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N-Terminal Truncations of α-Synuclein for Use in a Yeast Model of Parkinson’s Disease
Yoan Ganev and Ayesha Quraishi
Deparment of Biology
Lake Forest College
Lake Forest, Illinois 60045
α-synuclein is one of the main proteins implicated in Parkinson’s Disease, a prevalent neurodegenerative disorder. The protein’s N terminal has been proposed to mediate membrane binding, but this function has not been fully elucidated. Working with a budding yeast model of Parkinson’s, we created truncated versions of α-synuclein with first 19 and then 30 amino acids deleted. We hypothesized that the truncated fragments would exhibit the appropriate number of nucleotide base pairs and that they would be transformable in Saccharomyces cerevisiae. We successfully created both fragments by PCR and gene purification. We then sub-cloned these fragments into the pYES2 vector, transformed the DNA in E. coli, and identified the samples that were in the correct orientation. After sequence confirmation, we transformed the fragments into Saccharomyces cerevisiae. These steps allowed for the preparation of a tool kit that will be characterized by functional assays. Future tests could uncover the role of the N-domain in the onset of pathogenesis. We predict that reducing the size of the N terminal would reduce α-synuclein’s affinity for the cell membrane. This understanding would build on a growing set of data supporting the debilitating role of dysfunctional α-synuclein in PD.
Parkinson’s Disease (PD) is a widespread and debilitating neurodegenerative disease categorized as a hypokinetic movement disorder. PD is a continuum of diseases, in which environmental and genetic factors trigger sporadic or familial variants (Fahn, 2008). The symptoms most frequently associated with PD include tremors, bradykinesia, and muscle rigidity (Fahn, 2008). Nonmotor symptoms, such as depression, insomnia, and in severe cases, dementia, are frequently encountered (Fahn, 2008). PD is progressive and first strikes in the midbrain. Dopaminergic neurons in the substantia nigra die, and damage eventually spreads to the hippocampus and the amygdala (Petrucelli et al., 2008). Although different cellular abnormalities are detected, Lewy bodies (cytoplasmic aggregates of misfolded protein) are the main pathological aspect (Petrucelli et al., 2008).
Many mutations either increase a patient’s risk or lead to familial PD. Alterations in PARK2 and Leucine-Rich Repeat Kinase 2 may cause familial cases of PD (Ross et al., 2008). However, arguably the most important mutations associated with Parkinson’s involve α-synuclein (α-syn). α-syn is a small, flexible, and soluble protein of 140 amino acids (Reccchia et al., 2004). Proposed mechanisms of PD pathogenesis point to α-syn’s propensity to aggregate and its ability to interact with membranes (Snead et al., 2014). Familial versions of the illness involve six mutant forms of α-syn: A30P, E46K, H50Q, G51D, A53E, and A53T (Ross et al., 2008). The protein is also implicated in sporadic cases and early-onset PD in European families (Ross et. al., 2008).
α-syn is highly concentrated at the synaptic junctions of neurons. Mutant forms unfold in aqueous solution and aggregate in the cytoplasm (Lemkau et al., 2012). Misfolded α-syn is the main component of Lewy bodies (Baba et al., 1998). The normal function of the protein is not well understood, but research has revealed chaperone activities and roles in vesicle transport (Allendoerfer et al., 2008).
Studying the three domains of α-syn is a practical way to determine the protein’s functional properties. The N-domain is mainly responsible for membrane binding, the M-domain can mediate aggregation, and the C-domain has a role in solubility (Snead et al., 2014 and Vamvaca et al., 2010). Studying truncated versions of the protein reveals the impact of losing essential amino acids. The normal state of α-synuclein has an affinity for phospholipid membranes, and the N-domain (amino acids 1-60) is structurally linked to this function (Dikiy et al., 2012). In its unmutated form, the N-domain forms amphipathic α-helices, facilitating membrane binding (Vamvaca et al., 2009). Acetylation of specific amino acids stabilizes the protein and contributes to its membrane-binding capacity (Iyer et al., 2016). Truncating the N-terminal induces the protein to adopt a β-sheet conformation that loses membrane affinity and coils into aggregate fibrils. (Kessler et al., 2003).
The Biology 221 Lab at Lake Forest College undertook a 10-week project to make progressively shorter versions of α-syn by truncating increasing portions of the N-domain. We specifically made the 20-140 and 31-140 fragments, in which the first 19 and 30 amino acids, respectively, were removed. By shortening the N-domain, we might affect α-syn’s ability to bind membranes. There is no evidence for N-domain truncations occurring in nature, but studying them could help elucidate the normal function of α-syn. Previous work has demonstrated some properties of the N-domain, but experiments with these exact truncations have not been performed. The role the N-domain plays in PD remains to be investigated.
