rigidity, flexed posture, freezing phenomenon (feeling as if feet are glued to the floor), loss of postural reflexes, tremor-at-rest, and bradykinesia (Fayyaz, et.al, 2018). At least two of these symptoms should be present prior to diagnosis, with at least one of them being tremor-at-rest or bradykinesia. Symptoms typically begin on one side of the body and progress with time. PD can develop at any age, but likelihood increases with age, with a peak age of onset being around 60 years old (Elsevier Inc, 2008). PD is a neurodegenerative disease whose motoric symptoms have been shown to be related to dopamine deficiency in the striatum, which is caused by a decrease in the number of dopaminergic neurons in the substantia nigra pars compacta (Elsevier Inc, 2008). There are many genes that have been linked to PD, including parkin, SNCA, PINK, DJ-1, and LRRK2, and have been shown to cause both recessive and dominant forms of PD.
The hallmarks of PD are the loss of dopaminergic neurons in the substantia nigra pars compacta as well as the formation of Lewy bodies. It has been found that α-synuclein may be the main component of Lewy bodies, and is encoded by SNCA (Spillantini, et.al ,1997). Synucleins are abundant proteins that account for 0.1% of total brain proteins (Chandra, 2009). α-synuclein is a protein that localizes to pre-synaptic nerve terminals. Its functions are not totally understood; however, studies suggest its role in synaptic plasticity, inhibition of PCL2, and the development of PD (Bendor, et.al, 2018), (Jenco, et.al, 1998).
After sequence analysis of α-synuclein was performed (sequence of α-synuclein: GenBank ID L08850), a single base-pair mutation at position 209 in the fourth exon region of the genome of the protein results in an Ala to Thr amino acid substitution, which has shown to result in the development of PD (Polymeropoulos, et.al. 1997). It is predicted that this mutation, localized in a region of the protein with an alpha helix bounded by beta sheets, extends the beta sheet and disrupts the alpha helix (Polymeropoulos, et.al. 1997). Beta sheets have been linked to protein aggregation (Fink, 1998), (Hu, et. al., 2008).
α -synuclein is a 140 amino acid long protein that can be divided into three main regions, each with structural characteristics. The three domains are the amphipathic region (from amino acid 1-61), the NAC domain (from amino acid 61-95) and the acidic tail (from amino acid 95-140). The three missense mutations that have been shown to lead to early-onset PD are A30P, E46K and A53T, which all occur in the amphipathic region (Venda, et.al, 2003). The NAC domain is essential for α-synuclein filament assembly (Giasson, et.al, 2000). The C-terminal acidic tail is mostly negatively charged and generally unfolded, however, studies suggest that post-translational modifications to this region, such as oxidation and phosphorylation, can lead to α-synuclein aggregation and the formation of lewy bodies (Giasson, et.al, 2000). Phosphorylation at serine 129 increases fibrillation while phosphorylation at tyrosine 125 prevents fibrillation (Venda, et.al, 2010).
C-terminal truncations of α-synucleins increase aggregation (Venda, et.al, 2010)
However, α-synuclein is not the only synuclein protein involved in the development of PD. There are three known members of the synuclein families: α-synuclein, β-synuclein, and γ-synuclein, although α-synuclein is the most researched of the three (Rivers, et.al. 2008).
Contrary to α-synuclein, β-synuclein does not oligomerize (Gámez-Valero & Beyer, 2018). In fact, it has been shown to decrease the rate at which α-synuclein oligomerizes, which can slow the progression of neurodegeneration of PD. (Fan, et.al. 2006). However, it is not currently known how or why β-synuclein does this, and more research is being done to understand this mechanism and possibly developing therapies to encourage β-synucleins inhibition of α-synuclein aggregation (Leitao, 2018). A major difference in the structure of β-synuclein compared to α-synuclein is that it is only 134 amino acids long, and has an 11 amino acid deletion (from amino acid 73-83) in the amphipathic region of the protein. This could be why β-synuclein does not form fibrils. In general, synucleins seem to negatively regulate dopaminergic pathways, however, double knockout α-β-synuclein rats have decreased dopamine levels in striatal pathways (Chandra, 2009).
