- <div style="background-image:url(/live/image/gid/32/width/1600/height/300/crop/1/41839_V14Cover_Lynch_Artwork.2.rev.1520229233.png)"/>
Novel Mutations A53D, A53G, A53G, A53R, and H50D of α-Synuclein in Parkinson’s Dis- ease observed in budding yeast to deter- mine effects in aggregation by different amino acid properties
Viktoriya Georgieva & Julia Garcia
Department of Biology
Lake Forest College
Lake Forest, IL 60045
Parkinson’s disease (PD) is a neurodegenerative disorder that results from the loss of dopaminergic neurons in the substantia nigra of the brain. α-Synuclein (α-Syn) is the disease-associated pro- tein that is prevalent among PD patients. Aggregations of this protein result in the formation of Lewy bodies present in all PD cases. Famil- ial mutations of α-Syn: A53E and H50Q were recently discovered. Our study aimed to create new mutations to further explore the effects of the properties of each amino acid on aeration in each mutant. We cre- ated four mutations by changing the alanine amino acid on the 53rd position to an aspartic acid, A53D, glycine, A53G, and arginine, A53R; we also created a mutation of the histidine amino acid on the 50th position to an aspartic acid, H50D. We hypothesized that changing the chemical properties of the α-Syn sequence in the A53D and A53R mutations would aggregate aggressively, compared to the wild-type α-Syn, because of its impaired binding ability, much like the A53E mutation. We also hypothesized that there would be no changes to aggregation patterns in A53G mutants, because there are no chang- es in amino acid property between alanine and glycine. Lastly, we hypothesized an H50D mutation would also increase aggregation, behaving similarly to H50Q. We were able to successfully mutagen- ize and transform A53G into bacteria, amplify, purify & sequence the plasmid, then transform it into yeast. We have also completed prelim- inary spotting trials and found both A53G serial dilutions show less growth than galactose WT or A53E in yeast.
Parkinson’s disease (PD) is a progressive, hypokinetic motor disorder characterized by the loss of dopaminergic neurons in the substan- tia nigra, the brain structure associated with movement (Ross et al., 2011). PD negatively affects several regions of the brain that control balance and voluntary movement, thereby classifying the primary symptoms of this dis- ease. Patients with PD experience resting tremors, bradykinesia, postural instability, and rigidity (Jankovic, 2007). Along with the decrease in neural cells, surviving cells are afflicted by presence of Lewy bodies (Rutherford et al., 2014). Lewy bodies, which are proteins that abnormally clump to- gether in neural cells, are formed from the aggregation of the α-Synuclein protein. α-Synuclein (α-Syn) is a presynaptic protein, primarily expressed in the presynaptic terminals of the central nervous system (Recchia et al., 2004). The conformational flexibility of α-Syn allows the protein to misfold and form accretions, ultimately generating Lewy body lesions of neuronal degeneration. Cell death and toxicity, induced by these Lewy bodies, are the hallmark features of Parkinson’s disease (Proukakis et al., 2013; Petru- celli et al., 2002).
Two main types of PD exist: the sporadic form, which is caused by environmental factors such as toxins and epigenetic changes and ac- counts for 90% of PD cases, and the familial form, which is reported in 10- 15% of all cases (Papapetropoulos et al., 2007). Nine gene loci have been identified as being responsible for familial PD: SNCA, PARKIN, UCHL-1, PINK1, LRRK2, ATP13A2, DJ-1, GIGYF2, and HTRA1 (Klein & Westen- berger, 2012). The mutations and alterations of each of these genes in- crease the risk of α-synuclein misfolding. Moreover, SNCA is the gene that encodes for the production of α-Syn, the protein prevalent in both familial and sporadic PD aggregates. Through a series of experiments, Pasanen et al. (2014) showed that the areas of the brain with significant amounts of SNCA, the gene encoding for α-Syn, also had massive neuronal loss.
