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Eukaryon

Creating A29S α-synuclein C-Terminal Truncations at Amino Acids 95 and 110 for Investigation of Parkinson’s Disease in Yeast

Kenna Bailey, Jessica Day, Josie Klein
Lake Forest College
Lake Forest, Illinois 60045

SUMMARY

Parkinson’s disease (PD) is a neurodegenerative disease that can be influenced by α-synuclein mutation. α-synuclein is thought to contribute to PD because aggregation of the protein can create Lewy bodies, a defining characteristic of the disease. We conducted an experiment to create different gene truncations of A29S α-synuclein for yeast transformation. We created two  truncations:  one included the first 95 amino acids and the other included the first 110 amino acids of the α-synuclein protein. We hypothesized that we would be able to make the truncations successfully and that the fragments could be ligated into a plasmid and replicated for future study. Fragments were amplified via PCR and purified using gel extraction. The fragments were then subcloned into vectors and transformed into bacteria for growth. The orientation of the fragments was checked before we conducted a yeast transformation. Fragments that were in the correct orientation were sent for sequencing to genetically confirm; however, orientation checks and transformations were not successful. The successfully created truncations could be used to provide more information on how truncations in A29S α-synuclein could contribute to this neurodegenerative disease.

INTRODUCTION

Parkinson’s disease (PD) is a neurodegenerative disease that’s onset is thought to be related to a loss of dopaminergic neurons (Fahn, 2008). The symptoms of PD can include inability to retain memories and difficulty controlling body movement (Emamzadeh and Surguchov, 2018). In order to be diagnosed with PD a person must have a multitude of symptoms including tremors, bradykinesia, loss of postural reflexes, flexed posture, and a freezing phenomenon where the patient’s feet are “glued” to the ground (Fahn, 2008). There are many different parkinsonian states: primary, secondary, Parkinsonism-Plus Syndromes, and Heredodegenerative disorders (Fahn, 2008). Although it is not used as commonly, primary parkinsonism refers to the same condition as Parkinson’s disease, and it’s onset can be either sporadic or the influenced by genetic factors (Fahn, 2008). Familial Parkinson’s disease has been linked to many genes including SNCA, PINK1, UCH-L1, DJ-1, LRRK2, ATP13A2, HTRA2 and Parkin (Ross et al., 2008). PD is caused by a loss of dopaminergic neurons, specifically in the substantia nigra. The death of these neurons is believed to be caused by the aggregation of α-synuclein in Lewy bodies (Petrucelli and Dickson, 2008), and may also be caused by oxidative stress (Pimentel et al., 2012).

The protein α-synuclein has a wide range of functions, including the regulation of neurotransmission and responding to cellular stress (Benskey et al., 2016). This protein has three unique functional domains: an amino terminus, a hydrophobic region, and a highly negative carboxyl terminus (Allendoerfer et al., 2008). While this synuclein has been heavily researched, not much is known on its role in the mechanism of development of PD. Once its direct role is determined, many new therapy options are likely to become available (Lücking & Brice, 2000).

Although it is rare, SNCA - the gene that codes for α-synuclein - can mutate. Evidence suggests that this mutation can cause PD (Hoffman-Zacharska et al., 2013). It was first identified that the A53T mutation of the gene was associated with PD in the late 1990s (Polymeropoulos et al., 1997). Since this discovery, many other mutations in the gene have been identified as potential markers for familial and sporadic PD as well (Xu and Pu, 2016, Hoffman-Zacharska et al., 2013). Previous mutations have been greatly characterized in organisms, including A53T, which is linked to early onset PD (Kumar et al., 2017). Other papers have examined the lesser known mutations A18T and A29S, finding that A18T is highly aggregation prone, while A29S also caused aggregation, but at a slower rate than A18T and A53T (Kumar et al., 2017). This project focuses on the A29S mutation of the α-synuclein gene, which causes a change of the amino acid alanine to serine at the 29th position of the α-synuclein protein. An A29S mutation can lead to sporadic Parkinson’s disease; this type of PD often develops later in life, in individuals that are about 60 years old, and progresses rapidly (Hoffman-Zacharska et al., 2013). In the project, the α-synuclein protein coded for by A29S mutated gene was truncated from the C-terminal end to form fragments 1-95 and 1-110. Truncations from the C-terminal of α-synuclein have been observed to increase aggregation relative to the full-length protein, maintain the solubility of α-synuclein, and have also been shown to be important for the interactions of α-synuclein with other nervous system proteins (Xu and Pu, 2016).

