DEVELOPMENT OF SINGLE-WALLED CARBON NANOTUBES (SWCNT) AS GENETIC TRANSFORMATION VECTORS IN PICHIA PASTORIS X-33.
Pichia pastoris is an excellent microorganism for recombinant protein production due to its high expression capacity and post-translational modification capabilities. Genetic transformation in P. pastoris typically employs electroporation, which requires specialized equipment and incurs high costs....
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| Format: | Final Project |
| Language: | Indonesia |
| Online Access: | https://digilib.itb.ac.id/gdl/view/87840 |
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| Institution: | Institut Teknologi Bandung |
| Language: | Indonesia |
| Summary: | Pichia pastoris is an excellent microorganism for recombinant protein production due to its high expression capacity and post-translational modification capabilities. Genetic transformation in P. pastoris typically employs electroporation, which requires specialized equipment and incurs high costs. As an alternative, Single-Walled Carbon Nanotubes (SWCNT) can serve as a cost-effective solution, as they can bind to DNA and diffuse directly into cells without the need for specialized tools. Although this method has been successful in plants, mammalian cells, and bacteria, its application in P. pastoris remains limited. Additionally, the availability of specific plasmids for yeast poses a challenge; therefore, the plant plasmid pCAMBIA 1301 with the CaMV 35S promoter, known to be expressible in yeast, was utilized. This study aims to develop a method for using SWCNT as a genetic transformation vector in P. pastoris. SWCNT were dispersed using ultrasonication with the surfactant Pluronic F-127 to enhance solubility, followed by bioconjugation with Polyethylenimine (PEI) and incubation with pCAMBIA 1301. Transformation in P. pastoris using the SWCNT-PEI-DNA suspension was tested at various cell densities, and validation of the transformation system was conducted using chili pepper leaves (Capsicum annuum) through GUS assays. Transformation in P. pastoris and chili plants using Agrobacterium tumefaciens GV3101 was performed to validate the expression system of the pCAMBIA 1301 plasmid. The success of the transformations was confirmed through GUS assays. The results indicated that the modified synthesis of SWCNT yielded a relatively high concentration (OD600: 2.7). Transformation of P. pastoris with SWCNT at an OD density of 0.8 showed success, while lower OD densities yielded negative results. Additionally, the results with the A. tumefaciens method were also negative. Transformation of SWCNT and A. tumefaciens in chili plants showed blue coloration in the GUS assay, indicating that the expression system of the pCAMBIA 1301 plasmid can function in both P. pastoris and chili leaf tissues. This study demonstrates that SWCNT can be utilized as a genetic carrier system in P. pastoris. Further studies are recommended to use Green Fluorescent Protein (GFP) for quantifying gene expression and morphological analysis using Scanning Electron Microscopy (SEM).
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