Deleting residues 2-11 may decrease the formation of α-helices in α-synuclein (Vamvaca et al., 2009). Thus, both the 20-140 and 31-140 truncations could exhibit decreased structural integrity, leading to more pronounced aggregation. Mutating α-syn’s second amino acid increases the protein’s toxicity to yeast, showing that the N-domain may determine response to α-syn (Vamvaca et al., 2009). In the 31-140 fragment, the pivotal 30th amino acid is lost. This residue mediates an aggressive form of early-onset familial PD through the A30P mutation. The A30P mutant is more toxic than the wild-type version (Nielsen et al., 2013), aggregates more slowly (Cuervo et al., 2004), and is more likely to be degraded by the chaperone-mediated autophagy pathway (Lemkau et al., 2012). Deleting the 30th amino acid makes it impossible for the truncated protein to acquire the A30P mutation. This could make it easier for the protein to be broken down. Experimental evidence is needed to support such predictions of the properties of truncated α-synuclein.
Saccharomyces cerevisiae, a simple free-living eukaryote, is a powerful model organism for PD. Yeast is both low-maintenance and cost-efficient (Allendoerfer et al., 2008). They have a short generation time of 1.5 to 3 hours and can be readily transformed with foreign DNA (Goffeau et al., 1996). Every reading frame in the yeast genome could be manipulated (Orr-Weaver et al., 1983). PD and other neurodegenerative illnesses are characterized by the aggregation of misfolded proteins, and budding yeast can mirror this paradigm (Chiti et al., 2006). The in-depth understanding of the transcription, replication, and repair mechanisms of budding yeast makes the organism a prudent platform to model genetic alterations (Allendoerfer et al., 2008).
The human version of α-syn can be expressed in budding yeast (Chen et al., 2005). S. cerevisiae has confirmed that α-syn can bind to the cell membrane and form cytoplasmic inclusions (Outeiro et al., 2003). Yeast models have revealed that breakdowns in the secretory pathway might mediate the toxicity of α-syn to cells (Dixon et al., 2005). Yeast has also demonstrated that α-syn affects the trafficking of transport vesicles, downregulates phospholipidase D, and reduces cytoplasmic lipid droplets (Outeiro et al., 2003). Factors that contribute to toxicity, including oxidative stress and proteasomal impairment, have also been confirmed in yeast (Sharma et al., 2006). At the same time, budding yeast have modeled the familial mutations in PD, confirming that all six exhibit different toxicity and cellular localization (Sharma et al., 2006). These yeast findings provide an overview of the way α-syn could mediate dysfunction.
In this study, we aimed to create the 20-140 and 31-140 truncations of α-synuclein to be used for functional characterization in yeast. The first goal was to successfully truncate wild-type α-syn into the two desired fragments. Then, the DNA is to be purified, inserted into a vector, and transformed in E. coli for further amplification. If the orientation of the plasmids in the vector is correct and the sequence is confirmed, the two fragments can be transformed in yeast.
We hypothesized that truncating the N-domain of α-syn would induce aggregation in yeast. The loss of crucial amino acids on the N-domain could instigate this altered phenotype. For the 20-140 fragment, we expected that truncating the first 19 amino acids would leave a shortened version of the protein of 1,128 nucleotides. Similarly, for the 31-140 fragment, we predicted that 1,095 nucleotides would remain. We also hypothesized that both E. coli and S. cerevisiae would accept the two α-syn fragments.
Overall Project Design
Our first goal was to shorten WT α-synuclein tagged with eGFP into the 20-140 fragments and the 31-140 fragments by PCR. Subcloning the desired fragment into the pYES2 vector allowed us to then transform the fragments in E. coli for storage of the DNA and for further amplification. Our next task was to confirm the orientation of the plasmids within the vector by either direct plasmid or whole-cell PCR. The plasmids found to be in the correct orientation were sequenced at the University of Chicago. Finally, we transformed the DNA into S. cerevisiae for future functional assays (Figure 1A).
Primer Design and Testing
We began by designing custom forward primers that start PCR amplification of α-synuclein at the 20th and 31st amino acids for the 20-140 and 31-140 fragments, respectively. For both fragments, we used the eGFP reverse primer. The sequences of these primers are provided in Figure 1B. The 20-140 forward primer truncates the first 19 amino acids, while the 31-140 forward primer truncates the first 30 amino acids, thus creating progressively shorter variants of α-syn (Figure 1C). The 20-140 fragment will have 363 base pairs remaining from the sequence of α-synculein combined with 765 base pairs from eGFP to give a total of 1,128 base pairs. The 31-140 fragment will have 330 base pairs remaining from the sequence of α-synculein combined with 765 base pairs from eGFP to give a total of 1,095 base pairs. To check the primers’ annealing, Rosemary Thomas ran a gel in which all forward primers designed in the lab were paired with an eGFP reverse primer on a WT α-syn-eGFP template (Figure 1D). All primers, except the one for the 95-140 truncation (unrelated to this experiment), yielded strong bands that appeared progressively lower on the gel. The eGFP reverse primer, when paired with the standard Syn 1-FP, also yielded a good band. This success indicated that the 20-140 FP, 31-140 FP, and eGFP RP could be used for the next step.
