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Attempting to make G51A, G51R, and H50N mutants and analyzing the effects of the A53D mutant on toxicity in α-synuclein
Chelsea Evans and Paul Jones
Department of Biology
Lake Forest College
Lake Forest, Illinois 60045
Parkinson’s disease (PD) is one of many neurodegenerative diseases that are characterized by the death of certain neurons and formation of toxic aggregates of protein. In PD, these aggregates are called Lewy Bodies and are primarily composed of the protein α-synuclein (α-syn). Most cases of PD occur sporadically and are not associated with a genetic mutation or a familial history of PD; however, there are cases of early, aggressive PD in which a point mutation on α-syn is present. The goal of the project was to create amino acid substitutions (G51A, G51R, and H50N) on α-syn in order to study the properties of the mutation sites of the recently identified familial mutants using S. cerevisiae as a model. G51A is hypothesized to behave similarly to wild-type (WT) α-syn due to a similar, nonpolar charge between glycine and alanine. G51R is hypothesized to have a phenotype somewhat similar to G51D, as both arginine and aspartic acid are electrically charged. H50N is expected to have toxic properties similar to H50Q as both asparagine and glutamine have large, polar side chains. These mutants were not able to be created due to failed E. coli transformation, failed mutagenesis, and unsuccessful sequencing. However, another group created the A53D mutant, which was sequenced and transformed into S. cerevisiae, and toxicity was tested using a serial dilution assay. A53D grew somewhat worse than WT and about the same as A53E. A clearer and more definitive trend will appear with multiple spotting trials. Results would have been significantly improved with more experience with technique and procedure.
Parkinson’s disease (PD) is a brain-based, protein misfolding disease that commonly results in a dysfunctional gait and resting tremor (Obeso et al., 2010). If the brain of a patient with PD is analyzed, the normal dark pigmentation of the midbrain region that assist in motor coordination (Nicola, Surmeier & Malenka, 2000), the substantia nigra, is not visible and the dopaminergic neurons in this region have nearly all disappeared (Kopin, 1993). In almost all forms of PD, certain proteins have come out of solution and formed toxic aggregates called Lewy Bodies (Lansbury, 1997). Alpha-synuclein (α-syn), is found in abundance in these aggregates and is thus of great interest to PD research (Goedert, Spillantini, Del Tredici & Braak, 2012).
Although most cases PD are considered sporadic, there are nine genes that can possess mutations which are linked to an early, more aggressive form of PD that runs in families (Bendor, Logan & Edwards, 2013). These genes include PARK2, PINK1, DJ-1, LRRK2, ATP 13A2, and SNCA. As of now there are six amino acid substitutions that have been identified in SNCA¸ the gene which directly codes for α-syn, that are associated with PD (Nuytemans, Theuns, Cruts & Van Broeckhoven, 2010).
α-syn is a 140 amino acid long protein that contains three domains, each of which affects its properties and function. Although it is implicated in PD, the specific role of α-syn in the brain has yet to be identified. The N-terminus promotes membrane binding and leads us to believe that α-syn may have some role in releasing neurotransmitters (Crowther et al., 1998). This is further supported by α-syn’s affinity towards the presynaptic terminal. The M-domain is largely responsible for α-syn’s aggregative properties (Ritchie and Thomas, 2012). The C-terminus contains many acidic amino acids and thus promotes the solubility of α-syn (Crowther et al., 1998)
Three single amino acid substations in SNCA have been well studied and have given insight into the structure and function of α-syn as well as its implications in PD. All three point mutations lie in the N-terminus The point mutation A30P was identified over 10 years ago (Krüger et al., 1998) and is associated with increased fibrillization and aggregation of α-syn (Li, Uversky & Fink, 2001). A53T was identified at approximately the same time (Farrer et al., 1998) and was shown to have similar properties (Li, Uversky & Fink, 2001). E46K was identified in 2004 (Zarranz et al., 2004) and is shown to reduce helical conformation of α-syn (Wise-Scira, Dunn, Aloglu, Sakallioglu & Coskuner, 2013).
