Leslie Morales-21

Objective:

The purpose of lab today was to finalize our abstract and edit our poster so that they can be sent off to be printed.

Procedure:

1. Vote on logo for t-shirts and/or stickers
2. Collect sample ID
3. Enter metadata into spreadsheet and if PCR results were positive, move tube to appropriate rack and place soil bag in tub.
4. Go to computer lab and review comments on poster and make necessary edits.
5. Discuss abstracts and combine different ideas to form a final abstract.

Data:

Metadata

Group members: Leslie Morales, Holli Brown, Haleigh Inthavong

Section: 21

Group: 3

Soil ID: BIM21_3sp19

GPS location: -97.1168, 31.5502

Tree species: Texas Live Oak

BHD (cm): 49

pH: 6

Soil texture: Silt Loam

Extraction method: Silica bead

DNA concentration ng/µl: 598.2

~Volume: 15

PCR: +

Soil label on bag: HMB21S19

Poster title

The discovery of eDNA and the importance of ciliate biodiversity

Final Abstract

Soil ciliates are greatly understudied and play an important role in the rhizosphere. The objective of our study was to identify the presence of DNA within the soil sample collected from the Baylor University campus trees. The soil sample was collected from the O region of campus where the tree was classified as a Texas Live Oak, and the soil metadata, including a pH of 6, percent water of 6.024%, and soil texture of silt loam was observed and recorded. Following soil collection, students attempted to culture any observed ciliates, DNA extraction was performed by way of silica beads, the extracted DNA was purified through the use of a column and vacuum filtration, and PCR and gel electrophoresis was conducted utilizing the isolated DNA samples. The DNA sample had a 260/280 ratio of 1.42, which indicated that the DNA was relatively pure, and the sample had a positive PCR of approximately 400bp in comparison to the 1 kb ladder signifying that DNA was present within the sample. Because limited researched exists in regards to soil ciliates, the discovery of eDNA within campus trees can potentially lead to more future studies concerning the significance of ciliate biodiversity within the rhizosphere.

Final Poster 

Conclusion/ Future Steps:

Today’s lab dealt mostly with finalization. As the semester is coming to an end we are just trying to make sure everything is in place for presentations next week. In the future, we will practice presenting our posters and next Friday we will be in the atrium presenting the research that we have conducted.

April 26th, 2019

Rationale: 

The purpose of this lab was to revise our poster presentations as well as write our abstract drafts.

Procedure: 

• We began lab by editing our poster
  • The conclusion section was elaborated upon
  • The introduction was reorganized and shortened
  • A few of the figures were deleted and reconfigured
• Next, the abstract draft was written and submitted

Observations/Data:

Final Poster:

Abstract:

There are many standard methods for extracting Ciliate eDNA but not all are effective and cost efficient.

Ciliates are protozoa in the Kingdom Protista. They play many different roles within soil and aquatic ecosystems. There has been very little research done on ciliate behavior and interactions within the soil. Our study targeted ciliates within the rhizosphere. The rhizosphere plays a key role in the cycling of nutrients as well as plant growth. Ciliates shape the diversity of microorganisms within the rhizosphere, thus their presence is vital to a healthy ecosystem.

The purpose of this study was to investigate new methods for eDNA extraction of ciliates.

Creating this new method was achieved by researching different ways to extract eDNA. These different methods were then combined to maximize the amount of eDNA collected. Metadata was collected regarding the soil sample and the environment it was collected from. Gels were run to determine the amount of DNA collected. The samples that contained an abundance of DNA underwent PCR in order to amplify the strand so they could more easily be sequenced.

The samples were loamy sand, with a pH of 6.5. The concentration of DNA in the sample was 1125 ng/ul and the A260/A280 value was 1.33. The DNA sequenced not only belonged to ciliate species, but fungal and bacterial species as well.

These results gave reason to believe the use of charcoal and Silica beads was effective in the extraction of DNA.

Conclusion:

Our posters are ready to be presented. In order to explain our experiment, it is important that as a group we review our procedures and findings. We must understand our work completely so that we are able to communicate clearly with listeners the importance of our research.

Mackenzie Singer

4/25/19

Objective:

The purpose of this lab was to vote on a logo, create a final abstract, and complete our final poster so that they are ready to be presented.

Procedure:

1. Vote on a logo
2. Put soil metadata into the class spreadsheet
3. Label soil samples
4. Create a final abstract
5. Finish up the poster and make necessary changes

Data:

Abstract:

Ciliates play a major role in the food web which impacts all hierarchies of life. There is vast biodiversity in the rhizosphere of Earth, containing ciliates and other microorganisms (1). Little is known about the abundance of organisms that reside in the soil of terrestrial environments. This study was conducted to determine the diversity of soil ciliates in the rhizosphere on Baylor University’s campus. The process included collection, extraction, and amplification of ciliate DNA from the soil samples (2). The sample was retrieved from a Quercus Virginiana tree on Baylor’s campus in sandy loam soil. Our PCR for the 18S V4 primer resulted in a positive DNA band although the DNA concentration was not completely pure. Our results indicate that there is a large concentration of ciliates in the rhizosphere of trees on Baylor University’s campus.

Storage:

Soil tubes were placed in the rack.

Conclusion and future steps:

This lab was very productive in finishing up the posters. We finished them and submitted them for printing. Future steps will be to present these posters at the symposium. We also voted on a logo so that we can make cool stuff to wear.

