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Exploration of Ciliate Discovery in Waco€¦ · Exploration of Ciliate Diversity in Waco Kayla...
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Exploration of Ciliate Diversity in Waco Kayla Canava, Audrey Jacobs, Waverly Kundysek Baylor University Methods and Materials Abstract Acknowledgements Conclusion References Observations Weeks 1-2 In the first two weeks, soil samples were collected from locations in Waco. 10-15g were observed in a non-flooded plate using a compound scope. Observations were recorded. 5.0 grams of the wet soil were stored and in the second week, the soil was further investigated. Additionally, 5 mL of the soil samples were added to falcon tubes and vortexed. The following week, the water content, percentages of sand, silt and clay, pH, latitude/longitude, and temperature of sample site were recorded. Weeks 3-4 Using the non-flooded plates from week one, ciliates were extracted and cultured in 24 well plates with 900 microliters of Cerophyll media. If ciliates were not found, students used other’s soil samples. In the fourth week, a modified Chelex extraction protocol was used to extract ciliate DNA from the 24-well plates. Students added 5% Chelex to 500 uL of centrifuged ciliate culture, therefore protecting the DNA against enzymatic activity. The microtubes were then incubated at 56℃, boiled at 100℃, vortexed, and centrifuged. Remaining supernatant with DNA was transferred to new microtube. Weeks 5-6 In week five, Polymerase Chain Reactions (PCR) were performed on previously collected DNA samples using SSU ribosomal and universal primers. A reaction mixture was created using 5 uL of master mix, 1 uL of primer, and varying amounts of water. Unfortunately in week 6, the thermocycler broke and overheated the DNA samples, causing them to evaporate. Process was repeated to test new DNA samples. Agarose Gels were made by mixing 40 mL of Tris acetate EDTA with agarose gel, and mixing them with 2 uL of ethidium bromide. Gel electrophoresis box was filled with agarose gel and allowed to solidify. Gel Electrophoresis was performed by adding 10 uL of each PCR product to the wells and turning the power supply to 100 volts. DNA was viewed with UV light. The process of ciliate discovery is essential in learning about the diversity of soil in our world. The present experiment aims to teach students the scientific process of ciliate discovery and classification, through the soil collection, DNA extraction, Polymerase Chain Reactions, and Gel Electrophoresis. Because of malfunctions with the procedure machinery, the results were inconclusive. It is important to educate students about this process, so that they can continue discovering new ciliates to contribute to the current knowledge of biodiversity. Throughout the semester, students in the CILI-CURE laboratory have learned to pick and culture cells to begin the process of ciliate discovery. Over the course of several weeks, soil collections were performed and DNA was extracted from the ciliates found in the soil. Polymerase Chain Reactions were performed, with the intention of Gel Electrophoresis. Due to malfunctions with the thermocycler in the PCR process, the results were inconclusive. The thermocycler froze, thawed, and heated the DNA too many times, causing damage and evaporation to the DNA’s composition. Additional sources of human error would include miscalculations when creating the PCR master mix. The pre-mixed primer could have been incorrectly mixed or the DNA could have degraded prior to the PCR protocol. ( Figure IV: Observations Table: The data recorded during the entire experiment A special thanks to Dr. Tamara Adair, Michael Davis, and the incredible teaching assistants of the Department of Biology of Baylor University for making this experiment possible and leading our class with passion and purpose throughout the semester. Process Kayla (KAC04S17) Audrey (AMJ04S17) Waverly (WBK04S17) Non-Flooded Plates Found many nematodes, a few ciliates (about 3:1) Found many nematodes, a few ciliates (about 3:1) Didn’t find any life 24-Well Plates Transferred four ciliates, none survived the following week, no growth Transferred four ciliates in four different wells, different species, following week had 10-15 ciliates