One difficulty that comes with studying human neurodegenerative diseases is finding ways to closely examine internal pathology without harming a human being or crossing an ethical border. Scientists find many novel model organisms to solve this problem, including mice or Drosophila, and yeasts are also extremely useful for studying disease. Our experiment used Saccharomyces cerevisiae yeast as a model; we chose to use yeast as it is less expensive than other model organisms and it is easy to work with, as it grows in a short amount of time (Allendoerfer et al., 2008). This characteristic makes yeast a valuable tool to study mechanisms such as protein folding, quality control and degradation, and even cell death and survival (Franssens et al., 2013). Yeasts are also easy to manipulate genetically, which allows for outside DNA to be transformed into the cell and investigated more easily. Outside of PD, yeasts have given insight to eukaryotic cellular division, DNA replication, and RNA metabolism (Menezes et al., 2015). However, the yeast model has been shown to be helpful in identifying genes that regulate important mechanisms of PD, like protein aggregation and cellular toxicity as well (Franssens et al., 2013). Because yeast and human cells are both eukaryotic, they have many cellular mechanisms that are very similar. This allows yeast models to be able to take in and use human α-synuclein genes to express α-synuclein protein in order to learn about its function even though the protein is normally only found in humans (Menezes et al., 2015). Previously, yeasts have been used to demonstrate the cellular pathways affected by α-synuclein, cytotoxicity, and the effects of α-synuclein overexpression. Thus, yeasts have been, and continue to be, a novel method to study PD mechanisms that may eventually be applied to human patients.

 

In this experiment, the A29S mutation will be studied by truncating the C-terminal codons 1-95 in one sample and 1-110 in another. Based on the knowledge that C-terminal truncations of α-synuclein may be associated with increased aggregation of the protein, it is predicted that the 1-95 and 1-110 α-synuclein truncations will show increased aggregation of α-synuclein in yeast models, and will exhibit characteristic pathogenic features of PD.

 

This experiment aims to design primers in order to perform truncations at the 95th and 110th amino acids of A29S α-synuclein. The A29S α-synuclein fragments will then be amplified. In order to conduct later analysis, the 1-95 and 1-110 fragments will be isolated and purified. Then, these isolated and purified fragments will be inserted in a vector and the vector transformed into bacteria for amplification. The correct orientation of the A29S α-synuclein in bacteria will be confirmed using PCR and gel electrophoresis. The vectors in the correct orientation will be purified and their sequence thus determined. Lastly, these truncations will be put into yeast and evaluated. While a general function of this mutation is known, the specific effects of each segment of the protein are unknown and so truncation of specific regions may provide valuable knowledge about the function of A29S α-synuclein in PD. In combination, these steps create the overall aim of determining the function of the designated amino acid regions.

 

RESULTS

Overall Project Design

Our first goal was to establish a general experimental process to follow. We created two truncations of the A29S α-synuclein. One DNA fragment was from amino acids 1-95 and the other one was from amino acids 1-110. With these primers, we amplified the eGFP and α-synuclein fragments and subcloned them into a pYES2/CT vector. By subcloning into this vector, we were able to transform the fragments into E. coli, which allowed us to store and amplify the plasmids. We then checked if plasmids had the correct orientation, and those correctly oriented were sent to the University of Chicago for sequencing. Lastly, the DNA was transformed into yeast for further research (Figure 1A). For further explanation on the cells used, refer to the methods (See “Template DNA, Subcloning Plasmids, and Bacterial and Yeast Cells Used”).