Creation of the Fragments and Gene Purification
We then set out to create the two truncations by PCR reactions with the appropriate primers. Figure 2A shows the expected results. We repeated each PCR reaction with two different volumes of WT template (1 uL and 4 uL), and we evaluated success by gel electrophoresis. The 20-140 fragment produced strong bands between 1,000 and 2,000 base pairs for both volumes, while the 31-140 fragment produced a strong band only for the larger volume of WT template. As expected, the band for the 31-140 fragment was also between 1,000 and 2,000 base pairs, and it appeared lower than that for the 20-140 fragment (Figure 2B). The positive control (WT α-syn paired with 1-FP and eGFP RP) yielded an acceptable band. We also prepared a negative control with the reverse primer omitted and another without template DNA. The first negative control yielded an unexpected band, but the second remained blank, as predicted. In all cases, there were many extraneous bands above the bands for the desired samples.
We considered the PCR reactions yielding bands successful. We re-ran the four reactions representing the 20-140 and 31-140 fragments on a new gel to prepare the samples for gene purification. This enabled us to excise the stronger band for each fragment, thus separating it from an unwanted template or supercoiled DNA (Figure 2C). After gene purification, Rosemary Thomas ran yet another gel to confirm that the sample was not lost and that the unwanted bands had been eliminated (Figure 2D). DNA of the correct size was retained for both purifications. The 20-140 fragment exhibited one extra band at 2,000 base pairs, while the 31-140 fragment exhibited just one clean band of the correct size. We considered purification for both fragments a success.
Subcloning into the pYES2 Vector and Bacterial Transformation
Next, we subcloned the purified DNA into the pYES2 vector. A map of this vector, indicating the relative position of the sample, the AmpR, and the Ura3 genes, is shown in Figure 3A. After subcloning, we transformed the 20-140 and 31-140 fragments into E. coli for storage and amplification. We plated each transformation reaction on LB+AMP plates in 40 uL and 200 uL volumes. Even though colonial growth appeared in every plate, this result could not be interpreted as positive, because the negative subcloning control (using water instead of vector) and the negative transformation control both exhibited colonial growth, rendering the plates non-selective (Figure 3B). As expected, the positive transformation control (using DNA provided by the kit manufacturer) and the open vector control (containing no DNA but just pYES2 vector) both exhibited colonial growth. To make sure that sub-cloning was successful, we re-plated the 20-140 and 31-140 fragments on new LB+AMP plates. Again, they exhibited strong colonial growth (Figure 3C). When we re-struck the negative subcloning and transformation control, the plates remained clean (Figure 3D). This selectivity shows that sub-cloning and transformation were satisfactory.
Orientation Check by Direct Plasmid and Whole Cell PCR
Our next step was to select six well-separated E. coli colonies for both the 20-140 and 31-140 fragments and to grow them in liquid LB+AMP for isolation of the corresponding plasmids. Once we isolated the plasmids, we paired them with the Gal 1-FP and the eGFP RP in direct-plasmid PCR. Although we expected all samples to yield bands (Figure 4A), only plasmids 5 and 6 for the 20-140 fragment and plasmid 5 for the 31-140 fragment yielded bands between 1,000 and 2,000 base pairs (Figure 4B and Figure 4C). The positive control (WT-GFP α-syn paired with the aforementioned primers) failed.
We then ran whole-cell PCR on eight different colonies for each fragment as a backup (Figure 5A). None of the whole-cell PCR samples for the 20-140 fragment yielded positive results. Two bands appeared for the 31-140 fragment – colony 18 yielded a medium band, while sample 24 gave a very weak one (Figure 5B and 5C). Again, the WT-GFP positive control failed to give a band.
Our next order of business was to send the plasmids that yielded positive results to the University of Chicago for sequence confirmation. A second 31-140 sample from a different group of experimenters (plasmid 13) was also sent, rendering the whole-cell PCR results obsolete. The sequences for both samples contained the correct number of nucleotide base pairs (Supplemental Figure 1). For the 20-140 fragment, a glutamic acid at one position (highlighted in red) mutated to aspartic acid. Both sequences for 31-140 contained no unwanted mutations.
Our last step was to transform the plasmids into budding yeast. We plated the transformed 20-140 fragment plasmids 5 and 6 and 31-140 fragment plasmids 5 and 13 on both SC-URA glucose and YPD plates. All of the plates exhibited plentiful yeast growth on both types of media (Figure 6). We prepared a positive control using WT-GFP α-syn DNA. For the negative control, we excluded DNA. On SC-URA glucose, the positive control exhibited some colonial growth, and the negative control was clean. On YPD, we observed roughly equal growth for both controls (Figure 6).