One mutant of β-synuclein, V70M, is associated with lysosomal dysfunction. In this mutation, the 70th amino acid (valine) is changed (to methionine). This implies that the 70th amino acid is important for keeping the lysosomes functioning normally. If the 70th amino acid is lost we predict that lysosomal function may be affected.
In this experiment, we used S. cerevisiae as our model organism, as it is very easy and cheap to grow. Yeast also has a small genome, and is relatively simple. Although it is an elementary organism, yeast contains all of the cellular compartments that work in the same fashion as higher organisms such as humans. Yeast is also an excellent model organism for studying protein misfolding diseases as it is great for experimental manipulation. Yeast is versatile and can be grown as either a haploid or diploid. Therefore, one can cover or uncover the effects of mutations at any point. (Menezes, et.al., 2015)
Yeast, specifically S. cerevisiae, has been used in research of many different neurodegenerative diseases, including Alzheimer’s Disease, Huntington’s Disease, amyotrophic lateral sclerosis, and Parkinson’s Disease, as well as prion diseases, which are known to involve protein misfolding and aggregation. (Tenreiro, S., et al, 2013), (Polymeropoulos, M.H, et al, 1997). Much PD research has been done using yeast. Yeast models are useful in understanding the molecular mechanisms associated with PD. The expression of α-synuclein and its levels of toxicity on yeast cells has been studied. (Lazaro, D.F., et al, 2014). In one study, the expression of a-synuclein in yeasts has resulted in dose-dependent cytotoxicity. This goes along with the identification of familial forms of PD associated with duplication and triplications of the α-synuclein locus. Also, the expression of α-synuclein in yeast has resulted in the formation of intracellular inclusions. (Kain, 2018). In another PD study using yeast, α-synuclein was found to selectively associate with the plasma membrane, form cytoplasmic inclusions, inhibit phospholipase D, induce droplet accumulation, and affect vesicle trafficking. (Outeiro, T.F. and Lindquist, S., 2003).
In our experiment, we perform two truncations of β-synuclein: removing the first 61 amino acids in one truncation, and removing the first 80 in the other truncation. We predict that in both truncations, the proteins ability to bind with membranes will be affected, potentially causing the proteins to aggregate in the cytoplasm. This is because the N-terminal ends at amino acid 60 in β-synuclein, and the N-terminus is responsible for membrane binding and lipid interactions (Ducas, et.al., 2012). We also predict that the lysosomal function of the Δ80 truncation will be affected. This is because the 70th amino acid has been indicated to be important for keeping the lysosomes functioning normally (Wei, et.al., 2007). In the Δ80 truncation, this amino acid will be lost. We also predict that α-synuclein will be more likely to aggregate due to the hindered capabilities of β-synuclein, and it therefore may not be able to inhibit α-synuclein aggregation as well as wild-type β-synuclein. Finally, we predict that both truncations will successfully be transformed into the E. coli and S. cerevisiae yeast.
Our first aim was to isolate and exponentially amplify the two targeted DNA sequences of the desired truncations of β-synuclein from plasmids, using PCR. Our second aim was to purify the gene fragments (Δ61 and Δ80) via gel electrophoresis. The fragments were placed in wells, allowed to separate, visualized, extracted from the gel, then cleaned from the agarose. Our third aim was to subclone the gene fragments into TOPO pYES 2.1 vectors also containing an ampicillin resistance gene (Ampr). Our fourth aim was to confirm the correct orientation of the gene fragments into the plasmids in the E. coli. Our fifth aim was to purity the plasmid vectors with the correct orientation. Our sixth aim was to transform the plasmid vector into S. cerevisiae, to allow for further research on the effects of this modified version of β-synuclein on the yeast.