 

Primer Design and Testing

We began by designing forward and reverse primers for both truncations in order to begin PCR amplification (See “Primer Design and Synthesis” Method). In addition, the eGFP reverse primer was used to tag the modified genes. The same forward primer was used for both truncations, but each truncation had its own individual reverse primer. The sequences of such primers are given in Figure 1B. Fragment 1 only contained amino acids 1-95, and Fragment 2 had amino acids 1-110; the wild type (WT) α-synuclein gene has 140 amino acids each (Figure 1C).

 

Figure 1. Project design and primer design and testing(A) Steps of the project. The gene is fragmented into amino acids 1-95 and 1-110 (i) and then amplified by PCR (ii). The fragments were then purified using gel electrophoresis and a gene cleaning kit (iii). We then ligated the fragment into a PYES2 vector and transformed it into bacteria (iv). Lastly, we check the orientation of the gene fragment (v) verified the gene sequence (vi) before transforming the vector into yeast (vii) (B) The forward and reverse primer sequences for 1-95 and 1-110 truncation (C) A schematic of the truncation performed and used throughout the experiment. The red line represents the A29S mutation.

 

Making C-terminal Truncations Through PCR

Before running any gel electrophoresis, we set up seven PCR reactions using master mix, forward and reverse primers, template, and water. Of our seven reactions, one was a positive control, and two were negative controls, where one did not include the template DNA, and the other did not include the reverse primer. Two more contained the 1-95 fragment with either 1 μL or 4 μL template, and the last two contained the 1-110 fragment with either 1 μL or 4 μL template DNA. The reactions with the DNA fragments went through a “quick PCR purification” to exchange the buffers. The DNA was taken from PCR buffers to sac-1 buffer, then each PCR product sac-1 was restriction digested in sac-1 buffer (See “Plasmid-based PCR” Methods).

 

A29S α-synuclein 1-95 and 1-110 Extraction

In order to purify both our 1-95 and 1-110 DNA fragments we separated our PCR product by using gel electrophoresis. We made two .75% agarose gels, one with six wells and the other with 10 wells. The first gel (6 wells) contained a DNA ladder and the α-synuclein truncations of both the 1-95 and 1-110 fragments. After we ran this gel, we found that our 1-95 fragments (in lanes 2 and 3) were lower than the 1-110 fragments (in lanes 4 and 5); however, only one band showed up for the two 1-110 fragments (Figure 2B). Once we saw that we

 

had successfully isolated the 1-95 and 1-110 fragments, we excised the DNA from lanes 3 and 4 of the gel using a razor. For further procedure, refer to the methods (See “PCR Purification” Method).

A29S α-synuclein 1-95 and 1-110 Visualization

Next, we wanted to confirm we made the truncations successfully. We first ran a visualization gel that contained the same PCR products described in the above section and illustrated the expected products in a computerized gel (Figure 2C). When we ran the second gel, we found that lanes 5 and 6 contained no bands. These lanes were negative controls; this suggests the controls were successful.  Similarly, we observed a band in lane 9, which suggests a successful positive control (Figure 2D). We had bands show up for both 1-95 fragments (lanes 3 & 4) and a band show up for one of the 1-110 fragments (lane 7) (Figure 2D). Our peer teacher confirmed the primers would be successful in experimental conditions with a primer check verification, where all lanes showed bands except for lane 3, the negative control (Figure 2E). Our truncations were also verified by our peer teacher through a PCR verification, in which lanes 7 and 8 showed a band for the 1-95 truncations, and lane 9 had a band indicating one successful 1-110 truncation (Figure 2F). Our peer teacher then ran another gel extraction verification to ensure that we had successfully purified our PCR products. Bands appeared in lanes 4 and 5 around 1 kb. The expected products were about this size; the bands suggests that we were successful in creating both the 1-95 and 1-110 A29S α-synuclein truncations (Figure 2G).

Vector Transformation into E. coli

Following the purification of the α-synuclein DNA fragments, we ligated the fragments into a pYES2/CT vector for replication in E. coli. The vector was first sac-1 digested and treated with CIAP digest, and then it was purified by gel electrophoresis to prepare for ligation of the DNA fragments. We then ligated the previously sac-1 digested PCR product into the pYES2/CT vector.

