Abstract

Despite the significant time and money invested into its research, cancer has remained one of the most globally significant illnesses of our time. While many different mutations can lead to cancer, a significant amount are linked to mutations within the KRAS gene, a key regulator of cell growth and division. However, studying this in humans poses multiple problems due to its cost and complexity. Our lab has previously established that S. cerevisiae’s RAS1 gene is analogous to human KRAS with enough similarity to act as a model organism. This lab attempts to build on the previous lab’s work, with the eventual goal of constructing a vector containing Saccharomyces Cerevisiae’s RAS1 gene and stem loops to visualize mRNA transcription within the cell using the MS2 system. The previous lab’s attempts to amplify the RAS1 gene were unsuccessful. This lab attempts to troubleshoot this process by attempting to cultivate and isolate high enough concentration for plasmids to be usable. Despite testing a new Taq polymerase, improved DNA yields were not achieved, suggesting that new primers must be designed. With this and the proper plasmid, we expect fluorescence microscopy to allow us to visualize the mutations of the RAS1 gene in living S. cerevisiae cells.

Introduction
Almost everyone knows someone who has been affected by cancer. According to the American Cancer Society, more than ⅓ of Americans will experience some type of cancer during their lifetime [1]. While there has been a significant push for increased cancer research in recent decades, research on the disease should be further along. The American Cancer society predicts that in 2025, there will be 2 million new cancer cases and 618,000 deaths from cancer in the United States alone [2]. Cancer occurs when mutations in certain proteins lead to uncontrollable growth and division of the cell. Research is complicated by the fact that experimentation on humans and other complex animals is a costly and work-intensive endeavor. However, progress on science is not proceeding as quickly as one would hope given the time and funding. Since cancer is a phenomenon that occurs on the cellular level, changes that lead to uncontrolled growth and division can be studied in smaller model organisms. With this in mind, it may be more productive to investigate the changes that take place in each individual cell to return to the basics.

One gene that is involved in the development of cancer is KRAS. When functioning normally, KRAS produces proteins that regulate cellular growth. When mutations occur within the KRAS gene, it disrupts the cell cycle, driving the cell to become cancerous and replicate uncontrollably [3]. Saccharomyces cerevisiae, or baker’s yeast, is a common model organism with about ⅓ of its genome directly analogous to the human genome [3]. This includes an analog to KRAS known as RAS1.

Previously, our lab reviewed S. cerevisiae’s RAS1 gene to determine its utility as a model for studying the KRAS gene in humans. Overall, RAS1 shares approximately 40–50% overall sequence similarity with human RAS proteins, particularly in the N-terminal region, where essential signaling and regulatory functions are located [3]. The active site of the RAS1 gene is fully conserved, sharing 100% similarity between yeast and humans [3]. This indicates that mutations in RAS1 affecting GTP binding or hydrolysis would behave comparably to human RAS. Key regulatory and signaling residues are also conserved [9]. They additionally found an 80% similarity between the entire RAS genes, with 60% identical matches. Moreover, the yeast RAS1 contains a glycine (G19) analogous to a glycine in KRAS (G12) previously shown to be mutated in cancer cells (G12C) [3]. This indicates that S. cerevisiae is a viable model for studying the effect of this mutation within a cell but does not guarantee it will be able to be amplified or visualized under fluorescent microscopy.

Other labs have also studied S. Cerevisiae’s suitability as a model for cancer research and evaluated methods in which visualization of mRNA molecules using single-molecule fluorescence in situ hybridization (smFISH) could potentially benefit the research [5]. With this knowledge, our lab conducted electrophoresis on the DNA to determine whether the RAS1 segment could be cloned into a plasmid capable of visualizing the G19C substitution. Previous attempts to amplify a fragment containing the RAS1 gene were not successful. When the PCR reaction was separated by the Taq polymerase and put through gel electrophoresis, the lab found no bands of the expected size. The presence of small bands on the gel indicated the presence of primer dimers, which form when two primers self-anneal. Self-annealing is when the end and beginnings of the two primers bind to each other. The most common cause of this is primer-dimer formation, in which two primers bind to each other instead of the targeted DNA sequence. These results suggest that the conditions for PCR need to be changed, which could be done by redesigning the primers.

Using the pBY011 plasmid as a shuttle vector—meaning it can be propagated in multiple species and can move DNA between these cells—we attempted to amplify the RAS1 region of the genome. Plasmids pSP561 and pSP568 were inserted to allow the DNA to be visualized in the MS2 system; a stage was not reached within the span of this research.