Additionally, three new single amino acid mutations on SNCA have been recently discovered. These three mutations also lie in the N-terminus. G51D was identified in 2013 (Kiely et al., 2013) and has been shown to reduce aggregation and membrane binding in S. cerevisiae, unlike previous point mutations (Fares et al., 2014). H50Q was also identified in 2013 (Proukakis et al., 2013) and is shown to behave similarly to A30P and A53T (Rutherford, Moore, Golde & Giasson, 2014). A53E was identified near the beginning of 2014 (Pasanen et al., 2014) and has been shown to reduce aggregation and membrane binding (Ghosh et al., 2014). Only preliminary research has been completed on these three new SNCA point mutations.
Our lab continues the investigation by analyzing the effect and importance of the three amino acids at the three mutation sites. Our goal is to produce various new mutants in the place of each of the amino acids on the G51, H50, and A53 sites. The many mutants we have produced have either nonpolar, polar, acidic, or basic side chains. These new mutants will give insight into the three specific amino acid sites and the new familial mutants’ properties of the configuration, localization, and toxicity of α-syn.
Our particular lab group aims to produce the G51A, G51R, and H50N mutants. The G51A mutant converts the G51 amino acid from glycine to alanine. We predict that G51A will behave similarly to wild type as they both possess nonpolar side chains. However, this may not be true as glycine is the smallest amino acid. The G51R mutant changes glycine to arginine and will either disrupt a hydrophobic core or cause α-syn to not turn at the usual point (Fares et al., 2014). This mutation will completely change the structure of α-syn, much more than the G51A mutant. Hypothesizing about these particular mutants is particularly difficult because the G51D mutant is shown to actually reduce aggregation in vivo. Analyzing these mutants may give more insight into this property of G51D. H50N is expected to have toxic properties similar to H50Q as both asparagine and glutamine have large, polar side chains We have chosen to perform our analysis in S. cerevisiae because it is inexpensive and simple to modify, yet still possesses the protein making, folding, and degrading pathways that humans have. Thus, it is an excellent model for our particular research focusing on α-syn. Yeast have already provided some the most basic and essential insights into α-syn yet still have the capability to provide possible treatment routes for diseases such as PD (Caraveo et al., 2014).
In order to analyze these mutants in S. cerevisiae¸ the mutation must first be made in a vector containing Wild Type α-syn using specially designed primers. The resulting mutagenized vector will be transformed in E. coli to ensure no unmutagenized vector remains and that the mutagenized vector is successfully reproduced. Efficacy will be determined by gel electrophoresis and gene sequencing at an external lab. If the mutations are correctly made they will be transformed into S. cerevisiae and experimentation can begin.
Overall Project Design
The project’s schematic is summarized Figure 1A. The mutations, G51R, G51D and H50N, along with the original familial mutants and α-syn are depicted in Figure 1B. To create our specific mutant in S. cerevisiae, we will first mutagenize WT, full length α-syn tagged with GFP in pYES2 vector with carefully selected primers in a PCR reaction. If mutagenesis is successful, we will then transform our mutagenized α-syn into specialized E.coli that will degrade the unmutagenized, WT α-syn vector, leaving us with only the mutagenized vector. The vector will then be purified form E.coli and confirmed using gel electrophoresis. If the plasmid is present in the purification, the plasmid will be sent for sequencing. If sequencing comes back with the desired G51A, G51R, and H50N mutants with no other alterations, the plasmid will be transformed into S. Cerevisiae. Then, toxicity analysis will be performed using a spotting assay.
G51A and G51R Primer Design
Primers were designed based on specifications in DebBurman 21-25.
Figure1. Overview of Project
A. Project Schematic To create the intended mutants a mutagenesis PCR reaction will be run on WT α-syn. The efficacy of mutagenesis will be confirmed and plasmid will be transformed into E.coli. After E. coli has grown significantly, transformed plasmid will be extracted, confirmed for transformation efficacy and sent for sequencing. IF the sequence is as intended, the plasmid will be transformed into yeast and experimentation can begin.