After we ligated the 1-95 and 1-110 gene fragments into the vector, we then transformed the plasmid into E. coli and plated onto LB+AMP media. The ampicillin on the plates would prevent untransformed bacteria from growing; however, the pYES2/CT vector contains an ampicillin resistance gene, so the transformed bacteria, which accepted the vector, would still be able to grow. This means that if growth is observed on the plates with AMP, the vector must have been successfully transformed into some bacteria.

After one day, we observed the plates for growth. We initially transformed with the sac-1 digest, but there was no success for the transformations, so we switched to another vector called the TOPO vector. We observed growth on both the 1-95 α-synuclein (Figure 3A) and the 1-110 α-synuclein plates (Figure 3B). The positive control showed a large amount of growth and looked cloudy. The negative control showed no growth as expected (Figure 3A&B). The successfully transformed E. coli colonies were then analyzed further. For further description of actions taken, refer to the methods (See “Vector Subcloning and Bacterial Transformation” in Methods).

 

Isolating TOPO Plasmid from E. coli

In order to isolate the plasmid from the transformed E. coli, our peer teacher first grew colonies 5-8 from truncation 1-95 (Fig 3A) and 1-110 (Fig 3B) in liquid media overnight. The following day we collected the E. coli cells, and isolated and purified the plasmid (See Plasmid Purification in Methods). We then ran the purified plasmid samples on a .75% agarose gel to confirm the plasmids were not lost. All plasmids isolated from colonies 5-8 of the 1-95 and 1-110 truncations were present (Figure 4A).

Checking Orientation of α-synuclein Gene Fragments in Plasmid


After the plasmids were isolated and purified, we ran a PCR to determine the orientation that the plasmids were ligated into each vector. The plasmids  could either be inserted in the correct orientation, with the α-synuclein fragment following the GFP sequence, or in the incorrect orientation, with the α-synuclein fragment first. In order to check this orientation, we annealed a control forward primer that would bind to the Gal3 portion of the TOPO vector and the designed

 

reverse primers for the 1-95 and 1-110 fragments so that a positive result would only be observed if the fragment were in the right orientation. Ideally, we would see bands around 1kb for each 1-95 truncation colony tested, including the whole cell colonies (Figure 4B). However, when we ran the orientation checks on a .75% agarose gel, we did not observe any positive results (Figure 4C). There was a band at 2 kb in lane 8; however, this was not consistent with our expected products. The ideal result for the 1-110 truncations would be a band around 1kb for each of the colonies and a positive control band in the last lane of the gel (Figure 4D). Again, we saw no successful bands, and there was also no positive control band (Figure 4E). Bacterial Whole Cell PCR was utilized for this (Methods).

 

Yeast Transformation

The last step was to transform the plasmids into yeast. Because none of our E. coli colonies were in the correct orientation, we used plasmids from other groups in the class. The A29S α-synuclein 1-95 plasmids in the correct orientation came from Group 3’s colony one. The transformed yeast cells were plated on SC-Ura glucose media in 200 and 30 μL quantities so only cells with the Ura gene from the plasmid could grow. No growth was observed on the plates (Figure 5A). Another 30 μL of the transformants were plated on YPD media to ensure the yeast cells were still viable after transformation. We observed healthy growth on this plate (Figure

 

5A).

The A29S α-synuclein 1-110 truncations were also plated in 200 and 30 μL volumes on the SC-Ura glucose media. No growth was observed on these plates, aside from some contamination on the plate that used the 200 μL volume (Figure 5A). We also plated 30 μL of the transformants on YPD media to again check for yeast viability, and there was significant growth (Figure 5A). The positive control was the WT-GFP α-syn DNA, while the negative control did not have DNA, and both controls were put on SC-Ura glucose and YPD plates. There was no growth on the positive control or on the negative for SC-Ura glucose condition, while there was growth on both YPD controls indicating viable cells (Figure 5B). This was done by yeast transformation (Methods).