Here we examined whether we could amplify the RAS-containing fragment by changing the enzyme that had been previously used to amplify the DNA. While the standard Taq polymerase had been used before, we attempted to compare it to the DreamTaq polymerase. The DreamTaq is a genetically modified version of the Taq polymerase designed to improve the quantity and accuracy of results. This was attempted due to articles showing it provided higher yields, more accurate PCR product, and allowing amplification of a longer target sequence (the RAS1 sequence contains over 900 base pairs, making it long enough to pose problems during amplification), theoretically allowing labs to achieve higher quantity of the desired product.

Materials and Methods

1. SOB Broth

2.5 g tryptone, 2.5 g NaCl, 1.25 g yeast extract and 5 g agar were added to a beaker, with deionized H2O added until the measured amount reached 250 mL. They were mixed between a plastic 250 mL beaker and a glass 225 mg beaker, and occasionally vortexed to remove clumps from the bottom. The mixture’s pH was measured, and NaOH was added until the pH reached 7. The tube was sealed with aluminum foil and autoclaved for 45 minutes. Petri dishes were placed under the fume hood with a Bunsen burner running, where they were filled with broth to the ¾ line and left to harden.

2. Plasmid

The pBY011 plasmid sourced from DNASU, a company run by Arizona State University BioDesign Institute. Plasmids pSP561 and pSP568 were purchased from AddGene. PBY011 is a yeast expression vector that contains RAS1 while both pSP561 and pSP568 allow for fluorescent microscopy; pSP561 adds green fluorescent protein (GFP) while pSP568 inserts stem loops. All plasmids were stored at –20 °C.

3. Plating, Growing, and Storing Bacteria

3.1 Transformation into E. Coli

250 μL of glucose was added to the SOB in order to create SOC broth. After cells thawed on ice, 100 μl of competent cells and 1.7 μL B-mercaptoethanol were mixed into each tube. They were held on ice for another 10 minutes, being mixed again every 2 minutes. 1 μL of each plasmid was added to each tube, then stored on ice for 30 minutes. Tubes were heat-pulsed in a 42 °C water bath for 45 seconds, then placed back onto ice for 2 minutes. 0.9 mL of preheated SOC was added to the cells and incubated at 37 °C for 1 hour at 225-250 RPM in the water bath shaker.

3.2 Plating With and Without Antibiotics

Falcon tubes were removed from the shaker and centrifuged at 150 RPM for 2 minutes. Under the hood, the old broth was disposed of and replaced with 5 mL of filtered LB broth. Tubes were mixed by pipetting up and down until cloudy. For the plates with antibiotics, 20 mL of ampicillin was mixed into each plate. 100 μL of cell mixture was pipetted into prepared LB agar plates and spread evenly over their surface. Plates were then stored in an incubator at 37 °C for 24 hours.

3.3. Growing Bacteria

Under a fume hood with the Bunsen burner, 5 mL of filtered LB broth was added to each Falcon tube. An inoculator loop was used to retrieve one colony from the plate and placed into the broth, then mixed until integrated. Tubes were placed into a water bath shaker at 37 °C for 24 hours, then wrapped in parafilm and stored at 4 °C until used.

3.4. Storing Bacteria after Colony Growth

Tubes were removed from the 4 °C fridge and centrifuged for 2 minutes. They were transferred under the fume hood, where old broth was disposed of, and the pellets resuspended in 5 mL of filtered LB broth. 1 mL of this solution was transferred into an Eppendorf tube for future use.

4. Purification & Nanodrop

Plasmid DNA was purified using the Thermofisher Scientific GeneJET Plasmid Miniprep kit. 250 μL of resuspension solution was added to Eppendorf tubes before transferring the mixture to a microcentrifuge tube. 250 μL of lysis solution was added, and mixed by inverting 4-6 times, then 350 μL of the neutralization solution, mixed with 4-6 inversions. These were centrifuged at 10,000 RPM for 5 minutes. Excess liquid was discarded, with the microcentrifuge portion transferred into a GeneJet Spin Column. 500 μL of wash solution was added and centrifuged for 60 seconds at 10,000 RPM. This was then repeated 2 more times. Spin columns were transferred to 1.5 μL microcentrifuge tubes with 20 μL H2O. The tubes were incubated on ice for 2 minutes, then centrifuged for 2 more. The concentration of the plasmid in each sample was determined using the Nanodrop machine, then solutions were stored in a -20 °C fridge overnight.

5. Dilution

To prepare the samples for use in PCR, purified plasmid solutions were diluted to a final concentration of 50 ng/μL with autoclaved filtered water (AFW) added to reach a volume of 50 μL. AFW was added to dilute the plasmid to the appropriate concentration to be used as a template in a PCR reaction. Seven samples were made in total.