B. Mutagenesis Comparisons The three mutants that are being created are mutagenized from full length WT α-syn. The two familial mutants the mutations we are making are based on are G51D and H50Q. The mutations we intend to create are G51A, G51R, and H50N
C. Mutagenesis and Control Reactions The Mutagenesis reaction will produce a full copy of the pYES2 vector with mutated α-syn and GFP. Another set of reactions will be run to ensure the efficacy of the reactions. The forward primer reaction check will use our designed forward and a GFP reverse primer to give a DNA fragment of about 1000 bp pairs. The reverse primer reaction will use our designed reverse primer and α-syn forward primer to give a DNA fragment of about 170 bp pairs.
Figure 2. Primer Design Sequences
A. Wild Type α-syn
A short relevant sequence of WT- α-syn is given for comparison. The mutations sites 50 and 51 are colored red and purple respectively.
B. G51A Forward and Reverse Primers Sequence
The mutation from GGT to GCT results in a point mutation from glycine to alanine. The reverse primer is the opposite sequence except in the 5’ to 3’ direction.
C. G51D Forward and Reverse Primers Sequence
The mutation from GGT to CGT results in a point mutation from glycine to arginine. The reverse primer is the opposite sequence except in the 5’ to 3’ direction.
D. H50N Forward and Reverse Primers Sequence
The mutation from CAT to AAT results in a point mutation from histidine to asparagine. The reverse primer is the opposite sequence except in the 5’ to 3’ direction.
Three sets of primers were designed. The first two were used to mutagenize the 51st amino acid, glycine, of WT to alanine (A) and arginine (R). The forward primers contained 39 bases pairs and had the exact same sequence in the 5’ to 3’ direction as WT α-syn with the exception of the 51st codon changed from a GGT to CGT in G51A, and GGT to GCT in G51R. H50N had its 50th codon changed from CAT to AAT. Primer designs for G51A, G51D, and H50N are depicted in Figure 2. The reverse primer contained the exact same sequence of the forward primer but in the 5’ to 3’ complementary, direction. Primers were ordered from an external lab and are were confirmed efficacy using control mutagenesis reactions, shown in Figures 3B and 5B.
G51A and G51R Mutagenesis PCR
With primers designed as specified in Figures 2, mutagenesis reactions were carried out based on the instructions in DebBurman, 29. Additionally, a set of control reactions were performed to ensure the efficacy of our designed primers. The mutagenesis product was then loaded and ran on a DNA agarose gel according to DebBurman, 33. The idealized gel is depicted in Figure 3A. If mutagenesis was successful thick bands of DNA would have appeared in lanes 3 and 5 at approximately 6k base pairs. If the forward primers were effective, a band would appear in 8 and 10 at approximately 1k base pairs; the approximate distance between the 45-amino acid of α-syn and the end of GFP. Successful forward control reactions would appear at about 171 base pairs; the distance between first and fifty-seventh amino acids of α-syn, and would appear in 9 and 11. The entire lab’s positive control was also ran on our gel and should appear in lane 14. However, as Figure 3B shows, results were not as perfect as intended. Although G51A and G51R in lanes 2 and 4 were present, mutagenesis was weak at best. Additionally, G51A primers showed efficacy in lanes 8 and 9 but, G51R did not appear. Finally, the positive control appeared in both lanes 13 and 14. The efficacy of the G51R primers was shown by another group based on Figure 3C. Due to poor initial results, another set of mutagenesis reactions for mutants G51A and G51R was performed by Morgen
Figure 3. G51A and G51R Mutagenesis
A Idealized Gel for G51A and G51R Mutagenesis and Primer Checks 15 μL of each respective sample were loaded in each well. 5 μL of high mass ladder High Mass ladder was loaded into lane 1. 1:50 diluted WT Template was loaded into lanes 3 and 5 as a comparison for mutagenesis. G51A and G51D and mutagenesis are in lane 2 and 4 respectively. Successful mutagenesis reactions are expected to be significantly larger and darker than background as both had 1:50 WT template dilution. Amplisize ladder was loaded into lane 7. The forward and reverse primers for G51A are in lanes 8 and 9 respectively. The forward and reverse primers for G51D are in lanes 10 and 11 respectively. A lab-wide positive control was loaded in 14.