Grace Seevers

26 April 2019

Purpose:

The objective of this lab was to upload soil metadata results to the excel sheet and label DNA samples in order to choose one to be sent for sequencing. Our groups finalized the lab posters for the symposium next week. A final draft of the abstract was written and posted on Box.

Procedure:

1. Fill out the shared excel spreadsheet with soil metadata.
2. Vote on Cillicure logo design.
3. Obtain and label soil and DNA samples “DPS22_Sp19”
4. Complete final draft of abstract and upload to box.
5. Complete final draft of poster and upload to Box.

Data:

Abstract

Ciliates are unicellular eukaryotic organisms that reside in freshwater or soil ecosystems. Ciliates are an important factor within the ecosystem because they consume different bacteria and protozoa, and act as a source of nutrition for organisms at higher trophic levels. The enormous discrepancy between the amount of research conducted on ciliates living in marine biomes versus ones living in the soil is due to a general lack of interest and/or knowledge of their importance. This experiment was conducted over the course of fourteen weeks in an effort to fill the gap between the amount of information known about marine and soil biodiversity by determining the overall composition of ciliates living in the rhizosphere of different trees across Baylor University’s campus. The V4 region of the 18S rRNA was observed because it is conserved within all eukaryotes and is unique to each specific organism. Soil samples were collected from the rhizosphere, where the highest concentration of ciliates are presumed to inhabit because of its abundance of nutrients and gas availability. After collection, the soil was searched for the presence of ciliates. Once the ciliates were isolated, their DNA was extracted and purified. The pure DNA was amplified through a polymerase chain reaction and ran through gel electrophoresis to determine whether or not eukaryotic DNA was present. Although our sample contained a very minute amount of DNA, if substantial DNA had been found, it would be sequenced using an online bioinformatics platform- QIIME2, to determine the specific organisms in our sample.

Conclusion:

In conclusion, our group worked together to submit our soil and DNA results and finalized our abstract and poster in preparation for the symposium. Our group is prepared to present our research and answer questions about ciliate biodiversity in the soil on Baylor’s Campus. In the future, a sample will be chosen to be sent off and next years class will analyze the results.

Purpose: The purpose of this lab was to make changes on our poster one more time, work on our abstract and record our metadata on the spreadsheet.

Title: Modified techniques to extract and amplify soil DNA for studying ciliate biodiversity

Abstract:

Ciliates are some of the most abundant and diverse protozoans; however, they are relatively under researched and little is known about how they affect the environment. This study was conducted in order develop a way to consistently and confidently determine the ciliate biodiversity in soil. The goal was to use the results from the biodiversity profiles to make inferences about the relationship between ciliate biodiversity and soil health. Metadata was collected from each sample to determine the conditions of the soil such as percent water, soil texture, and pH. Then, DNA was extracted, purified, ran through gel electrophoresis, and underwent a PCR reaction. DNA was present in the soil and was measure on the spectrophotometry results; however, after the PCR reaction was conducted, the results were only positive for five out of eight samples. Due to DNA being present in all samples, it appears that the DNA collection method works. The negative PCR result may be caused by impurities in specific samples that were not present in all samples.

Final Poster:

Fimal Powerpoint Presentation (5)-1aqokoh

Metadata recorded:

Group Members: Myrnalid Zapata, Tyler Bewley

Section: 21

Group: 1

SOIL-ID: BZ21_1Sp19

GPS Location: -97.1191, 31.5444

Tree Species: Brazilian Bluewood

BDH (cm): 46

pH: 6.5

Soil Texture: Sand

Extraction Method: Silica Bead

DNA concentration: 169.6 ng/ul

Volume: Missing

PCR: Negative (-)

Soil Label on Bag: Myrnalid Zapata Section 21

Next Steps: Our poster are going to get printed and next week we will be presenting them.

Conclusion: We filled out the Soil eDNA Metadata Spring 2019 spreadsheet, label our tubes correctly, work on our abstract and we made corrections to our posters one last time and turn it in. The posters are going to get print and next Friday we will be presenting them.

Lauren langston

4/26/19

The purpose of this lab was to prepare ourselves for the CILI-CURE symposium and to finish our posters and abstracts.

Title:

Isolation and Purification of Ciliate DNA from Ilex Vomitoria

FINAL ABSTRACT:

Nearly 85% of ciliate diversity has not been accounted for, yet ciliates play a crucial role in soil ecosystems, ensuring the cycling of materials and energy flow of other microorganisms and serving as predators regulating the environment. This study was conducted to develop a well-defined methodology that could be used to explore soil ciliates on a molecular level via sequencing. Soil samples were taken from an Ilex vomitoria and DNA was extracted using the silica bead extraction method. Metadata anaylsis of the soil showed that the soil type was sandy loam and had a pH level of 6.5. Gel electrophoresis showed that crude DNA was extracted from the soil samples and PCR was able to amplify an 18S V4 region of the DNA. From the Nanodrop analysis, performed after the extraction of a crude DNA sample and DNA purification, the extracted DNA had a purity of 1.46 (A260/A280), which is the expected score of a pure sample. The utilization of this less costly and more efficient protocol can yield DNA samples of a good purity that can be used for future sequencing and taxonomic identification of organisms within a collected sample.

META DATA:

FINAL POSTER:

Conclusion and future goals

I was able to fix along with my group members the amount of words on my poster and reorganize several of my elements on my poster. in the future I wish to have a successful presentation at the symposium.