β-Synuclein Gene Fragment Amplification by PCR
The desired truncations of β-synuclein (Figure 1A) were created using designed primers, the sequence of which are shown in Figure 1B. The overall steps of our project can be seen simply in Figure 1C. An initial plasmid containing β-synuclein and GFP is used to create truncations using specially designed primers, creating the truncated β-synuclein and GFP DNA fragments. These truncated DNA fragments are amplified via PCR then subcloned into TOPO pYES2.1 plasmids, which have a 50/50 chance of subcloning the DNA in the correct orientation. The plasmids are transformed into E. coli. Then, the orientation of the plasmids in the plasmid and in the E. coli whole cell are checked via PCR and running the products through an agarose gel, the details of which are explained in more in the methods section. The plasmids with the correct truncation and orientation are finally transformed into S. cerevisiae. Next, samples of the plasmids were sent for DNA sequencing to confirm that the plasmid contained the correct orientation of the desired truncation of β-synuclein.
PCR Product Purification by Gel
The PCR product containing the amplified β-synuclein truncations were loaded into an agarose gel and run to see the truncations. This gel contained two PCR products from each truncation, along with DNA ladder. The four reactions included two with ∆61 (one with 1μL and one with 4μL), and two reactions with ∆80 (one with 1μL and one with 4μL). Figure 2A shows what the gel would have ideally looked like after running it. Pictures of the actual gel was taken after they were run are included in figure 2B. Next, the DNA bands from lanes 3 and 6 were manually extracted from the gel, as shown in Figure 2A& 2B. These lanes contained PCR products of the Δ61 truncation and the Δ80 truncation, respectively. Both of these lanes used 4μL of the template. No picture was taken of the gels after extraction. Another gel was run to visualize 4 reactions, along with two negative controls and one positive control (Figure 2C & 2D). Unfortunately, the visualization gel was let run for too long, and the DNA bands ran off the end of the gel (Figure 2D). Next, to double check that the designed primers were correct and that the DNA extracted is of the correct length, a primer check gel was run (Figure 2E). The bands in lanes 4 and 5 contain β-synuclein at truncations 61-135 and 80-135, respectively. The extracted DNA (Figure 2A & 2B) were then purified from the agarose to be ready for cloning in the next lab, the methods of which are explained in more detail in the methods section. Prior to freezing, 5μL of the purified extracted DNA were given to Chisomo, who ran another gel to verify that the gene fragments were purified correctly (Figure 2F). These lanes (lanes 2 and 4) contained samples of the extracted DNA from the bands that contained the products from reactions with 4 μL of template and primers for the respective truncations (Figure 2B).
Subcloning into TOPO pYES 2.1 Vector and Bacterial Transformation
Next, these gene fragments were subcloned into the TOPO pYES 2.1 vector. The TOPO pYES 2.1 vector contains Ampr an ampicillin resistance gene, and URA3, a gene that makes URA. This vector was then transformed into E. coli, the bacteria of choice in this study. The E. coli were then placed onto plates containing LB+AMP plates. Figure 3 shows pictures of the cultures taken roughly 24 hours after being placed on the plates. 8 colonies were selected from each truncation to be used in the next lab. The colonies used in the next lab were circled and numbered (Figure 3).
Gene Fragment Orientation Check in
Colonies 1-4 from each truncation underwent DNA extraction to purify the plasmid from the E. coli. To verify that DNA was extracted during this process, Chisomo ran a gel containing cultures 1-4 from the Δ 61 truncation and cultures 1-4 from the Δ80 truncation (Figure 4A). Then, two different PCR’s were conducted: on the purified plasmids and on the whole cell E. coli from cultures 5-8. The PCR products, from the plasmid-based PCR and the whole-cell PCR, underwent gel electrophoresis to confirm the orientation of the gene fragment in the vector. Although bands were expected for all of the plasmid-based PCR products for both Δ61 and Δ80 (Figure 4B & 4D), only two Δ61 plasmid-based PCR products produced bands (lanes 3 and 5, which contained plasmids from colonies 2 and 4, respectively), and no bands were produced in the Δ80 gel (Figure 4C & 4E). No whole cell PCR products yielded bands. The PCR products from the two successfully oriented plasmids from Δ61 were transformed into yeast.