B. Gel for G51A and G51R Mutagenesis and Primer Confirmation Mutagenesis Products in lanes 3 and 5 appear weak but significant enough to transform. G51A Forward and Reverse primer checks are in lanes 8 and 9 and visible, but G51R primers are not visible, implying that they may not be effective.
C. Gel for G51A and G51R Mutagenesis and Primer Confirmation This is another groups gel ran that analyzed the same G51R primers that were ran in our gel. Both forward and reverse primers appeared in lanes 10 and 11. Thus, the primers for G51R are effective
D. Idealized Gel for G51A and G51R Mutagenesis
This is ideal gel for the mutagenesis that Charles Alvarado and Morgen Marshall performed outside of lab. 5 μL of high mass ladder High Mass ladder was loaded into lane 1. 5 μL of each mutagenesis where loaded into each well.G51A, G51E, G51Q, G51R, and A53Q were loaded in lanes 2-6 respectively. 1:50 diluted WT Template was loaded into lane 7 as a comparison for mutagenesis.
E. Gel for G51A and G51R Mutagenesis and Primer Confirmation
Mutagenesis for G51R is successful. Mutagenesis for all other mutants including G51A and G51R rather weak but still noticeable compared to background plasmid. Thus, G51A and G51R are ready to be transformed into E. coli.
Marshall and Charles Alvarado. This reaction showed some efficacy as can be seen in Figure 4C.
G51A and G51R Transformations
Next, the G51A and G51R mutagenesis products from Morgan Marshall and Charles Alvarado were transformed into E. coli according to (DebBurman, 34-41). This transformation caused E. coli to replicate our mutagenized vectors and degrade WT unmutagenized vector. 30 and 200 μl of transformed cells with plasmid were plated on LB+Ampicillin agar. Ampicillin ensures that only E. coli with transformed vector that contains Ampicillin resistance is able to grow. A class wide positive control with a confirmed plasmid and E. coli and a negative control with water were also prepared by Morgan Marshall. Figure 4A shows the transformation results and the positive and negative controls. The positive results showed growth of E. coli colonies and the negative control showed no growth, as expected. There was no growth on any of the G51R or G51A plates. This implies that
Figure 4. G51A and G51R Bacterial Transformations
A. Bacterial Transformations.
Our mutagenized vectors were transformed into E.coli bacteria and grown on LB+AMP plates. 80μL and 20μL solutions of transformed bacterial cells were plated. The positive plate used a wild type plasmid with E. coli that is known to function correctly, while the negative control used sterile, deionized water. There was expected growth in the control plates, but no growth on any
of the G51A or G51R plates occurred.
there was no significant mutagenesis product. Thus, progress on the G51A and G51R could not be continued.
H50N Mutagenesis PCR
A mutagenesis reaction for H50N was prepared as the previous reaction was except with H50N forward and reverse primers. In addition, two control reactions were performed on H50N’s forward and reverse primers to ensure efficacy. The products were then loaded and analyzed using gel electrophoresis. The mutagenesis product should appear as a band at 6kb in lane 3. The H50N forward primer should appear at 1kb in lane 5 and
Figure 5. H50N Gel and Ideal Gel
A. Idealized Gel for H50N Mutagenesis and Primer Checks and A53E Plasmid Purification 15 μL of each respective sample were loaded in each well. 5 μL of high mass ladder was loaded into lane 1 to estimate the size of PCR product. H50N mutagenesis product is loaded into lane 4. 1:50 diluted WT Template was loaded into lane 3 as a comparison for mutagenesis. Successful mutagenesis reactions are expected to be significantly larger and darker than background as both had 1:50 WT template dilution. H50N forward and reverse primer reaction are in lanes 5 and 6 respectively. Plasmid Purifications were prepared using 13.5 μL of purified plasmid from and individual E. coli colony and 1.5 μL buffer. Plasmid purifications are in lanes 10, 11, and 12. These plasmids were obtained from another group with a successful mutagenesis and transformation.
