Missing Bacterial DNA

Unfortunately, I found out about two weeks ago that the DNA I had extracted from my unknown bacteria was missing. I know I had not misplaced it or threw it away because I remember putting it back where it always goes. I had not mentioned it before because I thought that it would eventually turn up. However, that was not the case and I am quite upset about it because I am almost certain that it was not a mistake I made. With that said, there was no further advancement in my research project this week. Instead, I was preparing and creating a PowerPoint about my research, along with adding more information to my final paper. As far as I am concerned, I will present this PowerPoint in front of other STEM interns and Bioscience faculty. I guess I am going to have to find out for myself in a couple of weeks!

Figure 1. Possible PowerPoint theme for the research project that was carried during the Fall 2017 S-STEM internship. It will be titled "Identification of Bacteria Isolated From Wild and Urban Larrea Tridentata Utilizing RS-16 Sequencing".

Additional Unknown Bacteria

From last week’s broth, the resulting broth that was left at room temperature over the weekend is seen in Figure 1. In addition, this week, I grew Samantha Faltermeier’s unknown bacteria in TSB broth. The bacteria that I am working with has been isolated on a TSA plate from a creosote bush. As a side note, this bacteria is quite interesting because they all seem to be different. For instance, some have a horrible stench, while others don’t. Also, some almost look like snowflakes and some look like a typical round bacterial colony. As mentioned last week, I performed DNA extraction on these unknowns. Next week, if I am able to, PCR and electrophoresis are the next steps!

Figure 1. The resulting TSB broth that was left at room temperature over the course of 5 days. These samples contained different unknown bacteria that was from a creosote bush.

Results... At Last!

On October 31, 2017, I ran my post-PCR DNA samples from October 26, 2017 under electrophoresis. Theses particular samples were different because I doubled everything, except the DNA. I added 20 microliters of Master Mix, 11 microliters of sterile water, 4 microliters of the forward primer, 4 microliters of the reverse primer and 1 microliter of DNA. All this totaled to an amount of 40 microliters. The primers used were 27F and 1492R. Once I ran these under electrophoresis, I finally got results (Figure 1)! I was so excited because at one point, I was beginning to think I was doing my procedures wrong. Nonetheless, I am happy to say that this change made a difference. Also, this week, I started growing bacteria in TSB broth (Figure 2) that was isolated by Samantha Faltermeier, an S-STEM intern. I will be performing DNA extraction and, eventually, PCR on these unknown bacteria!

Figure 1. The resulting gel after it was ran under electrophoresis. The post-PCR DNA in each row from left to right is as follows: MW Ruler, 1, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 & E. Coli. The particular DNA that amplified were 4, 9, 10, 11, 12 & 13. These were the ones that showed bright bands.

Figure 2. Unknown DNA samples from isolated bacteria in a Creosote bush. The samples were transferred from a TSA plate to these TSB broths. They are growing at room temperature and the samples are 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 & 14.

Project Background

What is currently known:

Currently, there are a wide range of research projects that have been conducted on larrea tridentata (creosote bush). In particular, a research carried by L.M. Nguyen and others, they were able to conclude that “the concentration of total bacteria 16S rDNA was similar in conditions of enriched and ambient [CO2]” (Nguyen, 2011). In other words, this particular DNA that is present in a creosote bush has a relationship with the amount of carbon dioxide. To support their findings, they noticed that “the decrease in representation of Gram positives and the similar values for total bacterial DNA suggest that the representation of other bacterial taxa was promoted by elevated [CO2]” (Nguyen, 2011). Another example involves an STD named herpes. Sandra Levy provided a the insight that there was a new product that was used to relieve symptoms. This product is Herp-Eeze, which “is a dietary supplement containing an extract of the plant Larrea tridentata” (Levy, 1999). In addition, it is also known that “arid ecosystems, which occupy about 20% of the earth's terrestrial surface area, have been predicted to be one of the most responsive ecosystem types to elevated atmospheric CO2 and associated global climate change” (Smith, 2000). Considering the fact the creosote bushes are located in a desert, this information does apply to it.

Importance to expand on this knowledge:

With the expansion of urban areas, figuring out what kind of bacteria grows on larrea tridentata is essential. This bacteria may or may not slowly kill a plant or seriously injure an animal or human. The same applies for wild larrea tridentata. However, there could be a possibility that the wild bacteria is beneficial to it and its surroundings. However, there is a chance that the certain bacteria is damaging to not only the plant but its environment as well. Figuring out which of the various species of bacteria are present will help lead to answer questions of interested. For instance, obtaining information about certain bacteria in urban and wild larrea tridentata can help explain why plant soil is toxic. An additional question of interest is whether there is a relationship between a species of bacteria and carbon monoxide. Could this potentially be one of the causes to the rise of carbon dioxide levels? Does the bacteria on these plants affect plants ability to obtain carbon dioxide? A study ran by L.P. Nguyen suggests that “the decrease in representation of Gram positives and the similar values for total bacterial DNA suggest that the representation of other bacterial taxa was promoted by elevated [CO2]” (Nguyen, 2011). Finding out whether or not carbon dioxide goes hand in hand with certain species of bacteria is valuable to know so that we can work together to reduce carbon dioxide levels and, thus, benefit living organisms (plants).

Larrea tridentata (creosote bush) ("Lady Bird Johnson Wildflower Center", n.d).

References

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Kuikman, P.J., & Van Veen, J.A. (1989). The impact of protozoa on the availability of bacterial nitrogen to plants. Biology and Fertility of Soils. https://doi.org/10.1007/BF00260510

Kembel, S. W., O’Connor, T. K., Arnold, H. K., Hubbell, S. P., Wright, S. J., & Green, J. L. (2012). Relationships between phyllosphere bacterial communities and plant functional traits in a neotropical forest. PNAS, 111(38), 13715-13720. https://doi: 10.1073/pnas.1216057111

Lady Bird Johnson Wildflower Center. (n.d.). Retrieved November 02, 2017, from https://www.wildflower.org/gallery/result.php?id_image=7711

Levy, S. (1999). Creosote bush in Arizona sprouts new herpes product. Drug Topics. Retrieved from General OneFile database.

Nguyen, L. M., Buttner, M. P., Cruz, P., Smith, S. D., & Robleto, E. A. (2011). Effects of elevated atmospheric CO2 on rhizosphere soil microbial communities in a Mojave Desert ecosystem. Journal of Arid Environments, 75(10), 917-925. Retrieved from Science Direct database.

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QuickExtract Bacterial DNA Extraction Kit. (n.d.). Retrieved from EpiBio website: http://www.epibio.com/docs/default-source/protocols/quickextract-bacterial-dna-extraction-kit.pdf?sfvrsn=8

Smith, S. D., Huxman, T. E., Zitzer, S. F., Charlet, T. N., Housman, D. C., Coleman, J. S., . . . Nowak, R. S. (2000). Elevated CO2 increases productivity and invasive species success in an arid ecosystem. Nature, 79-82. https://doi.org/10.1038/35040544

Stellwagen, N. C. (2009). Electrophoresis of DNA in agarose gels, polyacrylamide gels and in free solution. Electrophoresis, 30, S188-S195. https://doi.org/10.1002/elps.200900052

Steven, B., Gallegos-Graves, L. V., Yeager, C., Belnap, J., & Kuske, C. R. (2014). Common and distinguishing features of the bacterial and fungal communities in biological soil crusts and shrub root zone soils. Soil Biology and Biochemistry, 69, 302-312. Retrieved from ScienceDirect database.