We aimed to create the 20-140 and 31-140o α-syn truncations as part of a class project, in which portions of the N-terminal are progressively deleted. These fragments complete a tool kit for future functional assays. Such experiments would elucidate the N-domain’s role in aggregation and membrane-binding, two properties implicated in PD (Dawson et al, 2003). We successfully created both fragments. Each of the steps yielded data that confirmed our hypotheses. We created the truncations by PCR, purified the DNA, inserted it into a vector, transformed into E. coli, checked for orientation, sequenced, and transformed into yeast.
Creation of Truncated Variant and Gene Purification
We expected the 20-140 truncation to yield bands at 1,128 base pairs and the 31-140 – at 1,095 base pairs. Strong and unambiguous bands of the correct size appeared. The strength of the bands shows that PCR amplification was sufficient, as band thickness reflects the amount of DNA loaded onto the gel. The bands’ position indicates that the truncations were made successfully. Both fragments traveled more than the wild-type control. Short DNA moves further towards the positive end of the gel than longer samples. Both fragments represented α-syn that was shorter than the wild-type. In addition, the 31-140 fragment appeared lower than the 20-140 fragment, showing that it was shorter than the 20-140 fragment. The extraneous band that appeared in the first negative control might be attributed to spillover from the adjacent well or a failure of the control. The extraneous bands that appeared on the gels for both of the desired fragments likely represented unused template DNA. These bands justified the use of gene purification.
A round of gel electrophoresis after purification confirmed that the procedure had succeeded. The strong bands of the expected sizes showed that the DNA samples were not lost during treatment and elution. The 20-140 fragment exhibited an extraneous band slightly higher than 2,000 base pairs. This band might represent super-coiled DNA that traveled less than its counterpart. It is unlikely to represent remaining undesired DNA, as we excised only the band of the correct size from the original gel. The fact that the 31-140 fragment yielded a single band at 1,095 base pairs shows that the truncated gene was purified successfully.
E. coli Transformation and Orientation Check
After the sub-cloning and transformation procedures, an abundant number of colonies appeared in the LB+AMP plates. This result confirms that the isolated DNA was successfully inserted into the pYES2 vector. The vector contains the Ampr gene, which gives E. coli the ability to proliferate in ampicillin (present in the plates). Any bacterial colony thar survives must therefore have the vector and by association, the fragment. The sparse growth of the open vector control indicates that most colonies should contain the transformed product. The unexpected growth of bacteria transformed with water presented an obstacle to determining the success of subcloning. This growth could be attributed to several factors, such as the pYES2 vector closing in on itself and rejecting the fragments, contamination with resistant bacteria, or a failure of the ampicillin in the plates. The latter explanation is the most likely, given that we observed no growth for the negative control once the samples were re-plated on new LB+AMP plates. This re-plating established the success of both sub-cloning and transformation but gave no information about the orientation of the gene. Probability dictates whether the gene is cloned in the correct orientation or upside-down.
Direct plasmid and whole-cell PCR provided a way to check the orientation of the sub-cloned fragments. The key to this determination was the use of a forward primer, GAL 1-FP, binding to a promoter on pYES2 that falls immediately outside the sub-cloned gene. If the gene is correctly oriented, the eGFP reverse primer anneals at the end of α-syn’s tag, and PCR allows for amplification of the gene together with the promoter. If the plasmid is upside down, the GAL 1-FP still anneals at the promoter, but the eGFP RP anneals next to GAL1 on the same DNA strand. Both primers work in the 5’-3’ direction, and amplification is impossible. A band indicates amplification from PCR and identifies a sample in which the gene was correctly inserted. Whole-cell PCR yields a lower success rate because the presence of components from the ground-up cell makes it difficult for the primers to access the DNA.
Sequencing of both fragments was considered a success. The 20-140 fragment (plasmid 5) contained 363 nucleotides from the start of the truncated α-syn. This corresponds to the removal of the first nineteen amino acids, showing that the truncation was successfully made. The mutation of glutamic acid to aspartic acid was deemed unimportant. The two amino acids belong to the same class (negatively charged polar), so the alteration is unlikely to have far-reaching consequences on the structure of the protein. The sequence for 20-140 plasmid 6 was not analyzed due to an error from the sequencing facility. The sequences for the 31-140 fragment displayed 330 nucleotides, corresponding to the removal of the first 30 amino acids from α-syn.
The BY4741 strain of yeast has been engineered not to produce uracil, a nucleic acid essential for RNA synthesis. These yeast grown in a food source without uracil die as transcription breaks down. SC-URA selective plates provide all ingredients for cell growth, except for uracil. The pYES2 vector contains the URA3 gene, which rescues uracil production. The formation of yeast colonies in the selective plates implies that the cells can overcome the lack of nutrients and have therefore accepted the vector. The fact that colonies of yeast appeared on the SC-URA plates for both of the fragments and the positive control was consistent with the expected results. The negative control was blank as the yeast had no source of the plasmid and thus, no way to make uracil. All samples (including negative controls) exhibited growth on YPD plates, which provide uracil from the environment. α-synuclein toxicity could not have influenced the growth of the yeast cells. α-syn expression in the yeast is driven by a galactose-inducible promoter. Galactose was not present in any of the media, so the protein was not expressed.