B. Gel for H50N Mutagenesis and Primer Confirmation and A53E Plasmid Purification Template Background product is not visible on gel but did appear in a faint amount on scan in lane 3. No Mutagenesis Product appeared in lane 4. All three plasmid purifications for A53D in lanes 10, 11, and 12 show strong plasmid presence that can be prepared for sequencing.
the H50N reverse primer should appear at 171 base pairs in lane 6. The idealized gel in Figure 5A summarizes expected results. Actual results are shown in the gel in Figure 5B. Bands are present in expected location in
lanes 5 and 6 for the primer control reactions. However, no mutagenesis product appears in lane 4. Lane 3 contained very little, but identifiable template background plasmid. Thus H50N mutagenesis did not produce the point mutation as intended and progress cannot be continued, even though primers proved effective and functional.
A53D Plasmid Purification and Sequencing
Since G51D and G51R mutagenesis failed, we were given group 4’s A53D mutant which showed successful mutagenesis and transformation as can be seen in Figure 6. Mutagenesis and transformation was previously successful for this mutant so we continued the process and performed plasmid purification according to DebBurman, 42. Purified plasmid was then confirmed on a DNA gel. Plasmid purification is shown in Figure 5A in lanes 10, 11, and 12 from three different colonies. A complete mutagenized product of about 6 kb is expected. All three mutagenized plasmid shows successful purification as indicated by thick dark bands in Figure 5B. This plasmid was then prepared for sequencing by placing 5 μl of it in a parafilmed microcentrifuge tube and placing that tube into a parafilmed petri dish. This was then sent for sequencing at the University of Chicago.
A53D Sequencing Results
Sequencing preparation and procedure is described in DebBurman 46.The primers that were used in A53D mutagenesis are given in Figure 6A. The complete sequencing results for A53D are given in Figure 7A. As can be seen, mutagenesis was unsuccessful. Only one of three purified plasmid sent was readable and that sequence came out wild type.
Figure 6. A53D Group 4 Mutagenesis and Transformations
A. Primer Design for A53D
These are the sequences for the primers designed by group four for the mutagenesis of A53D. The first sequence is the forward primer and the second sequence is the reverse primer. The codon change, GCA to GAT, results in and aspartic acid at the 53rd codon.
B. Gel for A53D Mutagenesis
15 μL of high mass ladder was inserted into lane 1. 5 μL of 1:50 WT template dilution was placed in lane 2. 5 μL of A53Q, A53D, A53R, and A53G were placed in lanes 3, 5, 7, and 9. A53D is clearly present and much stronger than template background in lane 6 at about 6kb.
C. E. coli transformation
20 and 80 μL of cells transformed with A53D mutagenized plasmid were plated onto LB+AMP. Growth is clearly present on both conditions.
Figure 7. Sequencing Results
A. Sequencing Results for A53D: The start codon for α-syn is highlighted yellow. The mutation site is coded red. No mutation occurred and the sequencing came back wild type
B. Successful Sequencing Results for A53D Group 2: The start codon for α-syn is highlighted yellow. The mutation site is coded red. As can be seen GCA was mutagenized to GAT with no other random mutants. This mutagenesis is successful as the point mutation has been made and it is ready to transform into S .Cerevisiae.
However, as can be in Figure 7B, group four was able to successful create the A53D mutant. This mutant was given to our lab group to be transformed into S. cerevisiae.
A53D Yeast Transformation
The purified and sequenced plasmid was transformed into S. Cerevisiae cells according to (DebBurman, 49). The transformed mixture was then plated onto 30 μl and 200 μl SC-URA and 30 μl YPD. In addition, a positive control, consisting of untransformed S. cerevisiae plated onYPD, and a negative control consisting of sterile, deionized water plated on SC-URA was prepared. SC-URA acts as a selection factor to ensure that only transformed yeast can grow on the media, because untransformed yeast do not have the genes necessary for uracil biosynthesis. The vector being transformed contains a +URA domain that allows uracil to be
Figure 8. A53D Yeast Transformation
A S. Cerevisiae Transformation After the sequence for A53D was confirmed, purified plasmid was transformed into S. cerevisiae and plated on SC-URA and YPD media. 200μL and 30μL solutions of transformed yeast cells were plated onto SC-URA and 30 30μL was transformed onto YPD. There was growth on all the transformation plates as expected. Thus, A53D was successfully transformed into yeast and experimentation can commence.
