GENETIC TRANSFORMATION OPTIMIZATION IN SACCHAROMYCES CEREVISIAE USING SINGLE-WALLED CARBON NANOTUBES (SWCNTS)

• Main Author: Khairunnisa, Ranindya
• Format: Final Project
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/87653
• Institution: Institut Teknologi Bandung

Transformation is the process of internalizing exogenous DNA into living cells that allows organisms to acquire new genetic traits and adapt to changing environmental conditions. The yeast Saccharomyces cerevisiae is a model eukaryotic unicellular organism for genetic modification. In yeast cells, the most common methods used to perform transformation are the lithium acetate method and electroporation. The lithium acetate method tends to be easier to perform technically but requires quite a lot of reagents and has a lower transformation efficiency when compared to other methods. Another alternative is electroporation. This method can produce higher transformation efficiency, but the operational costs required are expensive. Recent research found that CNTs (Carbon Nanotubes) have the ability to support the process of gene and protein delivery in plants. SWCNTs (Single-Walled Carbon Nanotubes) is one type of CNTs with strong tensile strength characteristics to penetrate cell walls, good biocompatibility, and protection of cargo from cell metabolism and degradation so that it can act as an optimal carrier of genetic material and protein. The main objective of this study was to transform pCAMBIA1303 using SWCNTs in Saccharomyces cerevisiae and to determine the transformation efficiency of transforming pCAMBIA1303 into yeast using the SWCNTs method. For comparison, transformation using Agro bacterium was carried out, both in yeast and plants. The research design carried out first is the creation of a growth curve and standard curve of S. cerevisiae to determine the number of cells to be transformed. Next, the preparation of Agrobacterium transformant and SWCNTs as pCAMBIA1303 biocarrier in yeast cells and plant cells. The fourth stage is the genetic transformation using Agrobacterium and SWCNTs in yeast and plant cells. From the growth curve and the standard curve that has been done, the standard curve equation is y = 1.2919x + 6.148. To prepareAgrobacterium, transformation was carried out using the Freeze-Thaw method and selected with kanamycin antibiotics at a concentration of 50 µg/mL. Preparation of SWCNTs was carried out using PEI surfactant and obtained dispersed SWCNTs at a concentration of 55.61 mg/L. The transformation of pCAMBIAl 303 using SWCNTs in S. cerevisiae did not produce indigo color from the substrate X-gluc from the breakdown of beta-glucoronidase which should be expressed in yeast. The transformation results of SWCNTs and Agrobacterium showed positive results with the production of blue color in plant cells. SWCNTs successfully carried pCAMBIA1303 which was successfully expressed in plants, although it has not been successfully expressed in yeast. The unsuccessful transformation was thought to be due to the size of the SWCNTs used being too large, 15-30 nm, while those used in the reference article were 4-5 nm. The effect of a large amount of PEI as a surfactant in the first preparation stage may also affect cell survival. Additional optimization is needed to determine the optimal complex ratio of pCAMBIA with PEI-SWCNTs and develop a CNT-competent yeast cell manufacturing protocol