The truncation variants can be used in experiments to characterize their functional properties. An assay that is frequently utilized, the yeast-spotting assay, relies on serial dilution to assess the toxicity of altering a gene of interest in yeast (Dhungel et al, 2014). If the N terminal is responsible for keeping the α-syn bound to membranes, then continually shortening the protein should increase its toxicity to the cell, as dissociation from the membrane progresses. Another experimental assay, fluorescence microscopy, could be used to determine the localization of α-syn under different conditions (Outeiro et al, 2003). The eGFP tag attached to both of the fragments allows the researcher to track changes in the protein’s position over time. In the truncation variants outlined here, α-syn would most likely become diffuse throughout the cytoplasm of the cell as it loses its ability to bind membranes. A third experimental approach, the Western blot, might be used to show the level at which the mutant protein is expressed (Baba et al., 1998). Overexpression might be observed; the cell could compensate for the α-syn that has detached from the membrane by making more. The cell death assay could be used to investigate whether the truncated version of α-syn is forcing the cells to undergo apoptosis at an increased rate (Teng et al, 2011). Co-immunoprecipitation experiments could identify whether α-syn associates with other proteins and how the truncations alter this interplay (Strauss et al, 2005). Such experiments could reveal the role that the N-domain plays in the development of pathogenesis.
PD illustrates the biological problem of protein misfolding. Yeast is an adequate model organism for this dysfunction, so pertinent conclusions could be derived by characterizing α-syn variants. The N-domain’s interaction with the M and C domains remains to be described. Studying truncated variants of the protein might bring us closer to understanding its properties. The overall structure of the protein may be more important than the structure of the N-domain alone. An understanding of the cellular dysfunctions that lead to PD might pave the road towards identifying the molecular mechanisms that could yield a more viable treatment of PD.
To design the forward primers, we used WT α-syn sequence as a template. Each primer was 45 nucleotides long. For the 20-140 forward primer, we counted 45 nucleotides from the 20th amino acid on WT α-syn, and for the 31-140 primer, we counted 30 nucleotides from the 31st amino acid. The forward primers bind the template (3’-5’) strand, so the sequence for both was identical to the 5’-3’ strand of α-syn, from the appropriate position. There was no start codon on the fusion vector, so we added a Kozak sequence (ANNATGG) to each of the forward primers. The WT DNA had no stop codon to make a fusion with eGFP possible. To design the reverse primer, we counted 45 nucleotides backwards from the 5’-3’ strand of the eGFP sequence. We excluded the stop codon from the design of the reverse primer. Since the reverse primer binds the coding strand (5’-3’), we took the complement of each nucleotide (DebBurman, 2016, 24-25).
Template DNA, Subcloning Plasmids, and Bacterial and Yeast Cells Used
We used wild-type α-syn-eGFP isolated from E. coli cells as a template for both truncations. After PCR, the truncated DNA samples were sub-cloned into the TOPO pYES2 vector, suitable for both E. coli and S. cerevisiae. The vector contained the Ampr gene, allowing E. coli to grow in ampicillin and the Ura3 gene, permitting yeast to grow in the absence of uracil. We used TOP10 OneShot E. coli cells for bacterial transformation and S. cerevisiae cells of the BY4741 strain for yeast transformation. Bacteria were plated on LB+AMP plates, while yeast, on both SC-URA and YPD (DebBurman, 2016, 38-40 & 52).
We created the truncations by performing plasmid-based PCR using two different recipes per fragment. In one variation, we used 50 uL MasterMix, 43 uL sterile RNase-free water, 3 uL of forward primer (20FP for the 20-140 fragment and 31FP for the 31-140 fragment), 3 uL of reverse primer (eGFPRP), and 1 uL of WT α-syn template. In the other, we used 50 uL Master Mix, 40 uL sterile RNase-free water, 3 uL of forward primer (20FP for the 20-140 fragment and 31FP for the 31-140 fragment), 3 uL of reverse primer (eGFPRP), and 4 uL of WT α-syn template. We prepared a positive control using FP-1, eGFPRP, and the WT α-syn template. We also made negative controls: one with the forward primer omitted and one without template DNA (DebBurman, 2016, 30-31). We used the following PCR conditions: 30 seconds in 95°C, 30 seconds in 55°C, and 30 seconds in 72°C. The anneal-denature-replicate cycle was repeated 29 times. We then incubated the reaction at 72°C for 30 minutes and stored at 4°C indefinitely.