6. PCR

Following dilution, 50 ng of diluted RAS1 pBY011 solution was added to each of 16 PCR tubes. These were split into sets of 8, one receiving 5 μL Taq polymerase and the other with 5 μL DreamTaq polymerase, both purchased from Thermofisher Scientific. 1.0 μL each of the Forward-primer, Reverse-primer, dNTP, and MgCl2, was then added to each tube, along with 5 μL of the polymerase’s buffer. The F-primer sequence was GCG AGA TCT ATG CAG GGA AAT AAA TCA AC with a molecular weight of 8,968.9 μg/μmole, while the R-primer was GCG GCG GCC GCC TAA CAA ATT ATA CAA CAA CAA with a molecular weight of 10,094 μg/μmole. The amplified section of the RAS1 pBY011 contained 930 base pairs.

39 μL of autoclaved filtered water was added to reach 50 μL. Tubes were placed in the Thermofisher thermocycler at 95 °C for two minutes, which was then reduced to intervals of one minute. The temperature was lowered to 62 °C for 30 seconds, raised for a minute to reach 72 °C, and remained at 72 °C for two minutes. The products were then stored until needed in a 4 °C freezer to maintain viability.

7. Gel Electrophoresis

PCR products were separated using a 1.5% agarose TAE gel at 120 AMP with a BioRad gel electrophoresis kit. Agarose gel was made in lab, and a buffer of tris-acetate-EDTA was used to run the gel. Gel analysis was promptly performed using the GelDoc imager. The SYBR safe program was used to visualize the gel in order to determine the length of the PCR product. The molecular weight marker was a 1 kb DNA ladder.

Results and Discussions

Figure 1: Different plates collected during lab. A-B depicts contaminated plates, C-D depicts control plates, and E-F depicts experimental plates that show dense colony growth and lack of contamination

In order to isolate the plasmid, we transformed E. Coli cells with pBY011 yeast expression vector plasmid. While the experimental plates (Figure 1, E-F) contained ampicillin, control plates (Figure 1, C-D) did not. While ampicillin typically kills E. Coli, an ampicillin resistance gene is part of pBY011: colony growth on plates containing ampicillin shows that the bacteria successfully took in the plasmid (Figure 1, E-F).

During the first two attempts at plating cells, there were several instances of contamination that interfered with plasmid propagation on both ampicillin and control plates (Figure 1, A-B). However, Fungus and other bacterial species are always present in the air and can grow on a plate, where they may outcompete the target organism. To mitigate fungal and bacterial growth on the plates we implemented more rigorous sanitization procedures: thoroughly cleaning and replacing any lab equipment that may have been exposed to bacteria, stricter cleaning protocols when beginning and ending labs, more ubiquitous glove use and cleaning of gloves, more consistent autoclaving procedure, and the use of a fume hood with UV sanitization all allowed for further sterilization. Since then, we have observed a significant decrease in contamination. E-F displays successful experimental plates (Figure 1).

Table 1: The concentrations of the pBY011 (the yeast expression vector containing RAS 1 in seven samples, determined by Nanodrop spectrometry.

After contamination, E. Coli was replated and regrown. These bacteria were transformed with pBY011, which was isolated using the ThermoFisher Scientific GeneJet Plasmid Miniprep Kit. Purified plasmids containing RAS1 in the pBY011 yeast expression vector were analyzed using Nanodrop spectrophotometry to assess DNA yield. Our average plasmid concentration was 727.7 ng/μL, with the highest concentration at 1767.7 ng/μL and lowest at 312.4 ng/μL (Table 1). The measured plasmids had higher than expected ranges for downstream PCR applications, supporting the conclusion that amplification of the desired fragment may be possible. As the next step to construct a plasmid, we wanted to amplify a fragment of pBY011containing RAS1 through PCR and purify the PCR fragment for use in the MS2 fluorescent microscopy system, which would allow us to visualize mutations of RAS1 in real time.

Figure 2: Concentrations of DNA in each sample after PCR, for each sample number, colored in accordance with which polymerase was used. Sample numbers match Table 2.

We attempted to further the research by testing the DreamTaq polymerase. The polymerase was changed due to the previous lab’s problems with primer self-annealing. DreamTaq polymerase comes with buffers that are more suitable for longer DNA amplifications and produce more DNA without needing as much environmental optimization. Given the goal of making high volumes of PCR product, the possibility of higher yields and more accurate PCR product lended itself to choosing this polymerase. When compared with the concentrations of the previous Taq polymerase concentration, however, it proved to have no significant difference (Figure 2).

