Taters Gonna Tate

February 27, 2024

By Harper Gillespie & Kayleigh Cottrell

In the past few weeks, we have had exciting opportunities to expand our knowledge of osmosis, plasmolysis, and learn coding using R. These labs have been a lot of fun and helped us improve existing skills while gaining new ones. The goal of our osmosis demonstration using potatoes was to replicate hypertonic and hypotonic cells. Hypertonic cells occur when the substance outside of the cell has a higher concentration than inside the cell. As a result, water will move out of the cell to balance out the concentration. Hypotonic cells occur when the substance inside the cell has a higher concentration than outside. Consequently, water will move into the cell to balance it out. However, isotonic cells occur when the concentration outside and inside of the cell remains the same.

The osmosis demonstration experiment began by mixing solutions of various salt concentrations (1%, 3%, and 5%) with distilled water. Distilled water and the salt solutions were added to four tubes. Twelve potato cylinders were cut and three of them were then added to each test tube within their correct solutions. The beginning mass of the potatoes in distilled water, 1% salt, and 5% salt was 3.9g and the beginning mass of the potato 3% salt was 4.0g. The potato cores were then left within their given solutions for 96 hours, after which their color, flexibility, texture, and strength were analyzed. The potatoes varied in their flexibility and consistency. The mass changed in distilled water by –1.8g and +0.2g for the 1% salt solution. The mass of the 3% salt solution and the 5% salt solution changed by –0.8g. The data showed that distilled water caused the cores to lose the most weight due to hypotonicity; however, since the 96 hours exceeded the given time, the water built up within the potatoes and caused them to burst. The potatoes in the 5% salt solution should have lost the most weight due to the hypertonicity of their environment.

Figure 1. Harper observing the Elodea leaf cells in freshwater.

For the plasmolysis lab, we prepared a microscope slide using one Elodea leaf. Plasmolysis is when a plant cell loses water, and it causes the plasma membranes of the cells to shrink. To do this, we observed the leaf before, with regular pond water on it. The cells were not shrinking because they were at an equal concentration (isotonic). To create plasmolysis, we placed a paper towel at one end of the slide to draw out the freshwater and dropped salt water at the other end of the slide. The paper towel drew out the freshwater causing the salt water to move into the leaf. Then, on the monitor, we could observe that the plasma membranes in the cells were shrinking. This process would be considered hypertonic because the water was moving out of the cells to try to balance the saltwater outside. We repeated the process with a paper towel on the leaf slide to draw out the salt water and add fresh water again. We then were able to watch the plasma membranes expand as they took in water.

Figure 2. Kayleigh observing the Elodea leaf cells after salt water was added, noticing a lot of shrinkage.

Also, we had the opportunity to learn about biogeography using R, a programming language that helps us interpret and organize data. Each of us acquired our data on different species (Kayleigh- Syringa reticulata, Harper- Parthenocissus quinquefolia) and obtained geographical data for the species, finding their native occurrences. We then applied R to extract and analyze the data, correlating the coordinates of the species occurrences with climate data. The climate data we used included annual mean temperature, annual precipitation, and more. We analyzed these data points to determine whether our species was well-suited to its native range or if there was any unique or out-of-place data.

So far, our experiments have been successful. Understanding hypertonic, hypotonic, and isotonic using potatoes was interesting and beneficial. Additionally, the Elodea plant was particularly fascinating to observe, as we could physically witness the cells shrinking and moving due to the salt water. It was successful in displaying plasmolysis. Lastly, the use of R was initially confusing to us, but we hope to improve our skills in working with it.

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Potting Seeds In Soil And Working With R-Studio

22 February 2024

By Katelyn Gianni and Ash Waletski

Over the last few weeks, we have continued the seed germination experiment. Our group is currently working with Rubus idaeus, common raspberries, and Chamaenerion angustifolium, fireweed. We are observing the presence or absence of 5 grams of commercial mycorrhizal inoculant on the germination rates of these two species. When we started this experiment at the beginning of the semester, we had isolated seeds from store-bought raspberries, and we are eagerly awaiting their sprouting since the seeds we germinated and planted are from real fruit! After 4 weeks under C30 conditions (4 weeks in the fridge!), we were able to plant our seeds this week.

Figure 1. Katelyn labeling the four groups of planted seeds.