B S. Cerevisiae Transformation Controls
In addition, a negative and positive control were done. The positive plate used 30μL of S. cerevisiae transformed with empty vector on SC-URA, while the negative control used 30μL of sterile, deionized water on SC-URA. The negative control showed no growth, while the positive control showed noticeable growth.
Figure 9. A53D Spotting
A. Spotting Assay for A53D
S. Cerevisiae spotting for A53D. Lanes identified by vector and mutant type in red. Vector contains Gal promoter for α-syn so α-syn is only present in galactose condition. Glucose functions as a control to indicate that even and consistent amounts of cells were placed
biosynthesized in organism with that vector. Transformed S. Cerevisiae produce multiple colonies in both the 30 μl and the 200 μl conditions. Thus S. cerevisiae transformation was a success and experimentation could be performed on the A53D α-syn.
A serial dilution or spotting assay was performed with the new A53D to assess its relative toxicity. The A53D mutant was compared to empty pYes2 Vector, pYes2 Vector with GFP, WT α-syn, and the A53E familial mutant. The glucose spotting (no α-syn) was relatively consistent across all mutants, so it can be inferred that spotting was relatively even and the quantity of cells was consistent across all mutants. The cells in a galactose some weak trends. It is clear that S. cerevisiae with empty vector and vector GFP grew better than those with any α-syn. It also appears the A53D grows somewhat better than the A53E mutant. However, it appears that WT α-syn has the least growth of all. Galactose appears to have grown more, but this is because the images of the plates were taken at different time points. Glucose plates were imaged 28 hours after spotting while galactose plates were imaged 66 hours after spotting.
Although we failed to reach our goal of creating the G51A, G51D, and H50N mutants of α-syn in S. cerevisiae, we were able to contribute to the project by transforming the A53D mutant into S. cerevisiae and performing the first spotting assay analyzing it. The G51A and G51D had to be delayed due to failed E. coli transformation, and the H50N had to be delayed due to failed mutagenesis. The first A53D mutant that was given to use by group 4 failed to have the intended mutant and appeared as WT.
The G51D and G51A mutagenesis and control reactions did not appear to have any problems initially. It may be due to inexperience and poor coordination that the reactions did not succeed in producing a viable mutagenesis product, although this is unlikely due to the entire lab’s first mutagenesis failing. The issue with mutagenesis may either have been a result of the original technique or something done in lab preparation. The G51A’s control reactions worked properly in the first gel, but it appears G51R’s control reactions did not work. This is likely a result of an uneven gel that did not allow the control reaction dye mix to enter the wells. This was clearly seen by a significant amount a blue dye that escaped the wells and was scattered throughout the gel unit. Other groups gel confirmations of G51R further support this. Our second mutagenesis product also did not appear to have a strong possibility of success. Although we were told by Charles Alvarado and Morgan Marshall that mutagenesis had succeed, it appears that there is no mutagenesis product in lanes 2 or 5, especially when you compare it to template background. With such mutagenesis results, it was not a surprise that E. coli transformation failed.
H50N had nearly the same results as G51D and G51A. Mutagenesis appears to not have succeeded as no band could be seen by lane 2 of figure 5B. However, both the forward and reverse primers reaction succeeded. This issue is further compounded by no clear template background plasmid in lane 3. This may have resulted from the contrast between the purified plasmids and the mutagenesis reactions. It could also have resulted from poor technique when producing the 1:50 WT dilution. There remains no definitive reason why H50N mutagenesis failed.