Previous research discovered mutations in the α-Syn pro- tein, causing modifications in the protein’s cellular structure and genetic makeup. The regular function of α-Syn helps with neuronal signaling by regulating the release of dopamine and maintaining synaptic vesicles in the presynaptic terminal (Goedert, 2001); its three known domains - the N-domain, M-domain, and C-domain - each contribute to the protein’s main function. The N-domain, at the amino end of α-Syn, contributes to the protein’s membrane binding ability. The M-domain affects the protein’s ability to aggregate, and C-domain, containing the carboxyl end, allows the protein to remain soluble in solution (Baba et al., 1998). The following mutations have been discovered in the SNCA gene: A30P, E46K, H50Q, G51D, A53E, and A53T. Three of these mutations, A30P, E46K, and A53T, have been extensively studied in different model organisms and their pa- thology is well known. These pathogenic mutations were shown to affect membrane-binding of α-Syn in the A53T mutation (Athanassiadou et al., 1999), as well as in E46K, without results of neuronal toxicity (Zarranz et al., 2004). Altering the protein’s affinity for membrane-binding ultimately leads to aggregation, also shown in A30P, due to random coiling (Recchia et al., 2004).
Although H50Q, G51D, and A53E were only discovered with- in the past two years, researchers have already started to analyze their properties and phenotypes (Porcari et al., 2014; Rutherford et al., 2014; Pasanen et al., 2014). In H50Q, histidine gets mutated to an uncharged glutamine at the 50th amino acid, showing increased aggregation, com- pared to wild-type (WT) version of the protein. Its ability to strongly sta- bilize fibrils increases fibrillization (Porcari et al., 2014), further promoting Lewy body formation. If α-Syn is not binding to the membrane, then it will localize in the cytoplasm, leading to aggregation. The mutation also affects mitochondrial fragmentation, inducing neuronal cell death (Khalaf et al., 2014). For G51D, glycine gets mutated to a charged Aspartic Acid at the 51st amino acid, showing decreased aggregation; however, G51D induces neuronal toxicity when introduced to stress conditions (Rutherford et al., 2014). Lastly, A53E has the 53rd amino acid converted from a nonpolar alanine to an acidic, negative glutamic acid (Pasanen et al., 2014).
The discovered mutations of the α-Syn protein were predomi- nantly reported to have affected the membrane-binding functionality and aggregation properties of α-Syn; however, the effects of phenotypic chang- es in these mutations have not yet been fully examined. It remains un- known whether the increase in PD pathology occurs due to a change in the primary sequence or a change in the polarity and charge of the amino acid. More research is needed to further investigate the physical composition of these mutations and their potential role in the PD pathology.
The study investigated three novel mutations of the 53rd amino acid: A53D, A53R, and A53G as well as one novel mutation on the 50th amino acid, H50D, to better understand the physical properties of these mutations. The mutation A53E has been reported to cause α-Syn aggrega- tion due to its impaired membrane binding capability (Ghosh et al., 2014), so by altering the primary sequence, α-Syn’s function, localization, and tox- icity will be affected. We predicted that changing alanine to an acidic amino acid, such as aspartic acid (in the A53D mutation), would have resulted in the same α-Syn aggregation patterns as the A53E mutation. A mutation to change a nonpolar amino acid to a basic amino acid, like arginine (in the A53R mutation) is predicted to affect the protein’s membrane-binding abil- ity and aggregation tendencies. We expected to see no change in α-Syn’s original functions in the mutation A53G, because the glycine, which re- placed the alanine amino acid, is also nonpolar.
The mutation, H50Q, showed increased aggregation compared to WT version of the protein and causing fibrillary formation (Porcari et al., 2014), so we predicted that changing histidine to an acidic aspartic acid would have also increased aggregation.
In this study, Saccharomyces cerevisiae, budding yeast, was used as a model to evaluate the misfolding, aggregation, and toxicity-in- ducing ability of the four mutants (A53G, A53D, A53R, H50D). S.cerevisiae can be easily manipulated and its short generation time allows for model- ing of protein related diseases (Allendoerfer et al., 2011). Since PD is a protein misfolding disorder, yeast provides the ability to see how α-Syn is produced, expressed, and degraded. Yeast is also easily accessible and inexpensive, making it an ideal experimental model for undergraduate stu- dents to study PD.
The study aimed to create these mutations (A53D, A53G, A53R, H50D) to further explore the properties of these amino acid positions of α-Syn. To create these mutations, unique primers were designed to use in mutagenesis-based PCR. The mutated plasmids were then transformed into bacteria to produce large quantities of the mutated gene, and later, extracted from the bacteria, purified, sequenced, and inserted into yeast for observation. A spotting assay was performed to observe and compare the effects of the mutation of α-Syn and the WT version of α-Syn.