in two of the wells, and none in the other two wells Transferred five ciliates into five wells, a week later only one well still had living ciliates DNA Concentration (ng/ul) DNA Purity (260/280) 14.82 ng/ul 1.66 48.14 ng/ul 1.33 44.73 ng/ul 1.34 Introduction Soil Metadata AMJS1704 KACS1704 WBKS1704 pH 8 6.5 7.5 Soil Classification Silt Silt Loam Sandy Clay Loam Percent Water Composition 29.4 % 8 % 18 % Description of Location of Soil Collection Collected outside of the Baylor Science Building on Baylor Campus, in grass with no trees or plants near the site (Waco, TX) Collected outside of “Bear Park” on Baylor Campus, an area where grass was not growing (Waco, TX) Collected at Cameron Park, near a large tree and the Brazos River (Waco, TX) Ciliates Found? Yes Yes No - used AMJS1704 sample for the rest of experiment Figure III: Location where soil sample WBK04S17 was found Over the course of the semester, students in the CILI-CURE laboratory have learned important techniques in biology research, acquiring skills in micro-pipetting and culturing. Students practiced researching and reviewing scientific literature to apply to the growth mindset standard in the classroom. In the laboratory, students focused on the diversity of soil ciliates, which lead the path to collecting soil samples from different areas in Waco to identify different ciliate species. The purpose was to find commonalities in the soil metadata to conclude whether or not the characteristics affected which ciliates were found there. Figure I. Metadata Table: Information regarding soil samples collected D Lynn (2009) Ciliates. Encyclopedia of Microbiology. PDF. Figure IV: Ciliate Taxonomy displaying the various classes, orders, and families (Gao, F., et al.) Gao, F., Warren, A., Zhang, Q., Gong, J., Miao, M., Sun, P., . . . Song, W. (2016). The All-Data-Based Evolutionary Hypothesis of Ciliated Protists with a Revised Classification of the Phylum Ciliophora (Eukaryota, Alveolata). Scientific Reports, 6(1). doi:10.1038/srep24874 Metadata of Collected Sample Soils Observations from the Process Figure II. Map including locations of collected soil samples
Transcript of Exploration of Ciliate Discovery in Waco€¦ · Exploration of Ciliate Diversity in Waco Kayla...
Exploration of Ciliate Diversity in WacoKayla Canava, Audrey Jacobs, Waverly Kundysek
Baylor University
Methods and Materials
Abstract
Acknowledgements
Conclusion
References
Observations
Weeks 1-2In the first two weeks, soil samples were collected from locations in Waco. 10-15g were observed in a non-flooded plate using a compound scope. Observations were recorded. 5.0 grams of the wet soil were stored and in the second week, the soil was further investigated. Additionally, 5 mL of the soil samples were added to falcon tubes and vortexed. The following week, the water content, percentages of sand, silt and clay, pH, latitude/longitude, and temperature of sample site were recorded.
Weeks 3-4Using the non-flooded plates from week one, ciliates were extracted and cultured in 24 well plates with 900 microliters of Cerophyll media. If ciliates were not found, students used other’s soil samples. In the fourth week, a modified Chelex extraction protocol was used to extract ciliate DNA from the 24-well plates. Students added 5% Chelex to 500 uL of centrifuged ciliate culture, therefore protecting the DNA against enzymatic activity. The microtubes were then incubated at 56℃, boiled at 100℃, vortexed, and centrifuged. Remaining supernatant with DNA was transferred to new microtube.
Weeks 5-6In week five, Polymerase Chain Reactions (PCR) were performed on previously collected DNA samples using SSU ribosomal and universal primers. A reaction mixture was created using 5 uL of master mix, 1 uL of primer, and varying amounts of water. Unfortunately in week 6, the thermocycler broke and overheated the DNA samples, causing them to evaporate. Process was repeated to test new DNA samples. Agarose Gels were made by mixing 40 mL of Tris acetate EDTA with agarose gel, and mixing them with 2 uL of ethidium bromide. Gel electrophoresis box was filled with agarose gel and allowed to solidify. Gel Electrophoresis was performed by adding 10 uL of each PCR product to the wells and turning the power supply to 100 volts. DNA was viewed with UV light.