Plasmid Vector Transformation into Yeast
In the final step, one of the successfully oriented plasmids (from colony 2 of Δ61, shown in lane 3 of Figure 4C) was transformed into yeast. The yeast was then plated onto different plates. Two SC-URA plates were prepared. One contained only 30μL of the yeast and the second was prepared with 200μL. The third plate was prepared with 30μL of the yeast onto a YPD plate. All plates exhibited growth of cultures on both types of media (Figure 5A). More growth can be seen on the SC-URA plate with 200μL of the yeast than the plate with 30μL, which makes sense that the more yeast plated, the more is likely to grow. The lack of growth on the 30μL plate can then be attributed to there being less yeast to grow, and not due to any mistakes in the experiment or lack of correctly oriented and subcloned plasmids into the yeast. Positive and negative controls were done by Professor Wilcox (Figure 5B). The positive control plate was streaked with yeast that was transformed with a plasmid of a different protein (α-synuclein). The negative control plate was streaked with yeast that were transformed with water (no plasmid).
Plasmid Gene Sequencing
A sample of the correctly-oriented Δ61 plasmid from culture 2 that was transformed into the yeast was given to the University of Chicago to sequence the DNA of the plasmid. The sequence of the plasmid (Figure 6) was as predicted.
The goal of this experiment was to create successful β-synuclein truncations from amino acids 61-134 and 80-134, subclone them into plasmid vectors, and transform them into S. cerevisiae (Figure 1A-C). We were able to successfully isolate and purify both of these truncations from the original plasmid (Figure 2 A-F). These truncations were successfully inserted into plasmids and successfully transformed into E. coli (Figure 3 A-D). Only cultures 2 and 4 from ∆61 were successfully oriented (Figure 4C & 4E). Culture 2, containing the correctly oriented plasmid with the correctly
truncated ∆61 β-synuclein DNA was then successfully subcloned into S. cerevisiae (Figure 5). This project was done to provide materials for further research on the aggregation of β-synuclein in the yeast, which could hopefully elucidate the functions of β-synuclein in humans and its involvement in the development of Parkinson’s Disease. We were only able to successfully make the 61-134 truncation and transform it into the yeast. The Δ80 truncation was not oriented correctly in the plasmid and therefore not transformed into the yeast. The successful truncation and transformation of the ∆61 β-synuclein gene allows us to create a model of how truncated portions of β-synuclein would affect the S. cerevisiae. This could give indications about what functions the portions of β-synuclein truncated have, and its involvement in PD.
β-synuclein Gene Fragmentation via Designed Primers and PCR
From a template plasmid containing the wild type of β-synuclein, the truncated versions of β-synuclein were made. This was done by using specifically made DNA primers (Figure 1B) to induce replication after the first 61 amino acid for the first truncation and after the first 80 amino acids for the second truncation. The forward primers of the Δ61 truncation and the Δ80 truncation were designed based on the sequence of the wild type of β-synuclein. The reverse primer used for both truncations codes for GFP, a gene that encodes for green fluorescent protein obtained from Aequorea victoria (the desired DNA strands are therefore the truncated versions of β-synuclein attached to GFP). GFP will aid in the visualization of β-synuclein aggregation in the transformed yeast. The truncations made in this portion of our project were successful—they yielded the desired Δ61 and Δ80 truncation of β-synuclein in conjunction with GFP. We know it was successful due to the location of the bands on the gel. The band were around the 1kb level when compared to the DNA ladder. We know that the truncations with the GFP should be around this length because of the addition of the number of nucleotides per truncation with the nucleotides for the GFP gene. The ∆61 truncation of the β-synuclein contains 222 nucleotides and GFP contains 714 nucleotides, which adds to 936 nucleotides total for this truncation. The ∆80 truncation of the β-synuclein contains 165 nucleotides and GFP contains 714, which adds to 879 nucleotides total for this truncation. This is why the bands that appear around the 1kp length (1000 nucleotides bases) indicate successful results.