To visualize PCR results, we made 0.3% gels using 0.3 g of agarose dissolved in 40 mL of 1X buffer TAE. 1 uL of Ethidium Bromide was added to visualize DNA under ultra-violet light. We mixed each PCR reaction with 10.5 uL of 10X loading dye. In the gels used just for visualization, we loaded 15 uL of this mixture for each reaction and used a 10 kbp high-mass ladder. In the gel used for excision, we loaded 50 uL of the mixture for each reaction and used a two kbp Amplisize ladder. We imaged gels under UV light using ImageLab software (DebBurman, 2016, 32-33).
We performed gene-purification by excising a band containing the desired PCR product from the gel and placing it in a pre-tared tube. We added 100 uL of salt solution per 0.1 g of gel slice. We melted the slice at 55°C and added the mixture to a TurboCartridge in a two mL catch tube. We then centrifuged the tube, added a Turbo Wash/ethanol solution, and centrifuged the tube twice in succession. The cartridge was then transferred in a new catch tube, and we added 30 uL of the TurboElution solution. DNA eluted after a five-minute incubation followed by a one-minute centrifugation at maximum speed. We stored the samples at -20°C. (DebBurman, 2016, 35-37).
Vector Subcloning and Bacterial Transformation
We used the pYES2.1 TOPO TA Expression Kit, Version J to subclone both fragments into pYES2. We mixed 4.1μL of purified DNA, one μL of salt solution, and 0.9 μL of TOPO pYES2 vector. For the negative transformation control, we substituted the vector with water. To transform in bacteria, we added 2 uL of each sub-cloning reaction to a different vial of TOP10 OneShot E. coli cells. We incubated the reaction for 5 minutes on ice. We heat-shocked the cells, added SOC medium, and shook the reaction at 37°C for one hour. We plated 40 uL and 200 uL of each reaction on LB+AMP plates. After the first plating failed, we grew colonies from the plates in liquid LB+AMP and re-struck them on new plates. Rosemary Thomas ran a lab-wide negative transformation control, positive control, and open vector control, as described in the subcloning kit manual. (DebBurman, 2016, pg 38-41).
Plasmid Purification and Direct-Plasmid PCR
To check orientation, we isolated plasmid from 6 E. coli colonies for each sample. The cells were inoculated in liquid LB+AMP and grown overnight. We used the QIAGEN QIAprep Miniprep Kit to elute the DNA according to the manufacturer’s protocol (DebBurman, 2016, 43- 44). We ran direct-plasmid PCR on these samples using a reaction that consisted of 25 uL MasterMix, 21 uL sterile RNase-free water, 1.5 uL Gal 1 forward primer, 1.5 uL of eGFP reverse primer, and 1 uL of isolated plasmid. We employed the same PCR conditions described in “Plasmid Based PCR” (DebBurman, 2016, 42-45). We visualized results with a 0.4% agarose gel with 1 uL of ethidium-bromide.
Bacterial Whole Cell PCR
As a backup orientation check, we chose eight colonies from the transformation plates and dabbed half of each with a toothpick. We deposited the colonies in PCR mixtures that contained 25 uL MasterMix, 22 uL sterile RNase-free water, 1.5 uL Gal 1 forward primer, and 1.5 uL eGFP reverse primer. We employed the same PCR conditions described in “Plasmid Based PCR” (DebBurman, 2016, 42-45). We visualized results with a 0.4% agarose gel with 1 uL of ethidium-bromide.
The gene fragments that yielded successful direct-plasmid PCR results were sequenced at the University of Chicago DNA Sequencing and Genotyping Facility. We sent the Gal Forward Primer sent along with the samples (DebBurman, 2016, 51). We compared the results to the sequence of wild-type α-syn (Supplemental Figure 2).
We transformed the plasmids into the BY4741 strain of S. cerevisiae via a lithium-acetate transformation. We used yeast cells in YPD inoculated to a density of 5.0x106 cells/mL. The cells were centrifuged to remove the YPD and re-suspended in 0.5 mL of 100mM Lithium acetate. We washed the cells with 200 uL of LiAc, after which we prepared a transformation mix. We added 240 uL of PEG, 36 uL of 1.0 M LiAc, 25 uL of SS DNA, 46 uL DNA, and five mL of the appropriate plasmid. The cells were incubated and later heat-shocked in a 42°C water bath. We removed the transformation mix after centrifuging the cells and re-suspended each sample in 1 mL of water. We used WT-eGFP DNA for the positive control and prepared the negative control by omitting the plasmid altogether. We plated cells on both SC-URA and YPD and incubated them for 4.5 days at 30°C.