At this point we had no success in any of our reactions and could not proceed with our initially intended experiments. Thus we were given A53D from group four which had successful mutagenesis and E. coli transformations as can be seen in figure 6. The first three E. coli colonies showed strong plasmid purification success. However, two of these could not be identified during sequencing and the final sequence came back positive. We cannot determine any sources of error in our procedure, so the issue may have begun prior to our group receiving this sample. Again, we were given another sample of A53D from group two that was successfully sequenced and mutagenized. This was successfully transformed into S. cerevisiae with no apparent issue. There was clear colony growth in both 30 and 200 μl conditions in figure 7A. From this point, experimentation could begin. If our initial goal had been to produce the A53D mutant we would have had partial success. With an extra lab, we decided to begin experimentation of the A53D in S. cerevisiae. Our results showed some marginal success. We could conclude that we were at least marginally successful in putting even amounts of cells in each sample as can be seen by our relatively even glucose spottings in figure 8. Our galactose spotting were taken out a bit later than optimal, so our predictions are somewhat more inaccurate. It appears that A53D grew somewhat better than A53E, implying that A53D is less toxic. This is expected as aspartic acid is slightly smaller than glutamic acid and would cause less of a structural disruption when converted from alanine. This seems to imply that a smaller amino acid at this site is less toxic than a larger amino acid. However, we cannot infer anything about the differences in charges as both aspartic acid and glutamic acid are negatively charged. However, spotting results come in to question because both A53E and A53D actually show more growth than wild type α-syn. This conflicts with previous data which shows WT α-syn grows better than most A53E and most familial mutations (Fares et al., 2014). Thus, it would not be reasonable to make any remotely definitive conclusions about A53D without more data.
Many more experiments could be performed on A53D, now that it is successfully transformed into S. cerevisiae. Other forms of assays that assay toxicity could be performed such as various survival or viability assays using other vital stains or dielectrophoresis. Dieletrophoresis uses electrical fields to separate living S. cerevisiae from dead S. cerevisiae with rather high accuracy (Markx, Talary & Pethig, 1994). Additionally, an assay like western blotting could assay whether the various mutations cause a change in how much α-syn is produced and cleared with S. cerevisiae. Such an assay would collect all the α-syn present in a cell and stain it according to its quantity and size (Martins et al. 1988). Finally, the GFP present one the vector could allow α-syn to be visualize in vivo (Cabantous and Waldo, 2006. This could be done through fluorescence microscopy which allows the localization and quantity of α-syn to be determined directly by the presence of GFP (Giron and Salto, 2011).
Overall, although our project was a failure, there were moments of success. G51A, G51R and H50N never had a strong, successful mutagenesis. Our first mutagenized A53D plasmid did not show the intended mutant. However, once we were given a plasmid with that was sequenced we successfully transformed it into S. cerevisiae and began preliminary analyze which may show that A53D is less toxic than A53E. With continued research, this specific lab section’s work on may provide insight into the specific familial mutant’s point mutations. This will ultimately enhance our overall understanding of α-syn and PD, which will hopefully lead to more effective treatments and potential a cure for PD. Our particular lab project may be the smallest piece of the puzzle in understanding PD.
Materials and Methods
G51A and G51R Primer Design
Primer design was done according to DebBurman, 28. To design our primers, we compared the standard sequence of α-syn, which codes for glycine at the 51st amino acid. We created primers with the original sequence of α-syn in mind, using six codons on each side of the 51st amino acid, changing only a single amino acid in each mutant compared to wild-type α-syn from glycine to arginine, and from glycine to alanine (Figure 2- colored sequences changed). Both forward and reverse primers were created to produce a complete circular mutagenized vector. These plasmids were ordered in as oligonucleotides from Integrated DNA Technologies (IDT).
G51A and G51R Mutagenesis
The Mutagenesis reaction were done according to DebBurman, 29. G51A, G51D, and H50N sequences are given in figure 2. A53D sequence is given in figure 6A. Using WT α-syn tagged with GFP, which allows fluorescence microscopy, the designed primers, and a pYES2 vector, we attempted to mutagenize WT α-syn. We used six different combinations to attempt the mutagenesis. In each tube, we used 8.5 μL of sterile, RNAase free water and 1.0 μL of template plasmid. The mutagenesis reactions contained their respective primers and mutagenesis master mix. The forward primer confirmation contained the designed forward primer, GFP reverse primer, and Taq master mix. The reverse primer confirmation contained the designed reverse primer, α-syn forward primer and Taq master mix. These tubes also contained 32.7 microliters of sterile H2O and 13.4 microliters of a Taq or mutagenesis master mix which was as specified in DebBurman, 31. The positive control, formulated by Morgan Marshall, contained 34.1 microliters of sterile RNase-free H2O, 1.5 microliters of the control primer mix, 1 microliter of pUC19WHITE Control plasmid, 13.4 mutagenesis master mix, 10x Accuprime pfx Reaction Mix, 10x enhancer, DNA methylase, 25x SAM, and Accuprime Pfx. The results were ran on a gel and analyze using Image Lab 4.01 base on the specifications in DebBurman, 37.
