The plastid is an important organelle that allows eukaryotes to photosynthesize. The endosymbiotic event that led to the development of a primary plastid occurred more than one billion years ago. Since then the organelle has undergone a significant genome reduction, losing a large portion of non-essential genes whose function is covered by the eukaryotic host. The majority of the proteins needed for essential plastid function are transcribed in the nucleus and translated in the cytosol. These proteins are then translocated across the inner and outer plastid membranes. The difference in where proteins are translated and transported can be used to study plastid evolution, this can be done by examining what proteins are present in the red algae plastid and comparing it to proteins found in other algae groups. In this project, the exact placement of the proteins, mainly plastid proteins is of interest. In order to localize and identify proteins in the algal cell, an efficient method of cell lysis, both total and incomplete, where the preservation of organelles is essential needs to be developed. This thesis examines a set of different methods of cell lysis, both complete and incomplete, coupled with organelle fractionation, to achieve the isolation of proteins belonging to the different cellular compartments. I show that complete cell lysis with bead milling using 0.1-0.5 mm silica beads is more efficient than a method using a Dounce homogenizer. For incomplete lysis, I show that nitrogen cavitation at 750 psi for 15 min and 1,000 psi for 30 min provides the same level of cellular lysis, indicating that the nitrogen gas equilibrates within 15 minutes. Organelle fractionation with OptiprepTM gradient showed that the majority of the sample did not travel through the gradient, staying in the first layer, which also prevented revelation of the protein pattern on an SDS-PAGE gel, indicating insufficient centrifugation. A great deal of optimization is still required to make these methods as efficient as possible. The step that requires the most optimization is sample preparation for nitrogen cavitation and the use of an ultracentrifuge.
| Identifer | oai:union.ndltd.org:UPSALLA1/oai:DiVA.org:uu-533887 |
| Date | January 2024 |
| Creators | Kasparaviciute, Deimante |
| Publisher | Uppsala universitet, Institutionen för biologisk grundutbildning |
| Source Sets | DiVA Archive at Upsalla University |
| Language | English |
| Detected Language | English |
| Type | Student thesis, info:eu-repo/semantics/bachelorThesis, text |
| Format | application/pdf |
| Rights | info:eu-repo/semantics/openAccess |
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