Overall Project Design and Gene Amplification by PCR
To create these novel mutations, the overall schematic shown in Figure 1C was followed (DebBurman, 2015, 23). Full-length wild-type α-Syn underwent mutagenesis based PCR, using specific forward and re- verse primers to mutate the 53rd alanine into an aspartic acid (A53D), gly- cine (A53G), and arginine (A53R) (Figure 1A,B). The mutagenesis by PCR product was run on an agarose gel to verify that amplification occurred by comparison with a template background. The mutagenesis of H50D was also attempted; however, the amplification of the mutated plasmid yielded negative results (Figure 3B). As a result, the H50D mutant was abandoned. Using Mutagenesis based PCR, the specific mutations were created within the primary sequence, and the mutated α-Syn DNA was replicated. The mutated plasmid was transformed into bacterial cells to further amplify the mutant plasmid within E. coli. Following bacterial transformation, three bac- teria colonies were selected, and the α-Syn DNA plasmid was extracted and purified from the lysed bacterial cells. Gel electrophoresis was used again to verify that the plasmid had been extracted correctly from the gel. Once verified, the plasmid was sent out for sequencing. Following se- quence confirmation, the correctly sequenced plasmids were transformed into S. cerevisiae, allowing the finished product to be analyzed. A prelim- inary serial dilution-spotting assay was performed to see the effect of the mutant α-Syn against the WT and naturally occurring α-Syn mutant.
Figure 1. WT vs. Mutant Plasmid of Alpha-Synuclein and GFP cartoon, PCR Pairs Table, and Overall Schematic of Experimental Design. (A) Contrast of GFP-tagged WT a-Syn plasmid vs. Mutant (A53G, A53D, A53R, and H50D) plasmid, (B) PCR Primer Pairs Table, (C) Overall schematic of experimental design, showing what procedures will be used to transform yeast with newly made mutations.
As shown, the mutations, A53G, A53D, and A53R were created using spe- cialized forward and reverse primers containing the mutations on the WT α-Syn plasmid (Figure 1C).Three mutagenesis reactions were prepared, each containing one of the three desired mutations. Additionally, six PCR reaction tubes were created to serve as primer controls, ensuring that the primers would bind the α-Syn plasmid properly. An agarose ethidium bro- mide gel was run to verify the amplification of the mutagenesis product. The three mutagenesis reactions provided no positive results, because no significant amplification was seen between the mutagenized plasmid (lanes 3-5) and the template background (lanes 2 and 4) located at 6kb, and the two of the six primer (one forward and one reverse) reactions pro- vided negative results (lanes 10-15) with no bands at the 1000 bp and 200 bp respectively (Figure 2Bi), differing from the expected scenario (Figure 2A). It was worth noting that the particular primers do bind the plasmid, as shown by the darker colored bands in lanes 9 and 10, indicating that both primer controls of A53R worked (Figure 2Bii). Mutagenesis was performed again by the DebBurman lab, which yielded positive results for all three A53 novel mutations. In Figure 2C, there is a noticeable amplification of mutants A53D, A53R, and A53G plasmids in lanes 5-8 in A53E Gel Check. In G51 + A53Q Gels, mutants G51A, G51Q, G51R, and A53Q were identi- fied (lanes 3-6). When the G51 + A53Q reactions were repeated, the Deb- Burman lab was also able to identify G51E (lane 3). A fourth mutation to create H50D was also performed; however, this yielded no positive result, because no noticeable amplification of the plasmid was seen (Figure 3A). After that point, the H50D was no longer a part of the tool creation process.
Figure 2. Gel Electrophoresis and Bacterial Transformation. (A) Computerized Ideal Gel of Mutants A53D, A53G, A53R and expected weights; (Bi) Gel images of positive primer control reactions for A53D FP and A53D RP, A53R FP; (Bii) Gel Image displaying positive primer control reactions for A53R mutation as labeled in lanes 9 and 10, performed by colleagues (C) Gel Images by DebBur- man Lab for A53E, G51, and A53QMutations – each indicating positive results for A53D, A53R, A53G (lanes 5,7,9 in first gel image, G51A, G51Q, G51R, A53Q (lanes 3-6 in second gel image), and G51E (lane 3 in third gel image); (D) Bacte- rial Transformation Plates: 20μL and 80 μL in LB + Amp plates for A53G, A53D, and A53R mutations (i.) Before Overnight Observation and (ii.) After 24 Hours – there appears to be no growth in A53R plates and minimal growth in A53D plates. Four colonies were labeled in A53G plates, but only three colonies were selected for extraction; (iii.) Positive and Negative Controls of Bacterial Plates – no growth shown in either of the two positive controls (LB + Amp plates with chemical X-gal), nor the negative control (sterile water), as expected.
