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Abstract
The goal of this experiment was to isolate and sequence the GAPDH gene from Lilac. To extract, amplify, and transform the genomic DNA, we used PCR and nested PCR. This data was observed with gel electrophoresis and plating, which allows us to analyze the effectiveness of the ligation. pJET1.2 provided an essential restriction site that included eco47IR that killed the expressed genes in combination with the Bl/II gene that provided ampicillin resistance. The PCR was unsuccessful, as the DNA did not appear with the right base pairs during gel electrophoresis. The DNA sequence indicates that the GAPDH we extracted from Lilac leaves was derived from a species of deciduous tree: Fraxinus Quadrangulata.
Introduction
Many organisms undergo a step during cellular respiration called glycolysis. During glycolysis, energy is extracted from glucose by dividing it into pyruvates. This process occurs in the cytosol of the cell and the enzyme GAPDH is involved in the release of energy. GAPDH is oxidized in an exergonic reaction, thus releasing the energy that is used for phosphorylation (Khan Academy). Syringa Vulgaris, commonly known as Lilac, is a flowering shrub that was the source of DNA in this experiment (Gardenia).
The GAPDH from the lilac leaves was extracted and amplified by polymerase chain reaction (PCR) using Taq DNA polymerase. Gel electrophoresis was used as a way to observe and examine the results of PCR, with Arabidopsis used as a control group in comparison to the amplified DNA. Arabidopsis is a small plant often used as a model for genetic research due to its small genome sequence (TAIR, 2005). pJET1.2 contains a restriction site where the needed gene was introduced, while also containing eco47IR; this gene is what gives Escherichia coli (E. coli) its toxicity. Beta-lactamase (Bl/II), also encoded in pJet1.2, provides the genes with an antibiotic resistance to ampicillin through detoxification. The pJET1.2 was encoded with IPTG, which allows the lac operon to induce protein expression for the Bl/II enzyme. When plated, the E. coli that have adapted the plasmid DNA will survive due to containing the proper Bl/II genes that allow ampicillin resistance (Piispanen, 2019). The goal of this experiment was to extract, isolate, and sequence the GAPDH genes from Lilac. Sequencing showed GAPDH from a species of tree called Blue Ash, although there could be a genomic relationship to Lilac.
Methods and Materials
All steps including the centrifuge were spun at 13,000 x g for the instructed time unless stated otherwise. The instructions for the Bio-Rad nucleic acid extraction kit were followed using Lilac leaves. The Bio-Rad GAPDH PCR kit was used, following only initial PCR instructions using Taq DNA polymerase. Gel Electrophoresis was used to observe the separation of PCR products. 5 ul 5X Orange G loading dye was pipetted into each microcentrifuge tube with 20 ul of the PCR products into the respective microcentrifuge tube and mixed. 10 ul of the 500 bp molecular weight ruler was loaded into lane 1 of the gel with 20 ul of each initial PCR product diluted in Orange G into the gel. The electrophoresis was run at 110V for 30-40 mins, then visualized with the Gel-Doc. Nested PCR was conducted with the Bio-Rad GAPDH PCR kit and the templates created during the initial PCR after being treated with Exonuclease I. Instructions for purification followed the Bio-Rad kleen purification kit. Ligation was conducted using the Bio-Rad ligation and transformation kit. The QIAprep Spin Miniprep Kit was used for plasmid isolation. Transformation was carried out following the Bio-Rad ligation and transformation kit. Gel electrophoresis was conducted again to observe the results. The samples were then sent to the science lab at Dartmouth College for sequencing.
Conclusion/Discussion
Syringa Vulgaris contains GAPDH that is, in part, responsible for areas of glycolysis. The genomic DNA was extracted, amplified, transformed, and ligated in an attempt to isolate and sequence the GAPDH genes. The experiment was ultimately unsuccessful, as the results from gel electrophoresis showed that the PCR did not work in transforming and ligating the genomic DNA. Base pairs correctly appeared for the gDNA and Arabidopsis gDNA, although it was expected for there to be more base pairs at around 600. The plated DNA for pJET1.2 and pGLO was also unsuccessful, with little bacteria on the ampicillin resistant plate, and excessive growth on the non-ampicillin resistant plate. This could have been due to bad or expired ampicillin, but a second attempt at plating was also unsuccessful. The sequence of DNA connected to Blue Ash, which could have been caused by incomplete or altered sequences. This is possible if the DNA was contaminated, or due to a genomic similarity between Fraxinus Quadrangulata and Syringa Vulgaris (Frazer et. al. 2003).
References
- Frazer, K. A., Elnitski, L., Church, D. M., Dubchak, I., & Hardison, R. C. (2003). Cross-species sequence comparisons: a review of methods and available resources. Genome research, 13(1), 112. doi:10.1101/gr.222003
- Gardenia. Syringa Vulgaris (Common Lilac). (n.d.) https://www.gardenia.net/plant-variety/syringa-vulgaris-common-lilac
- Khan Academy. Cellular Respiration: Glycolysis. (n.d.) https://www.khanacademy.org/science/biology/cellular-respiration-and-fermentation/glycolysis/a/glycolysis
- Missouri Botanical Garden. Fraxinus Quadrangulata. (2019.) http://www.missouribotanicalgarden.org/PlantFinder/PlantFinderDetails.aspx?kempercode=a869
- Piispanen, A. (2019). DNA Extraction from Plants [Class Handout]. BI211 Genetics, Franklin Pierce University Rindge, NH.
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