We would like to thank our professor, Dr. Shubhik DebBurman (Lake Forest College), Dr. Virginie Bottero, and our lab peer mentor, Rosemary Thomas ’18 (Lake Forest College), for providing extensive training, for giving us all of the materials necessary to complete the experiments, and for their guidance in writing this report. We would also like to thank Emily Ong ’17 for her peer teacher workshops. We express gratitude to everyone involved with Biology 221 for providing a supportive atmosphere and the resources that made this work possible. The Biology 221 course was supported by the Lake Forest College Department of Biology.
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A. Experimental Overview
B. PCR Primer Sequences
C. Progressive truncations of α-synuclein
D. Primer Checks
Figure 1: Experimental Design and Primer Synthesis
(A) WT α-synuclein (I) is first to be truncated into the 20-140 fragments and the 31-140 fragments by PCR (II). Both fragments are then to be subcloned into the pYES2 vector, which with its AmpR and Ura3 genes is suitable for both E. coli and C. cerevisiae (III – one generic fragment is shown). Next, the truncations are to be transformed in E. coli for further amplification (IV), and the orientation of the plasmid within the vector is to be checked via PCR (V). The DNA that is in the correct orientation is then to be isolated from the bacteria and is to be sent for sequencing (VI). Upon confirmation of the correct sequence, the DNA is to be transformed into yeast, for future functional assays (VII).
(B) The forward and reverse primers used to make the 20-140 fragments and the 31-140 fragments of α-syn via PCR. We used the eGFP reverse primer for all reactions.
(C) A comparison of WT α-synuclein (I), containing all 140 amino acids on the N, M, and C domains, with the two N-terminal truncations (II-III). In (II), the first 19 amino acids from the N terminal have been removed to create the 20-140 fragment, while in (III), the first 30 amino acids have been truncated, yielding the 31-140 α-synuclein fragment. In all versions, the enhanced Green Fluorescent Protein tag remains unaltered.
(D) The designed forward N-terminus truncation primers (red) were tested for proper annealing to the template DNA when paired with standard reverse primers (140-RP) in lanes 2-8. As truncation number increased, the bands appeared at lower kilobase pairs when compared to the 2kb ladder in lane 1. Both the 20-140 and 31-140 FPs yielded strong bands (lanes 3 and 4). In addition, eGFP reverse primer (red) with standard forward primer (1-FP) was tested in lane 9, and a band appeared between 1.5 and 1 kilobase pairs.
A. Expected PCR Results
B. Actual PCR Results
C. Post-Excision Gel
Figure 2: Creation of Truncations by PCR and Gene Purification
(A) These computerized images reflect the theoretical outcomes of the PCR reactions performed. In (I), 50 uL of the designated reactions were loaded into the wells. The 20-140 fragment was expected to produce a band at 1128 base pairs, while the 31-140 fragment was expected to produce a band at 1095 base pairs. The positive control was predicted to appear at 1185 base pairs. No difference was expected between the different template volumes used for each reaction. In (II), 15 uL of sample were loaded into each well. Lanes 1, 2, 5, 6, and 7 were expected to be identical to the first five lanes in (I). Lanes 3 and 4 on the bottom gel exhibit no bands, as reagents crucial to PCR are missing.
(B) In (I), 50 uL of each sample were loaded into each well. Lanes 1 and 2 correspond to the 20-140 fragment of α-syn after PCR using 1 and 4 uL of template (respectively). Lanes 3 and 4 represent the 31-140 fragment of α-syn after PCR using 1 and 4 uL of template (respectively). The fifth lane is a positive control. In (II), lanes 1 and 2 were left empty. 15 uL of the 20-140 sample (using both template volumes) were loaded into lanes 3 and 4. Lanes 5 and 6 were reserved for the negative controls. In lanes 7 and 8, both PCR reactions involving the 31-140 fragment were run, while lane 9 was reserved for the positive control.
(C) This is an image of the gel from Figure 2a (I) after the DNA from lanes 2 and 4 was excised to be used for the purification protocol.
(D) The Gene-Clean purified truncated α-syn DNA for the entire Biology 221 class was run on a gel to show that purification of the fragment was successful. The bands in lanes 4 and 5 of the gel (highlighted in green in the table) represent the 20-140 and the 31-140 fragments described in this study.
A. Map of the pYES2 Vector
B. E. coli Transformation Results
C. Transformation Results after Re-Plating
D. Re-Plated Controls
Figure 3: E. coli Transformations
(A) This map of the vector DNA used to accept the PCR product shows the properties that make transformation and future assays possible. The URA3 and Ampicillin Resistance genes, as well as the Gal1 promoter, are to be of special interest to this study.
(B) In (I), 40 and 200 uL respectively of the E. coli cells transformed with the 20-140 fragment were pipetted and spread onto LB+AMP plates. In (II), 40 and 200 uL respectively of the E. coli cells transformed with the 31-140 fragment were pipetted and spread onto LB+AMP plates. (III) represents a series of controls. The 40 and 200 uL Negative Control samples represent cells transformed with a subcloning reaction containing water instead of vector. The negative transformation control represents a transformation in which no subcloning reaction was added. For the positive transformation control, a vector known to work efficiently in E. coli was introduced. Finally, the open vector control is the product a subcloning reaction in which the empty pYES2 vector was transformed into the cells.