 

DISCUSSION

This study aimed to create two truncations of the A29S α-synuclein gene. The two fragments should have been a gene with codons 1-95 and another with codons 1-110. These fragments provided the basis for further steps in experimental design. Both fragments were successfully created, but not all the steps completed yielded data to support our hypothesis. We created the truncations by PCR, conducted DNA purification, and inserted them into a pYES2/CT vector for growth successfully; these truncations can be used in future experiments to examine the effect of specific parts of the C-terminus of A29S α-synuclein on the development of Parkinson’s disease in yeast. However, we did not see positive results with bacterial transformation, orientation checks, and yeast transformation despite this success.

Gene Truncations and Gene Purification

We expected both the 1-95 and 1-110 truncations to yield bands. The gel confirmed these findings, as the bands were dark and clearly visible at the correct sites. We expected that the bands from the 1-95 fragments would be around 1 kb, and slightly lower than the1-110 fragments, because they are smaller and containing less  nucleotides. This led us to conclude that the PCR amplification process was successful, due to the presence of the band. The position also indicates that the truncations were successful, as they all travelled below the wild-type control. This showed that the α-synuclein fragments were shorter. Any extra bands observed could be attributed to the template that remained unused or primers that did not anneal.

The unused template observed when performing gel electrophoresis led us to conduct gene purification. This purification process allowed us to see if we lost any product during the DNA purification process. Our peer teacher ran a gel explicitly to show that we purified the DNA, and we concluded from this gel that we successfully purified the gene fragments without losing any product.

Bacterial Transformation and Orientation Check

For our initial bacterial transformation with the pYES2/CT vector, we had many colonies that grew (image not included), which may be attributed to a mistake in the plates. We suspect the plates did not contain ampicillin, which resulted in all E. coli cells to be able to grow rather than only transformants. When we switched to the TOPO vector, transformation of 1-95 and 1-110 truncations into E. coli was successful, and we were able to analyze the colonies further.

Unfortunately, we did not have any plasmids in the correct orientation. Because the positive control did not show on the gel, we suspect there was human error in the making of the PCR reactions. We may have used primers incorrectly , or we may forgotten to include ingredients. Other PCR reactions in the class were successful which indicates the thermocycler did not have any issues. There is a slight possibility that the enzymes in the master mix we used became unstable because the reaction tubes were left off of ice for a few hours while they contained the enzymes. However, the Taq polymerases in the master mix should be stable at extremely high temperatures (Dotson, 2018), which suggests this is an unlikely  source of error. If we could repeat this experiment, in the future we would ensure all enzymes are kept on ice and that we visibly saw each ingredient go into the PCR reaction. Due to unsuccessful orientation results, we were unable to send any samples for sequencing.

Yeast Transformation

We expected to see healthy growth on the SC-Ura glucose plates with the transformant yeast colonies, but this was not observed. Because the positive control was also not successful, we suspect there may be an important ingredient missing from the SC-Ura glucose plates that would enable the yeast colonies to grow. If the plates lacked glucose, then the yeast cells would not have the nutrients they need to make energy and thrive, which may explain the absence of growth on the transformation plates. It may also be possible that the yeast were not given enough time to grow before we moved the plates into the fridge. Yeast cells can take between 3-5 days to grow, and we only allowed two. However, it is important to note that we previously observed yeast cells to be capable of growing in two days, so this potential error seems  unlikely. If the plates contained all ingredients and the yeasts were given enough time to grow, there is always the possibility that human error caused the transformation to fail. However, no group in our class got successful results; this suggests that there may have been a larger issue in the materials we used. There could potentially be a problem in the plasmid where the cells are unable to express the URA gene that would allow them to survive in the -Ura condition on the plates. This would require further trouble shooting after attempting the transformation again.

Future Directions

In order to determine the effect of these α-synuclein truncations in a yeast model, we can use fluorescence to phenotypically visualize if α-synuclein aggregates in the yeast cells in a way that is similar to the Parkinson’s pathology seen in humans with the disease. The α-synuclein protein is already tagged with a GFP sequence, which would easily enable images to be captured. This method is implemented in a number of other studies on α-synuclein function, specifically in the pathological influence of α-synuclein (Outeiro and Lindquist, 2003). These cells containing the truncated segments of α-synuclein can be compared to the cells that have WT α-synuclein to determine the differences in localization of the protein. Western blotting (Kurien and Scofield, 2015) can also be used to examine the relative amounts of α-synuclein protein that is expressed in cells with WT versus those with truncated gene. This would allow us to observe the effect α-synuclein truncations have on the overall expression of the protein.