G51A and G51R Transformations (Mutagenesis Lab)
The E. coli were done according to DebBurman, 34. Using gel electrophoresis and the PCR products from the mutagenesis lab, we transplanted our plasmid vectors onto an agarose gel. We synthesized this gel using 0.4 agarose powder and 40 ml 1X TAE buffer. The gel was left to dry for 30 minutes and then we added 10x loading dye to our PYES2 plasmid vector to visualize the bands on the gel electrophoresis. We separated the DNA by sizes using the agarose gel at an electric current of 100V, allowing the smallest fragments of DNA to travel the furthest, and the most amplified DNA to have the thickest bands- see ideal bands of Figure 3. This allows for the amount and size of DNA to be compared to other samples.
The Mutagenesis reaction were done according to DebBurman, 29. We attempted to perform the same mutagenesis reaction on a different α-syn mutant- H50N. The procedure for this step was the same as with the G51A and G51R mutations. A mutagenesis reaction with H50N forward and reverse primers and Pfx master mix was performed. Additional a forward primer check with the H50N forward primer, GFP reverse primer, and Taq master mix and a reverse primer check with H50N reverse primer, α-syn forward primer and Taq master mix was performed. Upon performing PCR, we then attempted transformations using our G51A and G51R mutants, need to review process for this- talk about heat shock and removing methylase DNA (Figures 4 and 5).
H50N Plasmid Purification and Sequencing
Plasmid purification was done according to DebBurman, 42. Upon transformation of our mutant PCR results to cell cultures, we then collected cells to extract and transfer to a plasmid. The H50N sequence was sent to an outside lab for sequencing.
A53D from group two Yeast Transformation
This step was done according to DebBurman, 49. The goal of this portion of the lab was to transfer mutated α-syn into S. cerevisiae yeast by means of electroporation and spheroplasting. In the electroporation, we were using electricity treatment with lithium acetate to increase the permeability of the cells, then we used the enzyme zymolase to spheroplast (digest the cell wall) (Figure 7). Because of the lack of success with the previous mutations, we now were using the A53D mutation, as this mutation was successful in other groups’ mutagenesis. Upon transformation, the peer teacher combined these with YPD to incubate over a 24 hour period to then be able to measure the amount of cells that successfully proliferated.
Spotting Assay was given emailed from lab with no information to cite. To measure the amount of success of the transformation into the S. cerevisiae, we took the cells that were grown overnight in the YPD medium and diluted them to a specific cell concentration. To measure the concentration, we counted the cells on a hemocytometer by eye, then averaged the totals of all six cell types of the empty vector, wild-type α-syn, the original A53E mutagen, two different A53D mutants, and a GFP. Upon creating a uniform concentration of 2x10^7 cells/mL in each cell type, we then created serial five 1:5 dilutions of the cells of in SC-URA Glu and Gal. Glucose does not cause α-syn to be produced and acts as a loading control to ensure that equal amounts of cells were added to each lane. Galactose causes α-syn to be produced due to a Gal promotor for α-syn in the pYES2 vector. We then incubated these media over 72 hours to measure the difference in growth between mediums via the amount of growth visible on our serial dilution petri dishes (Figure 9).
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W\e would like to thank Dr. DebBurman and Dr. Wilcox for their assistance and direction throughout the duration of this project. Additionally, we would like to thanks Charles Alvarado for his assistance in lab preparation and Morgen Marshall for her advice and assistance and nights spent in the lab on behalf of our class. Finally, we would like to thank Lake Forest College for providing us with the means to perform these experiments.
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