After verifying the PCR product, the newly mutagenized plas- mid was transformed into competent DH5-α E. coli cells. The transformed bacterial cells were plated onto LB + Amp media to ensure that only trans- formed bacterial cells were selected. Two volumes (80μL and 20μL) were plated for each mutant to contrast growth in each medium (Figure 2Di), along with two positive controls plates 7 and 8) and one negative control (plate 9) (Figure 2Diii). Bacterial transformations were successful for both the A53G (plates 3 and 4) and A53D mutants, showing transformed col- onies (plates 5 and 6) (Figure 2Dii). In contrast, the A53R mutant did not yield a successful transformation, providing zero colonies. Strategically, it was difficult to interpret non-control rates. It was also worth noting that neither of the positive controls (LB + Amp plates with chemical X-gal) had observable colonies growing. The negative controls (sterile water) had no colonies as expected, but given the state of the positive controls, it was hard to validate the results seen on the negative control plate (Figures 2D).
The bacterial cells underwent plasmid prep to isolate the plasmid from the cells. Later, gel electrophoresis was run to ensure that the plasmid was purified correctly (Figure 3B). The three dark bands in lanes 7-9 indicated a positive purification of the three plasmids; however, only one plasmid was sent to the University of Chicago DNA Sequencing & Genotyping Facility for sequencing. The A53G mutation was successful, as seen in Figure 3C.
Figure 3. Gel Image of H50D Mutagenesis and Control Primers and DNA Se- quencing Results. (A) Computerized Ideal Gel of H50D Mutagenesis Reactions and Control Primers with weights; (B) Actual Gel Image indicating positive re- sults for purified plasmid (shown by dark colored bands) (C) DNA Sequencing of A53G Mutant – labeled start codon in Yellow “ATG” and positive result for mutation A53G in the 53rd codon: “GGA” in red.
Plasmid Transformation into Yeast
After receiving a positive sequence result from the lab at Univer- sity of Chicago DNA Sequencing & Genotyping facility, the mutated plas- mid was then transformed into yeast. Three plates for the mutant plasmid were created. Two volumes (200uL and 20uL) were plated onto SC-URA Glucose plates, which selected for the plasmid in the yeast cells. Both of these plates had yeast colonies. The third plate was the positive control on YPD to ensure that the cells were viable (Figure 4A). The negative controls in saline water showed no cell growth in the 200uL (plate 4) and minimal growth in plates 5 and 6 for both the empty vector and the WT, probably indicating contamination. As expected, yeast appeared to grow in the YPD plates 8 and 9 for both the empty vector and WT; however, there was also growth for the negative control (water) in plate 7, possibly indicating con- tamination (Figure 4B).
Figure 4. Yeast Transformation Plates with Positive and Negative Controls (SC- Ura v. YPD). (A) Positive control in YPD in plate 3 shows yeast growth, indicat- ing successful transformation. Little to no growth in plates 1 and 2 indicate that yeast were barely able to survive (as they are somewhat seen in plate 2) in SC-Ura medium. Only yeast that grow (plate 2) indicate that they’ve been successfully transformed; (B) negative controls in saline water showed no cell growth in the 200uL (plate 4) and minimal growth in plates 5 and 6 for both the empty vector and the WT, indicating contamination. As expected, yeast appeared to grow in the YPD plates 8 and 9 for both the empty vector and WT; however, there was also growth for the negative control (water) in plate 7, pos- sibly indicating contamination (Figure 4B).
A53G Toxicity Spotting
With the A53G plasmid in budding yeast, a serial dilution spotting assay was performed to compare the toxicity of the mutant α-Syn A53G to the WT and A53E α-Syn. A 1:5 dilution of our (1) empty vector, WT α-Syn, original A53G mutant, then 2 samples of A53G mutant were added in six wells (Figure 5A and 5B). Two glucose plates served as growth controls because α-Syn is not expressed when yeast is grown on glucose, meaning no difference in growth should have been seen between the variations. The plates showed a lot of variation in the seven samples that were spotted, with a definite trend shown in the plates. A53G showed less growth than anything else including WT and A53E.