Another  method that could be used to examine the effect of the truncated α-synuclein segments on cell viability is serial dilution spotting (Thomas et al., 2015). If α-synuclein has detrimental effects on the cell’s ability to function, we would expect to see a smaller number of colonies the more dilute the cells become.

In this experiment, we were able to successfully create α-synuclein truncations at the 95th and 110th amino acids by PCR. These gene fragments were successfully ligated into a vector and replicated in bacteria to increase the concentration of the plasmid rapidly. The plasmid containing the gene fragment was successfully isolated and purified from the bacterial cells for further study. We did not successfully check the orientation of our gene fragment and did not send it for sequencing. We also did not successfully transform our plasmid into yeast for potential future studies listed above. However, the 1-95 and 1-110 truncations can be used to determine whether the C-terminal end of A29S α-synuclein has any effect on the development or progression of Parkinson’s disease in yeast. Any results from these further studies may be applicable to humans which could assist in future treatment development.

 

MATERIALS/METHODS

Primer Design and Synthesis

The sequences of these primers can be found in Figure 1B. The forward primer was created from the GFP sequence attached to the α-synuclein sequence (1). We began at the beginning and counted about 40 nucleotides in. A Kozak sequence was also included in this primer to help begin transcription of the GFP + α-synuclein fragment in PCR and when in the plasmid.

For the reverse primer, we counted about 45 nucleotides backwards from the desired truncation location on the A29S α-synuclein, either at the 95th or 110th nucleotide. Because the reverse primer binds the coding strands, the reverse complement of each nucleotide was found.

1- DebBurman, S. (2013). Amplification of Truncated Gene by PCR. In S. DebBurman (Ed.), BIO 221 molecules, genes, and cells, laboratory manual (pp. 22-31). Lake Forest, IL: Lake Forest College.

Template DNA, Subcloning Plasmids, and Bacterial and Yeast Cells Used

For the template DNA we used an eGFP-α-synuclein 1-140 gene with the A29S mutation. When subcloning plasmids we used the pYES2 plasmid vector for yeast and the pRY121 plasmid vector for bacteria. The bacteria cells we used were E. coli and the yeast cells we used were S. cerevisiae.

Plasmid-based PCR

We used plasmid-based PCR to create the gene truncations. Each fragment had a different recipe. Both recipes included 50 uL of master mix, 43 uL of sterile water, 3 uL of forward primer, 3 uL of the truncation specific reverse primers, and either 1 uL or 4 ul of plasmid DNA. A positive control was done, including everything in the PCR recipe (1). We made a negative control by omitting the plasmid, and another negative control by omitting the forward primer.

1- DebBurman, S. (2013). Amplification of Truncated Gene by PCR. In S. DebBurman (Ed.), BIO 221 molecules, genes, and cells, laboratory manual (pp. 22-31). Lake Forest, IL: Lake Forest College.

PCR Purification

We excised the bands that were successfully produced by PCR and placed them into tubes (1). We then recorded the mass and added 100uL of turbo salt solution per every 0.10 gram of the gel. We then melted the solution and added the mixture to a tube for centrifuging. We then washed it before the TurboElution solution was added and the DNA incubated at room temperature for 5 minutes. We then centrifuged at maximum speed for one minute. Lastly, we stored the samples at -20°C.

1- DebBurman, S. (2013). Purifying Desired Fragment by Gel Electrophoresis. In S. DebBurman (Ed.), BIO 221 molecules, genes, and cells, laboratory manual (pp. 32-37). Lake Forest, IL: Lake Forest College.