We decided to pot our plants in soil rather than tape them to the window, which is another common way to start seeds. We chose this because we are working with fungi, specifically a mycorrhizal inoculant that is supposed to create a symbiotic relationship between the plant and the mycorrhizae of the fungi. The symbiotic relationship between fungi and plants is described as fungi being able to help the plant by extracting nutrients from soil and/or water; in return, the plant helps the fungi to also receive nutrients by obtaining sugar from the plant. In some cases, the fungi can also help to protect the plant against harmful organisms. By choosing to pot our plants in soil rather than taping them to the window, we plan to watch whether or not the symbiotic relationship actually helps germinate our seeds faster or not. Our prediction when we first started this experiment was it was not going to help germinate our seeds faster. We predicted this because our seeds were previously in a plastic bag and we thought that the mycorrhizal inoculant would not help because there are no nutrients for the fungi to pull out from the plastic bag enclosure our seeds were germinating in.

Another project we have been working on is learning to use R, the coding language used to analyze biogeographical data. Katelyn chose Quercus rubra, the Northern red oak, and Ash chose Quercus Grisea, the Northern gray oak. We found data on each tree and where it grew, then we took that data, downloaded it, and are now working on translating that data into R to push out some useful information having to do with the species-specific niches in their environment. This information will help us to better understand the species and will also show us how to do it on future species.

Figure 2. Ashley working with her species data and R-studio.

Overall, every week has been extremely interesting and insightful so far! Learning how to code on R has definitely been quite the experience and has challenged us technology-wise. Although it’s been difficult, we’ve learned some basic R coding techniques and tricks in the process of many mistakes. On the bright side, our experiment concerning the symbiotic relationship between the germination of Rubus idaeus (common raspberries) and Chamaenerion angustifolium (fireweed) and the mycorrhizal inoculant (fungi) is going considerably well. Again, through some troubleshooting and factors that were out of our control, we learned that experiments are experiments for a reason and that not everything has to be perfect or go according to plan. As we continue our journey through this research program, we will take our experiences and our newfound knowledge to the future challenges we might face.

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Primer Design, PCR, and Gel Electrophoresis of KCNJ4 and NR1D2 Genes

By Sarah Oberstar and Kaitlyn Shaffer

Figure 1. Kaitlyn pipetting water into her samples creating a negative control.

23 February 2024

Over the last couple of weeks, we have been working on designing and testing primers for our project. We are studying the expression of certain genes between breeding and non-breeding Anolis carolinensis, a species of small lizard. We picked two genes, the KNCJ4 and NR1D2 genes to study. The KCNJ4 gene provides instructions for making a protein that acts as a channel in cells, specifically in the heart and brain. The NR1D2 gene helps synchronize the biological circadian rhythm. It also influences the metabolic processes of the body.

A couple of weeks ago we designed three primer sets for our selected genes. We analyzed the primers to ensure they did not have hair-pin structures, self-dimers, and hetero-dimers. This made sure the primers we chose had a high success rate. When the primers arrived, they came as powder. We had to reconstitute them. We added water to the primers to dilute them. We could then use the primers in our PCR (Polymerase Chain Reaction).

Figure 2. Sarah pipetting DNA samples into gel.

We created three solutions using the KNCJ4 primers, and three solutions using the NR1D2 primers. We created a final solution with beta actin. The beta actin solution acts as a control while we work with our designed primers because we know the beta actin primers work. These solutions were then used to make fourteen for the gel electrophoresis. Each of the original solutions were split and either cDNA or water was added. This gave us a positive and negative control. We then ran our solutions in a thermocycler for two hours. This copies the DNA. We had to make sure we labeled everything well now that we were working with multiple different solutions to test. We got better at planning ahead by drawing and writing in our notebooks. We became more efficient and gained a better understanding of the process the more we did it.

Once the PCR was done, we then moved on to gel electrophoresis. We created our own gel and put it into the gel box. We pipetted our solutions into the wells and let the gel run. When the DNA was done moving, it could be imaged. Using the BIORAD Gel Doc™ XR+ Gel Documentation System, we were able to take a picture of our gel. Unfortunately, the results came back contaminated and contained primer dimers. We were unable to see if our primers worked. However, our results told us that we should use a temperature gradient and use different cDNA next time.

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Exploring the genes of the green Anolis carolinensis and sharpening our PCR and gel electrophoresis skills!

Figure 1: Corbin heating up the agarose gel mixture

16 February 2024

By: Ashley Bass and Corbin Carlson

Throughout the last couple of weeks, we have been exploring numerous genes of the green anole lizards (Anolis carolinensis), specifically ZBTB11 and BID.  The ZBTB11 gene regulates transcription, and BID regulates apoptosis. So far, their role in green anole seasonal reproduction is unknown.  We have made immense progress on gene research, and we have ordered and received our primers that we have designed in lab.  During this time, we have also been sharpening our PCR skills in the laboratory, in which PCR allows us to replicate a specific segment of DNA. Additionally, we have been aspiring to improve our gel electrophoresis skills and continuing to practice that in lab.