PCR Product Purification by Gel
The truncated DNA strands from the previous step were then amplified exponentially via PCR. Then, these PCR products were placed in an agarose gel and run to separate the DNA (Figure 2A & 2B). The bands at the 1kb level appeared, which is the correct size of the desired truncations. The gene fragments were shorter than the wild type β-synuclein. Other bands were also found on the gel that could be excess DNA from the PCR. These bands were discarded. The desired bands were extracted as shown in Figure 2A&2B. The next gel that was made contained PCR products of four reactions, along with two negative controls and one positive control (Figure 2C & 2D). The four reactions included two with ∆61 (one with 1μL and one with 4μL), and two reactions with ∆80 (one with 1μL and one with 4μL). Ideally, bands will be present at the correct length (around 1kb) for all of the lanes except those for the negative controls, which were in lanes 5 and 6. Unfortunately, the gel was let run for too long, and the DNA bands ran off the end of the gel, so no conclusions can really be made from this gel. Next, to check the effectiveness of our primers, a primer check gel was run (Figure 2E). Comparison between the primer check gel and the bands from the extracted gel supported that the extracted DNA was truncated β-synuclein at 61-135 and 80-135 because the bands in lanes 4 and 6 (which contained ∆61 and ∆80, respectively), were around 1kb. After the DNA was extracted, they were purified from the agarose gel. Samples of the extracted and purified DNA were taken and run in another gel to test that they were purified correctly (Figure 2F). This gel verified that the truncations were purified correctly and yielded the desired β-synuclein truncations because of the bands visible in lanes 6 and 7 (truncations ∆61 and ∆80 respectively) are at the correct length of 1kb when compared to the DNA ladder in lane 1. So far, we have successfully created β-synuclein truncations at Δ61 and Δ80, and verified this.
Subcloning into TOPO pYES2.1 Vector and Bacterial Transformation
The β-synuclein truncations with the GFP gene were then subcloned into the TOPO vector, which contains Ampr, the gene for ampicillin resistance. This vector was transformed into E. coli, which was then plated onto LB+AMP plates, ensuring that only E. coli that were successfully transformed were able to survive, because they need the Ampr gene in the plasmid. There was abundant growth in all of the plates, indicating successful subcloning of the ∆61 truncation (Figure 3A-D). We know that growth in the LB+AMP plates indicate that the TOPO pYES2.1 vectors were successfully subcloned into the E. coli due to them being able to transcribe Ampr to survive the environment of the medium.
Gene Fragment Orientation Check in
The plasmids from cultures 1-4 from each truncation were extracted; samples of these extracted plasmids were given to Chisomo to run, and based on the results (Figure 4A), the plasmids were extracted successfully. This gel verifies that DNA was extracted, yet no conclusions regarding the orientation of the β-synuclein fragments within the plasmids can be made. Essentially, we know we extracted DNA, but not if the truncation was inserted in the correct orientation in the plasmid. This is because the DNA has a 50/50 chance of being integrated into the plasmid in the correct orientation. In order to check for the orientation of the plasmids, PCR was run, and the PCR products were checked for correct orientation via gel electrophoresis (Figure 4B-4E). Only products that have the correctly oriented plasmid are able to exponentially replicate their DNA under PCR, because if the truncations were placed incorrectly in the plasmid, the directionality of the DNA and the direction of the primers prevent replication. So, if there are products in the gel, this indicates that the plasmid was correctly oriented. Cultures of E. coli chosen from the plates (Figure 3) all have the plasmid, but have a 50/50 chance of having the plasmid in the correct orientation. Ideally, all of the plasmid-based PCR products in lanes 2-5 would have the correct orientation and two of the whole cell PCR in lanes 6-9, for both fragments would have the correct orientation (Figure 4B& 4D). Out of both the plasmid-based PCR and the whole cell PCR tests, only two were successful: the bands in well 3 and 5 of the Δ61 truncation (Figure 4C). No bands were yielded in the Δ80 gel (Figure 4E). A possibility for the plasmid-based PCR not yielding DNA at the correct length could be because our plasmids did not include the β-synuclein and GFP DNA segment in the correct orientation, and we have a 50/50 chance of picking the correct ones. Another possible source of error could be the fact that the Δ80 gel had to be moved after the wells were loaded. The wells were loaded before the gel was rotated in the correct position for gel electrophoresis. Although we carefully removed the gel, rotated it to the correct position, and carefully placed it back in the gel electrophoresis tank, the movement of the gel could have moved the DNA out of the wells and prevented the formation of bands in the Δ80 gel.