The process of ciliate discovery is essential in learning about the diversity of soil in our world. The present experiment aims to teach students the scientific process of ciliate discovery and classification, through the soil collection, DNA extraction, Polymerase Chain Reactions, and Gel Electrophoresis. Because of malfunctions with the procedure machinery, the results were inconclusive. It is important to educate students about this process, so that they can continue discovering new ciliates to contribute to the current knowledge of biodiversity.
Throughout the semester, students in the CILI-CURE laboratory have learned to pick and culture cells to begin the process of ciliate discovery. Over the course of several weeks, soil collections were performed and DNA was extracted from the ciliates found in the soil. Polymerase Chain Reactions were performed, with the intention of Gel Electrophoresis. Due to malfunctions with the thermocycler in the PCR process, the results were inconclusive. The thermocycler froze, thawed, and heated the DNA too many times, causing damage and evaporation to the DNA’s composition. Additional sources of human error would include miscalculations when creating the PCR master mix. The pre-mixed primer could have been incorrectly mixed or the DNA could have degraded prior to the PCR protocol.
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Figure IV: Observations Table: The data recorded during the entire experiment
A special thanks to Dr. Tamara Adair, Michael Davis, and the incredible teaching assistants of the Department of Biology of Baylor University for making this experiment possible and leading our class with passion and purpose throughout the semester.
Process Kayla (KAC04S17)
Audrey (AMJ04S17)
Waverly (WBK04S17)
Non-Flooded Plates Found many nematodes, a few ciliates (about 3:1)
Found many nematodes, a few ciliates (about 3:1)
Didn’t find any life
24-Well Plates Transferred four ciliates, none survived the following week, no growth
Transferred four ciliates in four different wells, different species, following week had 10-15 ciliates in two of the wells, and none in the other two wells
Transferred five ciliates into five wells, a week later only one well still had living ciliates
DNA Concentration (ng/ul)DNA Purity (260/280)
14.82 ng/ul1.66
48.14 ng/ul 1.33
44.73 ng/ul 1.34
Introduction
Soil Metadata AMJS1704 KACS1704 WBKS1704pH 8 6.5 7.5Soil Classification
Silt Silt Loam Sandy Clay Loam
Percent Water Composition
29.4 % 8 % 18 %
Description of Location of Soil Collection
Collected outside of the Baylor Science Building on Baylor Campus, in grass with no trees or plants near the site (Waco, TX)
Collected outside of “Bear Park” on Baylor Campus, an area where grass was not growing (Waco, TX)
Collected at Cameron Park, near a large tree and the Brazos River (Waco, TX)
Ciliates Found? Yes Yes No - used AMJS1704 sample for the rest of experiment
Figure III: Location where soil sample WBK04S17 was found
Over the course of the semester, students in the CILI-CURE laboratory have learned important techniques in biology research, acquiring skills in micro-pipetting and culturing. Students practiced researching and reviewing scientific literature to apply to the growth mindset standard in the classroom. In the laboratory, students focused on the diversity of soil ciliates, which lead the path to collecting soil samples from different areas in Waco to identify different ciliate species. The purpose was to find commonalities in the soil metadata to conclude whether or not the characteristics affected which ciliates were found there.
Figure I. Metadata Table: Information regarding soil samples collectedD Lynn (2009) Ciliates. Encyclopedia of Microbiology. PDF.
Figure IV: Ciliate Taxonomy displaying the various classes, orders, and families (Gao, F., et al.)
Gao, F., Warren, A., Zhang, Q., Gong, J., Miao, M., Sun, P., . . . Song, W. (2016). The All-Data-Based Evolutionary Hypothesis of Ciliated Protists with a Revised Classification of the Phylum Ciliophora (Eukaryota, Alveolata). Scientific Reports, 6(1). doi:10.1038/srep24874
Metadata of Collected Sample SoilsObservations from the Process
Figure II. Map including locations of collected soil samples
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