(C) Cells containing the 20-140 fragment (I) and the 31-140 fragment (II) were scraped from the original plates, grown in liquid LB+AMP, and re-struck on new LB+AMP plates. Colonies of bacteria were observed for both of the fragments.
(D) The procedure described in Figure 3c was repeated for the negative control (I), the open vector control (II), and the negative transformation control (III). The negative control and the negative transformation control exhibited no cellular growth (the plates were blank). The open vector control exhibited some colonies, but they were much fewer than those in the experimental conditions.
A. Expected Direct Plasmid PCR Results
B. Direct Plasmid PCR Results
C. High-Exposure Pasmid PCR results for 31-140
Figure 4: Direct Plasmid PCR Results
(A) Gel-electrophoresis is expected to yield a band whenever the orientation of the α-syn fragment is correct. Here, the top part of the gel represents the 20-140 fragment while the bottom corresponds to 31-140 (thus, the bands are lower). For 20- 140, bands should appear slightly higher than 1,128 base pairs, for 31-140 – higher than 1,095, and for the positive control, higher than 1,185 bp. In this idealized result, every sample yields a band, but in reality, the success rate is likely to be lower. The orientation of the sub-cloned gene is entirely left to probability.
(B) Thick and strong bands appeared for fragment 20-140, samples 5 and 6. using the direct-plasmid PCR technique. These successful plasmids could be sent for sequencing. The band for the positive control is very weak. A faint band appeared for fragment 31-140, sample 5.
This plasmid could also be sent for sequencing.
(C) The exposure of the bottom half of the gel in Figure 4b was manually adjusted to reveal the weak band.
A. Expected Whole-Cell PCR Results
B. Whole-Cell PCR Results
C. High-Exposure Whole-Cell PCR results for 31-140
Figure 5: Direct Plasmid PCR Results
(A) As for the direct-plasmid technique, whole-cell PCR is expected to yield a band for both fragments whenever the plasmid is in the correct orientation. This expectation is reflected in the idealized gel, showing the 20-140 fragment on top and the 31- 140 fragment on the bottom. In reality, the success rate of whole-cell PCR is likely to be even lower than that for direct-plasmid PCR, dictated by both the probability of finding the correct orientation and difficulties associated with the technique itself.
(B) None of the whole-cell PCR samples for the 20-140 fragment yielded bands. The positive control was also blank. Two bands appeared for the 31-140 fragment – sample 18 yielded a medium band, while sample 24 gave a very weak one.
(C) The exposure of the bottom half of the gel in 5b was manually adjusted to reveal the bands for fragment 31-140 samples 18 and 24.
Figure 6: Yeast Transformations
Lithium acetate yeast transformations of the 20-140 and 31-140 fragments and controls were performed. The positive control on SC-URA glucose exhibited some yeast growth, while the negative control exhibited none (I). Cells containing the 20-140 fragment plasmids #5 and #6 were plated in SC-URA glucose, in 30 uL and 200 uL volumes for both (II). Strong colonial growth appeared for all samples. Cells transformed with plasmids #6 and #13 for the 31-140 fragment in SC-URA glucose (again plated using 30 and 200 uL) also exhibited strong colonial growth (III). 200 uL samples of both fragments were also plated on YPD, and again, many yeast colonies grew, although fragment 20-140 plasmid #5 was slightly contaminated. (IV). 200 uL samples of the positive and negative controls were plated on YPD, and colonies grew in abundance for both samples (IV).
I) Sequence for Fragment 20-140 Plasmid 5
II) Sequence for Fragment 31-140 Plasmid 5
II) Sequence for Fragment 31-140 Plasmid 13
Supplemental Figure 1: DNA Sequences for the 20-140 and 31-140 fragments
(I) The sequence for the 20-140 fragment contains 363 nucleotides from the start of α-syn. The first 45 nucleotides of α-syn (including the Kozak sequence) correspond to the 20-140 forward primer and are highlighted. The unwanted mutation glutamic acid to aspartic acid (GAC instead of GAG) is highlighted in red. The blue highlight represents the last six amino acids of α-syn. Note the intentional absence of a stop codon. The sequence for 20-140 Plasmid 6 could not be analyzed due to a formatting error from the sequencing facility.
(II) The sequences for the 31-140 fragment plasmids 5 and 13 contain 330 nucleotides from the start of α-syn. Again, the first 45 nucleotides and last 6 amino acids are highlighted. We found no extraneous mutations in either sequence.
Supplemental Figure 2: WT α-syn sequence
The un-truncated wild-type sequence of human α-syn against which we compared the truncation sequences. Note that the sequence is 140 amino acids (420 nucleotides) long.
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