Plasmid Vector Transformation into Yeast
In the final step, the successful Δ61 plasmid from culture 2 was transformed into yeast, then plated onto plates with SC-URA medium (Figure 5A). This ensures that only yeast with our specially designed plasmid can grow, because the plasmid contains URA, a gene to create uracil, which is essential for yeast survival. The growth seen on the SC-URA plates were yeast that had the correctly oriented plasmids, allowing them to make uracil. Although there was no visible growth on the plate with only 30μL of the yeast transformation solution, there was noticeable growth on the plate that was streaked with 200μL of the yeast (Figure 5A). Three control plates were made (Figure 5B). The negative control was streaked with yeast that was not transformed with a plasmid, so there wasn’t supposed to be growth, and there wasn’t. This was to make sure that only yeast with plasmid containing the Ampr would be able to grow on the LB+AMP plates. The second plate was a positive control, which contained yeast with plasmid with α-synuclein, was not successful, which would have been helpful if none of our plates had growth, but they did, so results of the positive control isn’t very significant here. The third plate was yeast streaked on YPD plate, and growth on this plate ensured us that the yeast is capable of growing at all.
Plasmid Gene Sequencing
The correctly truncated, correctly oriented, successfully made plasmid from culture 2 of the Δ61 truncation was sent to the University of Chicago for gene sequencing to verify the success of this project. The results show the sequence of the plasmid with the truncation (Figure 6). This is the predicted sequence of the ∆61 β-synuclein sequence (Figure 1B). This means that the truncation was successfully made and successfully subcloned into the plasmid, as well as successfully transformed into the yeast.
Review of Hypothesis
We predicted that both truncations would successfully be transformed into the E. coli and S. cerevisiae yeast. However, only one of the successfully oriented plasmids (from culture 2 of Δ61) was transformed into yeast. A reason for this could have been due to errors made in lab such as rotating the gel in the loading tray after already loading the DNA in the wells during the gel electrophoresis.
Serial dilution spotting, is a way to test the effects of a mutation on the growth and survival of cells. Serial dilution spotting has been done with yeast. Applying this to this project, serial dilution spotting with yeast would allow us to see how a mutated protein (in this case, how truncated β-synuclein) would affect cell survival. This method would be applicable to pure cultures as well as liquid samples. (Thomas et al., 2015). Fluorescent microscopy could also be used in the future. Fluorescent microscopy is used to visualize the location of protein expression of the protein tagged with the GFP protein. Since the truncated β-synuclein we made is also attached to GFP, fluorescent microscopy could be used to visualize the location of β-synuclein in the cell. More specifically, this method would allow us to see potential aggregation of β-synuclein. This is important because aggregation of β-synuclein is linked to Parkinson’s Disease. (Pringle et al., 1989). Western blotting is another method that may be used in the future. Western Blotting is a way to visualize protein expression in cells. Therefore, comparing β-synuclein expression in mutated yeast (yeast with the truncated β-synuclein) with wild type yeast would allow us to compare levels of β-synuclein expression. So, knowing if β-synuclein is overexpressed in the mutated cells would allow us to understand how levels of β-synuclein can affect Parkinson’s Disease pathology, since overexpression of β-synuclein could lead to Parkinson’s Disease.