Figure 5. Toxicity Spotting. (A) Growth Controls in Glucose + SC-Ura Plates First Row – Empty Vector (1 – undiluted, 2 – 1:5, 3 – 1:25, 4 – 1:125, 5 – 1:625, 6–1:3125)SecondRow–WTa-Syn(1–undiluted,2–1:5,3–1:25,4–1:125,5 – 1:625, 6 – 1:3125). *Each Column follows the same dilution, (B) Top 2 Plates – SC-Ura + Galactose media with all six dilutions including Empty Vector and WT a-Syn in the first two rows, followed by Third Row – Mutant a-Syn 1 (prepped by colleagues*), Fourth Row – Mutant a-Syn 2, and Fifth Row – GFP. Bottom 2 Plates – SC-Ura + Glucose media with all six dilutions, First Row – Empty Vec- tor, Second Row – WT a-Syn, Third Row – Mutant a-Syn 1, Fourth Row – Mutant a-Syn 2, and Fifth Row – GFP.
Problem & Main Findings
The goal of this experiment was to create mutations (A53D, A53G, A53R, and H50D) of the α-Syn gene and transform them into yeast to further study their properties and functions. Changes in the conforma- tional shape of the original amino acid could cause genetic variations, so we hypothesized that changing the alanine amino acid on the 53rd codon of the α-Syn sequence to an acidic amino acid like aspartic Acid (A53D), would result in similar aggregation patterns, similar to those seen with the A53E mutation. A mutation to change a nonpolar amino acid to become a
basic amino acid, like arginine (A53R) was predicted to affect the protein’s membrane-binding ability and aggregation tendencies. We also predicted that we would see similar functions, as the wild-type version of α-Syn in the mutation A53G because Glycine and the original amino acid, Alanine, are both nonpolar. Finally, we predicted that H50D would cause cell toxicity, due to its change from a basic amino acid to acidic. As Figure 4A showed, we were only able to successfully mutagenize and sequence the A53G mu- tant, so more studies should be done to observe this mutant’s properties and functions. Poor lab technique and miscommunication could have cer- tainly affected the results of this study; however, our lack of lab experience, especially in interpreting experimental, non-control results, could have also resulted in overlooking our data.
Mutagenesis of the α-Synuclein
In this experiment, we attempted to create the A53D, A53G, A53R, and H50D mutations through PCR-based mutagenesis, and then further tested the success of these mutations through gel electrophoresis. As demonstrated in Figure 2B, gel electrophoresis failed to produce any of the mutant bands in the expected areas; however, four out of the six control primers appeared on the gel. This could have been the result of a faulty PCR machine, or having overexposed PCR preps before transferring over to the gel. Among the four, the forward and reverse primer controls for the A53D mutation were evident. This figure also showed positive results in the reverse primer control for the A53G mutation and the forward primer control for the A53R mutation (Figure 2B). Our colleagues, who had also performed experiments with the A53G gene found that both of their reverse and forward primers worked for A53R, indicating that if the PCR reactions had worked correctly, then the mutagenesis product should have also ap- peared on the gel as slightly darker than its primers. The gel electropho- resis procedure failed to produce any observable bands in the expected areas for any of our mutants; therefore, we concluded that neither one of our four mutagenesis reactions worked. Though some of our primers were able to bind to the plasmid, as indicated by the darker colored bands, over- all, there were consistent negative results in the mutagenesis reactions of our colleagues’ gel images. The DebBurman lab repeated the PCR prep to recreate the mutations, and successfully isolated the three mutants, which were confirmed by their gel images (Figure 2C), appearing at 6000kb on the agarose gel for the A53R, A53G, and A53D mutants. Despite having performed two PCR based mutagenesis reactions with the H50D mutation, H50D was consistently not appearing in the gel images. This could have also been a result of poor PCR prep; regardless, no further experiments were made with the H50D mutant. The inconsistent results from the gel images reinforced the idea that gel electrophoresis is a difficult assay to work with and analyze, which could lead to misinterpretations of the data.
Using the positive mutagenesis results expressed in our peer teachers’ gel images, we transformed this DNA into E.coli cells. This was confirmed by the bacterial colonies labeled on the LB + Amp plates (Fig- ure 2D). The old plasmid was degraded by the bacteria, which contained a special enzyme that degrades methylated DNA. Because the plasmid contained the gene for ampicillin resistance, the cells would not have been able to survive in the LB + Amp plates; therefore, the growth that was shown in these plates meant that bacteria were transformed. However, only the A53D and A53G had colonies, and even then, the colonies were very small and very few. The A53R mutant had no colonies at all. It was later discovered that the water bath used to heat shock the bacteria was not at 42 degrees Celsius, but instead, it was in the mid 30s which could have affected the quality of the transformation and reducing the quantity of colonies seen.
