Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Undergraduate Category:Engineering and Technology Degree Level: Bachelors of Science Abstract ID# 1213 Developing Plant Tissue Cultures for the Production of Pharmaceuticals 1Dept. Camille A. 2, Martin *, Dr. Erin of Chemical Engineering / Bioengineering, INTRODUCTION GOAL 3 Cram , 2Chemistry Dr. Carolyn & Chemical Biology, and To investigate how the production of valuable pharmaceutical compounds from Catharanthus roseus are regulated. Figure 1. Chemical structure of the anti-cancer compounds5 Figure 2. Catharanthus roseus The biosynthetic pathway in C. roseus consists of approximately 30 steps and is tightly regulated by a number of transcriptions factors. Several studies have been conducted to determine crucial steps within the pathway including overexpressing enzymes in plant tissue cultures. However, these attempts were unsuccessful in increasing alkaloid production in C. roseus. Figure 3. Hairy root culture (above)4 Figure 4. TIA biosynthetic pathway (right)3 The Lee-Parsons Group is investigating the transcriptional regulation of the TIA biosynthetic pathway utilizing transgenic hairy root cultures. Hairy roots are produced via Agrobacterium-mediated transformation. Hairy root cultures are useful for studying the biosynthesis of TIAs in C. roseus and is scalable, genetically stability, and can be cultivated independently of environmental conditions. ACKNOWLEDGEMENTS Noreen Rizvi, PhD, and Jessica Weaver, PhD Figure 5. Pictured left to right are the steps for developing transgenic hairy root cultures. 1. Seed sterilization and planting. 2. Engineered plasmid is inserted in the plant genome via Agrobacterium-mediated transformation during the infection step. 3. Root excision. 4. Agrobacterium elimination and selection. 5. Liquid media adaption followed by several rounds of subculturing. 4 Waters. How Does High Performance Liquid Chromatography Work? Digital image. Waters.com. Waters, 2016. Web. 17 Mar. 2016. van der Heijden, R., et al. Current Medicinal Chemistry, 2004, 11, 607-‐628 Goklany, S., et al., Biotechnology Progress, 2009, 25, 1289-‐1296 Rizvi, N., et al., Journal of Plant Biotechnology, 2015, 120, 475-‐487 Tikhomiroff, C. and Jolicoeur, M. Journal of Chromatography A, 2002, 955, 87-‐93 Hairy root culture were extracted from ~50 mg of dried plant material in methanol. Samples are analyzed via HPLC with a C18 column and Empower software. 1 3 2 1 Figure 9. Chromatogram analyzed at 254 nm; peak 1: strictosidine, peak 2: ajmalicine, peak 3: serpentine GENOMIC INTEGRATION Figure 10. The UV spectra of ajmalicine (left) and serpentine (middle), strictosidine (right) Figure 6. The results of the genomic integration analysis of 17 hairy root culture lines.4 Figure 7. Diagram of Agrobacterium rhizogenes used for gene transformation4 In order to ensure that the correct genes have been inserted into the DNA of C. roseus, genomic integration was performed. The first step was to extract the genomic DNA of each line. PCR was then used to amplify the following genes: • Rps9: Housekeeping gene specific to C. roseus, PCR control • Gfp: Gene of interest, confirming integration of desired gene • HygR: Hygromycin resistance gene, crucial for root selection • VirD2: Virulence gene, indicative of Agrobacterium contamination • RolC: Hairy root control, indicative of root generated by Agrobacterium The amplified genes were separated on a 2% agarose gel. A UV transilluminator was used to view and confirm the expected size of the desired PCR products. ALKALIOD PRODUCTION ANALYSIS We study the alkaloid profile in the hairy roots by High Performance Liquid Chromatography (HPLC). We can identify a number of alkaloids based on their characteristic ultraviolet (UV) absorbance spectra and retention times. Northeastern Departments of Chemical Engineering, Bioengineering, Chemistry & Chemical Biology, and Biology 1. 2. 3. 4. 5. RESULTS 1 National Science Foundation – CBET AWARD NUMBER 1033889 REFERENCES 3Biology METHODS TRANSGENIC LINE DEVELOPMENT Catharanthus roseus, also known as Madagascar periwinkle, is a medicinal plant that naturally produces over 130 alkaloids including the terpenoid indole alkaloids (TIAs), vinblastine and vincristine. Vincristine and vinblastine are chemotherapy drugs that fight a variety of cancers, including leukemia and Hodgkin’s disease by disrupting tumor development. The natural production of vincristine and vinblastine by C. roseus is inefficient, requiring approximately 500 kg of plant material for 1g of isolated alkaloids. In addition to low production, the extraction of these key compounds is extremely expensive and strenuous. Therefore other methods of producing these compounds have been explored. 1,2 Lee-Parsons Figure 8. Scheme of high performance liquid chromatography1 Alkaloid concentration (mg/g DW tissue) Victoria 1, D’Agostino *, 3.4 3.2 3.0 2.8 2.6 2.4 2.2 2.0 1.8 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 Ajmalicine Serpentine Strictosidine Figure 11. Diagram of TIAs isolated from C. roseus hairy cultures CONCLUSIONS & FUTURE DIRECTIONS C. roseus is known to naturally produce useful alkaloids, including the pharmaceutical compounds vincristine and vinblastine. By modifying the metabolic pathways of the plant, tissue cultures can be produced which overproduce these useful alkaloids at higher production rates. In the future, tissue cultures will be developed in which the desired target gene is engineered, and the resulting alkaloids profiles can be monitored and quantified (as shown above). The goal of this research is to optimize the rate of pharmaceutical production from C. roseus.