In this project, we were able to create ∆61 truncations of β-synuclein attached to GFP, subclone them into plasmids, and successfully transform them into S. cerevisiae. This allows us to create a model of how truncated portions of β-synuclein would affect the S. cerevisiae. This could give indications about what functions the portions of β-synuclein truncated have. If these modifications of β-synuclein lead to an increase in protein aggregation in S. cerevisiae, then this could give indications about how this protein is involved in the development of Parkinson’s Disease. We know that β-synuclein is involved in Parkinson’s Disease, it is just not clear how. If, somehow, these truncations of β-synuclein lead to less aggregation, that could mean that the part of β-synuclein that was cut out could be the portion responsible (or linked with) the aggregation of β-synuclein. This could give further indications about how this protein, and synucleins in general, are involved in Parkinson’s Disease.
Primer Design and Synthesis
In order to design the forward primers, we used a WT β-synuclein sequence as a template. Each primer was 30 nucleotides long. For the 61-135 forward primer, we needed to find the 5’ boundary on the sequence, and the primer is the complimentary nucleotides to it (Figure 1B). The same procedure was done to design the forward primer for 80-135. Since there was no start codon, we added a Kozak sequence (ANNATGG) to each of the forward primers. We designed the reverse primers by finding the 3’ boundary of the GFP and taking the complimentary nucleotides of the sequence (Figure 1B). Template DNA, subcloning plasmids, and bacterial and yeast cells were used. DebBurman, S. (2018). Primer design and synthesis.
- DebBurman (Ed.), BIO 221 molecules, genes, & cells laboratory manual (pp. 21-26). Lake Forest, IL: Lake Forest College.
To separate, identify, and purify our DNA fragment, we prepared and ran a PCR. This was done by creating a 50uL Master Mix which contained MgCl2, Buffer, Taq DNA Polymerase, and Dntp. Then we pipetted 43uL of RNAse-free H2O, 3 uL of forward and reverse primers for β-synuclein, and 1 uL of purified plasmid into a PCR tube. We also created a positive and negative control using the β-synuclein template. The following PCR conditions were used: 30 seconds in 95° C, 30 seconds in 55° C, and 30 seconds in 72° C. The cycle was repeated for a total of 29 times, then incubated at 72° C for 30 minutes and stored in 4° C indefinitely. DebBurman, S. (2018). Plasmid-based PCR.
- DebBurman (Ed.), BIO 221 molecules, genes, & cells laboratory manual (pp. 30-31). Lake Forest, IL: Lake Forest College.
PCR Product Purification by Gel Electrophoresis
To obtain PCR results, we created and separated our PCR products on an agarose gel through electrophoresis. The agarose gel was prepared by first heating and pouring a mixture of agar, TAE solution, and 10 uL of ethidium bromide into a gel tray and embedding depressed wells to contain PCR products or plasmid mixtures. A charge was run through the gel that makes the side closest to the DNA negative and the side opposite the wells positive. This pulls the negatively charged DNA to the positive side. The DNA fragments that are the smallest travel the quickest through the gel and appear lower on the gel after the reaction is over. Once the gel was ready, we analyzed the gel under ultraviolet light, which made the bands visible due to the fluorescence of ethidium bromide. Pictures were taken of all of our gels.
- DebBurman, S., BIO 221 molecules, genes, & cells laboratory manual (pp.32 - 35). Lake Forest, IL: Lake Forest College
Vector Subcloning and Bacterial Transformation
Using the pYES2.1 TOPO TA Expression Kit, Version J, we transformed our previously prepared and purified PCR product into the plasmid TOPO pYES2.1 vector, which was then subcloned into. cerevisiae. This reaction was done by creating a reaction tube with 4 uL of purified PCR product, 1 uL of salt solution, and 1 uL of TOPO vector. For the negative control, water was used instead of the vector. After the cloning reaction, the vector was transformed into bacteria and grown onto LB+AMP plates containing ampicillin. 30 uL and 200 uL from each transformation were incubated overnight at 37 °C. DebBurman, S. (2018) Vector subcloning and bacterial transformation.
