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Title: Diatom silicon transporters : from protein function to biomimetic silica synthesis
Author: Senior, Laura
ISNI:       0000 0004 5923 838X
Awarding Body: University of Bristol
Current Institution: University of Bristol
Date of Award: 2014
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Biomineralisation is the synthesis of inorganic materials in biological systems. Many biominerals - such as bone! teeth, and shells - are high-performance composites synthesised with extreme precision under physiological conditions. Understanding biomineralisation is expected to inspire 'green' methods for the manufacture of novel materials. Diatoms are eukaryotic algae that mineralise an external cell wall, or frustule, composed of hydrated silica. Silicification depends upon the uptake of soluble silicon (silicic acid) from the local environment by specific silicic acid transport proteins (SITs). This unusual family of integral membrane proteins are relatively uncharacterised. This project aimed to express and purify 5113 from the diatom Thalassiosira pseudonana (TpSIT3) for further characterisation in vitro, and to explore whether synthetic SIT3 proteoliposomes could be used as a model mineralisation system with potential applications in nanotechnology. TpSIT3 was successfully overexpressed in yeast and purified in the solubilising detergents Fos-choline 12 and octyl glucoside. The purified protein was successfully reconstituted into synthetic liposomes and silicic acid uptake was assessed using two fluorescent assays including a novel method which utilised zinc silicate fluorescence. This method was used to determine that silicic acid transport by TpSIT3 displayed Michaelis-Menten kinetics with a Km of 6.1 ± 2.7 μM, similar to silicic acid uptake studies in diatom cultures. The structure and function of a silicifying cationic peptide were also characterised for the first time. Peptide-mediated silicification only proceeded at ≥2 mM silicic acid when pH was >6.4 and peptide concentration was ≥2.5 mM. These results underpinned efforts to synthesise silica within the int erior lumen of peptide-loaded SIT proteoliposomes. Preliminary electron microscopy and elemental analysis suggested that such an approach was feasible. This thesis thus establishes a series of novel methods that can be used to study silicic acid transport and silica mineralisation in vitro
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available