Furthering this experiment, the plasmid was extracted from these bacterial colonies, and purified, then verified in another round of gel electrophoresis. This part of the experiment was essential to our ultimate goal of transforming the carefully designed mutations into yeast. The three dark bands in lanes 7-9 shown in the gel image (Figure 3B) indicated posi- tive purification of the three plasmids; however, only one plasmid was sent to the University of Chicago DNA Sequencing & Genotyping Facility for sequencing. The sequencing results were successful in that our desired mutation of A53G was present.
Plasmid Transformation into Yeast
After receiving a positive sequence result from the lab at the University of Chicago DNA Sequencing & Genotyping Facility, the mutated plasmid was transformed into yeast. Two volumes (200uL and 20uL) were plated onto SC-URA Glucose plates, which selected for the plasmid in the yeast cells. The yeast vector had a URA3 gene, which allowed the uracil deficient BY4741 yeast strain to produce uracil if it received the plasmid. So, any colony grown on SC-URA glucose plate had the plasmid and was successfully transformed. Both of these plates had yeast colonies grown on the plate. The third plate was the positive control for the plasmid grown on YPD to ensure that the cells were viable (Figure 5A). All three plates showed positive results, indicating yeast growth. The three negative con- trols showed no cell growth (Figure 5B). Having successfully transformed A53G, the mutant was ready to be analyzed.
A53G Toxicity Spotting
A 1:5 serial dilution spotting assay was performed to compare the toxicity of the mutant α-Syn A53G to the WT and A53E α-Syn. This method perfectly illustrated the mutant’s toxicity by the amount of growth in yeast cell per dilution. Had this procedure been performed properly, we should have been able to see the difference in yeast growth under varying conditions. Glucose was used a positive control, because the promoter that controls the expression of pathogenic effectors, are repressed by glucose. Conversely, galactose activates this inducible promoter, so we hypothe- sized to see less growth in undiluted sample of A53G in galactose plates compared to glucose plates. Poor technique showed a lot of variation in the seven samples that were spotted. This made the two galactose plates, which induce α-Syn expression, extremely difficult to interpret. No proper comparison could be made between the glucose and galactose colonies. Also, there was some contamination seen on one of the plates, but it was due to spotting being done one colony at a time, forcing the media to be exposed for a long time to outside contaminants.
Future experiments will be conducted to properly examine the familial mutant A53G in yeast models. More specifically, further spotting assay trials need to be performed to evaluate the toxicity of the mutant. Understanding the toxicity patterns is important in examining cell survival. Although we had predicted that there would be no change in alpha-synu- clein original function in this A53G mutation, extending the spotting assay trials will show whether or not this novel mutation actually induces the on- set of Parkinson’s disease. Along with spotting, a western blot analysis will be performed to check for the level of protein expression for the particular mutant. This assay separates proteins by its size, so performing this exper- iment will help contrast the accumulation patterns of the A53G mutant and WT alpha-synuclein. With western blot analysis, given that the results from further spotting trials showed increased toxicity in the A53G mutant, we hypothesize to see thicker bands showing more protein in the A53G muta- tion compared to the WT alpha-synuclein. Third, fluorescence microscopy should be performed to check for the localization of the protein given the change in the amino acid sequence. The mutations of alpha-synuclein in this experiment were tagged with GFP, fluorescence microscopy will high- light where this mutant is located in the cell. This will help understand and contrast the localization pattern of the A53G mutant. We predict more ag- gregates when examining the A53G mutant in the cell. Lastly, growth curve analysis should be used to measure the growth of cells (Hall et al., 2013). This procedure will keep track of the growing number of cells during its time of incubation, delineating its growth. It could also help with contrasting the number of cells with WT a-Syn and the mutant a-Syn, A53G, to see how this mutation alters cell growth rate. We predict to see less growth in cells with mutant a-Syn present.
We would like to acknowledge Dr. DebBurman for teaching the Biology 221 course, as well as Dr. Wilcox for instructing the laboratory sec- tion at Lake Forest College. We would also like to thank our lab mentors, Morgan Marshall and Saul Bello Rojas, for assistance in analyzing the data and the rest of the DebBurman lab for preparing the plates and the media.
Eukaryon is published by students at Lake Forest College, who are solely responsible for its content. The views expressed in Eukaryon do not necessarily reflect those of the College.
Articles published within Eukaryon should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.