- DebBurman (Ed.), BIO 221 molecules, genes, & cells laboratory manual (pp.38-41). Lake Forest, IL: Lake Forest College
Next, Figure 3 shows pictures of the cultures taken roughly 24 hours after being placed on the plates. 8 colonies were selected from each truncation to be used to confirm gene fragment orientation in vector in bacteria. We conducted PCR using plasmids purified from bacteria as well as using whole bacterial cells. This was done to identify the correct orientation of the subcloned gene fragment. We used the QIAGEN QIAprep Miniprep Kit from E. Coli bacteria that was grown in LB liquid media overnight to isolate bacterial plasmid from our colonies. We prepared and cleared the undesired lysate from pelleted bacteria by absorbing the DNA in the QIA prep membrane, then washed and eluted the plasmid DNA. A Master Mix of 25 uL was created then 21 uL of sterile RNAse-free H2O, 1.5 uL of forward primer (Gal FP), 1.5 uL Reverse primer (GFP RP), and 1.0 uL of plasmid was added. We prepared a positive control using the original plasmid that served as our template for our PCR conducted in our first gene fragment. We then loaded 9 reactions into the PCR machine. The following PCR conditions were used: 30 seconds in 95° C, 30 seconds in 55° C, and 30 seconds in 72° C. The cycle was repeated for a total of 29 times, then incubated at 4° C for 30 minutes and stored in 4° C. DebBurman, S. (2013). Plasmid purification.
- DebBurman (Ed.), BIO 221 molecules, genes, & cells laboratory manual (pp. 42-43). Lake Forest, IL: Lake Forest College
Bacterial Whole Cell PCR
The following week we set up a whole cell PCR using the colonies on our growth plates labeled 5-8. We prepared a 25 uL master mix, and then added 22 uL of sterile RNASE-free H2O, 1.5 uL of Forward Primer (Gal FP), 1.5 uL of Reverse Primer (GFP RP), and a very small amount of each colony dabbed from our growth plates using a sterile toothpick. To determine if we obtained PCR products, from the plasmid-based PCR and the whole cell PCR, both underwent gel electrophoresis to confirm the orientation of the gene fragment in the vector. Although bands were expected for all of the plasmid-based PCR products for both Δ61 and Δ80 (Figure 4B & 4D), only two Δ61 plasmid-based PCR products produced bands, and no bands were produced in the Δ80 gel (Figure 4C & 4E). No whole cell PCR products yielded bands. The PCR products from the two successfully oriented plasmids from Δ61 were used in the following lab. DebBurman, S. (2013). Bacterial whole cell PCR.
In DebBurman, S., BIO 221 molecules, genes, & cells laboratory manual (pp. 44-50). Lake Forest, IL: Lake Forest College
Next, we sent in our gene fragment from the Δ61 truncation to be sequenced by the University of Chicago DNA Sequencing & Genotyping Facility. 5 uL of the plasmid was prepared in a separate 1.5 mL microfuge tube and sealed with parafilm. Results of the gene sequencing are included in Figure 6.
DebBurman, S., BIO 221 molecules, genes, & cells laboratory manual (p. 51). Lake Forest, IL: Lake Forest College
One of the successfully oriented plasmids (from culture 2 of Δ61) was transformed into yeast. Electroporation, which uses electricity, and treatment with lithium acetate (LiAc) was used in order to make the cell wall permeable and allow the plasmid to enter. The yeast was then plated onto SC-URA and YPD plates with 30 uL on one Sc-Ura glucose, 200 uL one one Sc-Ura glucose, and 30 uL on YPD. All three plates exhibited growth of cultures on both types of media (Figure 5A).
DebBurman, S. (2013). Yeast transformation. In S. DebBurman (Ed.), BIO 221 molecules, genes, & cells laboratory manual (pp. 52-54). Lake Forest, IL: Lake Forest College
This project would not have been possible without the help of our peer teacher Chisomo Mwale, my lab partner Wendy Gross, our lab professor Alexander Wilcox, and our lecture professor Shubhik DebBurman.
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