After conducting several PCR trials, we have gained a better understanding of the specific details within the protocol.  Similarly with gel electrophoresis, we have exercised our skills in making gels, pipetting, gel imaging, and effectively analyzing our gels. We have been repeating these two processes and protocols with the hope of continuing to improve. We have also just started our PCR protocol for our primers that we had designed.

Figure 1: Ashley pipetting DNA samples into agarose gel wells.

As we enter the following weeks of conducting PCR trials with our specific ZBTB11 and BID genes, we hope to use our learned skills of PCR reactions and gel electrophoresis to jumpstart our next goal. Unlike the β-actin gene, with its universal expression across eukaryotic cells and well-understood amplification conditions, the ZBTB11 and BID genes present us with a new challenge. Our task has now evolved from simply conducting successful PCR and gel electrophoresis to meticulously determining the unique conditions required for the successful amplifications of these specific DNA sequences in preparation for next semester. This challenge, while seemingly daunting, is an opportunity to deepen our understanding and refine our problem-solving skills through the rest of the semester, and onward to future years in our scientific education.

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Seed Germination and Stomatal Pore Index

February 13, 2024

By Lily Flaherty and Savanna Newkirk

We began the semester by designing and conducting our seed germination experiment. To begin the experiment, we chose two seed packets out of a selection of native Minnesotan plants. The seeds we chose were that of the spider milkweed (Asclepias viridis), which has a germ code of C30 and the wild hyacinth (Camassia scilloides), which has a germ code of M. Seeds with a germ code of C30 need to germinate in a cold, moist environment for 30 days before being planted, and seeds with a germ code of M are best planted outdoors in the fall.

For our experiment, we followed the steps for germ code B with half the seeds from each packet, 24 seeds for the spider milkweed and 44 seeds for the wild hyacinth. For germ code B, you pour boiling water over the seeds and then leave them to soak in the water for approximately 24 hours. Due to scheduling, we allowed the seeds to soak for two days instead of 24 hours. After letting the seeds soak, we followed the steps of germ code C30 for all of the seeds, placing the seed groupings, one group of the germ code B treated seeds and one untreated from each plant, into a coffee filter and dampening it with distilled water before placing it in a sealed plastic bag and allowing them to germinate in the refrigerator. Our hypothesis was that the boiling water would negatively impact the seeds of the spider milkweed, while the wild hyacinth would benefit from the treatment.

We checked every week for any changes to the seed groupings, as well as changing the coffee filters as needed to prevent mold growth. However, no changes have been noticed thus far.

Figure 1: Seed Germination Groups

Figure 2: Savanna Observing Seed Group

We also had the opportunity to measure stomatal densities and aperture lengths. Stomata are small pores in plant tissue that allow gas exchange to occur, which is necessary for life. Aperture lengths are the lengths of the open pores. By learning about this information, we were able to determine how plants and their stomata characteristics help them survive in their environment.

For our experiment, we chose two species of leafy plants from the greenhouse here at MNSU to measure. The first species we chose was a weeping fig (Ficus benjamina), and the second species was an angelwing jasmine (Jasminum nitidum).

To begin the process, we picked three leaves of each species. We then painted a small strip of clear nail polish onto the leaves. We were careful to avoid large, prominent veins. We tried to avoid the veins so that our samples would have an accurate stomata count, as veins do not contain stomata. After leaving the paint to dry, we took a piece of clear tape and attached it to a small part of the nail polish. We slowly peeled the nail polish off the leaf – carefully so that we did not touch the polish – and placed the polish on a slide. After sticking it to a slide, we secured the sample by using more tape. We were then able to observe the stomata through a light microscope.

Through the microscope, we were able to count the number of stomata that were seen in a given area. We took the averages of all three samples of each leaf and found the densities for both species. For our first species, – Ficus benjamina – the leaves had an average stomatal density of 143.543 stomata per mm² and an average aperture length of 0.03630 mm. For our second species, – Jasminum nitidum – the leaves had an average stomatal density of 276.018 stomata per mm² and an average aperture length of 0.04720 mm. After several other calculations, we determined that the SPI (stomatal pore index) for the Ficus benjamina was 0.18915 mm². We also calculated the SPI for the Jasminum nitidum and obtained a result of 0.61492 mm².

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