Figure 3: Electrophoresis results from the selected plasmids measured in Figure 3. There are 13 lanes total. Lanes 1 and 13 were 1 kb DNA ladders, lanes 2-5 were each dedicated to a single sample 1-4. Lanes 9-11 each hosted sample numbers 5, 6, and 7, respectively.

Each of the PCR product samples measured in Figure 2 were loaded onto agarose gel, along with 1 kb molecular weight markers, and separated by electrophoresis, with each sample being given its own lane. However, our electrophoresis results did not reveal bands of the expected sizes (Figure 3).

While it is only one possibility, we suspect that this is due to the palindromic repeats in the sequence; the DNA sequence we’ve chosen contains matching base pairs on both the beginning and ends of the sequence, which can lead to them folding in on themselves rather than binding to our target area. This is called primer-dimer formation and is a common failure when running gel electrophoresis. However, the electrophoresis results lacked the blurry band that is typically found in primer-dimer.

Figure 4: Custom plasmid, constructed and visualized by past lab members. It will require optimization and streamlining to further research due to the possible formation of primer dimers associated with the addition of the previous plasmid.

These results indicated that plasmid extraction and purification proctors were working as intended and that observed downstream failures were unlikely to be caused by insufficient DNA concentration or degradation. The use of two different polymerases did not resolve this issue, indicating that the polymerase choice alone was insufficient to overcome the apparent primer self-annealing. These findings demonstrate that primer optimization is a critical prerequisite for successful amplification of RAS1 constructs for MS2 tagging (Figure 4).

Despite the negative findings related to the research, there was still significant information gathered from the research completed thus far. The results demonstrate that plasmid extraction and purification protocols consistently produce high quality DNA at concentrations suitable for downstream applications. This means that DNA yield or degradation are not limiting factors. This suggests that the difficulty with amplifying the fragment is due to problems related to enzyme performance or plasmid quality. Future steps involve attempts to optimize other conditions of the PCR reaction, such as designing new primers and adjusting temperatures during the annealing step of the PCR reaction.

These findings narrow the scope for future troubleshooting and provide clear direction for future studies, such as obtaining a custom plasmid to avoid future occurrences of primer dimer formation (Figure 4).

Conclusions

This study evaluated the possibility that previous attempts at amplification were not successful due to either a lack of sufficient plasmid concentration used for the PCR or an appropriate polymerase. Our initial attempts to isolate the plasmid were impeded due to contamination of the experimental plates containing bacteria. After changing laboratory hygiene procedures, we were able to grow these bacteria without contamination, which allowed us to isolate our plasmids. Nanodrop measurements confirmed high yields of plasmid concentrations, indicating that improved laboratory contamination prevention measures were effective. We attempted to amplify the fragment of the plasmid containing RAS1 using two different thermostable polymerases as templates which showed that there was no difference in plasmid yield between the two. Unfortunately, electrophoresis results did not show amplification of a band of the correct size. While movement towards visualization of RAS1 in the MS2 system was not achieved, these results provided important support and insight of the limitations in this research.

Future Directions

Immediate future work should focus on optimizing the PCR conditions, or by constructing or acquiring a new custom plasmid (Figure 4). One approach could be to design other primers to amplify the RAS1 region. This would reduce primer dimer formations, potentially by minimizing 3’ complementary sequences, adjusting primer concentrations, increasing annealing temperatures, or using hot-start DNA polymerases to prevent chemical reactions such as non-specific amplification and primer dimer formation from occurring until a specific temperature is reached, usually around ~95 °C [9]. The use of no-template controls and melting curve analysis may further improve validation of properly behaving primers. A 𝚫G analysis of the sequence may be able to confirm whether a new custom plasmid would eradicate primer dimers. A 𝚫G value equal to or greater than -2.0 kcal would indicate a higher likelihood of self-annealing by creating an exergonic reaction at the 3’- terminal dimers. Any primer below this threshold should be avoided [10].

Acknowledgements

Thank you to our mentor, Professor Joseph C. Sparenberg, as well as Dr. Hannah Pie, Dr. Kathryn S. Jones, Dr. Brendan Diamond, and all URSC mentors for their continued support. We’d also like to dedicate this paper to Rayhan Stewart. In the short time Rayhan was part of our undergraduate research group in the last cohort, he was an active and dedicated member who contributed meaningfully to our project. He was always eager to learn, grow, and share his knowledge with others. We would not be where we are without him. As a team, we would like to express our deepest gratitude and extend a special thanks in the memory of Rayhan Stewart.

Contact: elie.park@howardcc.edu, irene.allbritton@howardcc.edu, jsparenberg@howardcc.edu