Vector Subcloning and Bacterial Transformation

In order to begin a bacterial transformation (1) of our 1-95 and 1-110 gene truncations, we first had to ligate our gene fragments into the pYES2/CT vector. To do this Sac1 digested pYES2/CT, then CIAP treated digested pYES2/CT, then gel purifies digested and phosphate pYES2/CT. For the DNA ligation we used 5 μL of water, 1 μL of Sac1 digested PCR product, 2 μL of 5X ligase buffer, 1 μL Sac1 digested pYES2/CT vector, and 1 μL of T4 DNA ligase. Then we were able to begin our bacterial transformation. We started by taking our created ligation solution and adding it to the thawed competent cells, and then we incubated them on ice. Next, we placed the mixture in 42°C water for thirty seconds as a heat shock. Then we added S.O.C to mediate the transformation and incubated the cells in a 37°C shaker for one hour. We then plated 30 μL and 200 μL of the cells on LB+Amp plates and let them incubate overnight at 37°C.

  • DebBurman, S. (2013). Subcloning into a TOPO vector and Bacterial transformation. In S. DebBurman (Ed.), BIO 221 molecules, genes, and cells, laboratory manual (pp. 38-41). Lake Forest, IL: Lake Forest College.

Bacterial Whole Cell PCR

For the bacterial whole cell PCR, we took colonies from the plates that were created previously from our bacterial transformation and added them to a mixture of 25 μL Master Mix, 22 μL sterile RNase-free water, 1.5 μL forward primer, and 1.5 μL reverse primer (1). Then we took the reactions and put them in the PCR machine.

1- DebBurman, S. (2013). Confirm gene fragment orientation in vector in bacteria. In S. DebBurman (Ed.), BIO 221 molecules, genes, and cells, laboratory manual (pp. 42-46). Lake Forest, IL: Lake Forest College.

Plasmid Purification

From the bacterial transformation, we took the plasmid from 8 different colonies (1). We then made a liquid culture from these using LB+Amp media. After allowing these plasmids to grow overnight, we then ran PCR again on these samples. We used 25 μL master mix, 21 μL of sterile water, 1.5 μL of Gal forward primer, 1.5 μL GFP reverse primer, and 1 μL of the respective isolated plasmid. We visualized the results from this using gel electrophoresis again.

  • DebBurman, S. (2013). Confirm gene fragment orientation in vector in bacteria. In S. DebBurman (Ed.), BIO 221 molecules, genes, and cells, laboratory manual (pp. 42-46). Lake Forest, IL: Lake Forest College.

DNA Sequencing

The fragments that  were found to be in the correct orientation in the vectors were sent to the University of Chicago for analysis (1). We did not send any colonies to University of Chicago because none were determined to be in the correct orientation, so there was no data to report.

1- DebBurman, S. (2013). DNA Sequencing. In S. DebBurman (Ed.), BIO 221 molecules, genes, and cells, laboratory manual (pp. 51). Lake Forest, IL: Lake Forest College.

Yeast Transformation

The yeast were first cultured overnight by our peer teacher to have enough cells to work with during the lab. The cells from culture were then aliquoted and distributed to the class (1). The cells were centrifuged to remove the supernatant and resuspended in LiAc. The transformation mix was prepared with 240 μL PEG, 36 μL 1.0 M LiAc, 25 μL ssDNA, 46 μL Water, and 5 μL Plasmid DNA. The plasmid DNA was borrowed from other groups in the class (see results section). The pellet was vigorously resuspended in the mixture and the reaction was incubated at 30°C for 30 minutes. The cells were then heat shocked in a water bath at 42°C. The transformation mix was removed, and the cells were resuspended in water for plating.

  • DebBurman, S. (2013). Plasmid vector transformation into yeast. In S. DebBurman (Ed.), BIO 221 molecules, genes, and cells, laboratory manual (pp. 52-54). Lake Forest, IL: Lake Forest College.

ACKNOWLEDGEMENTS

We would like to acknowledge the Lake Forest College Biology Department for providing the resources for this study, Dr. Alexander Wilcox and Dr. Shubhik DebBurman for instruction in the laboratory and lecture, and Ariane Balaram, our peer teacher for the class, for her assistance in lab. We would also like to acknowledge our peers in the class who allowed us to share their materials in the later transformation steps.

 

 

 

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