Genetic and Environmental Manipulation of Diatom Frustule Shapes (in vivo biosilicification)
The overall goal of this thrust is to understand how genetic, biochemical, and environmental factors control the shapes and fine features of silica structures in diatoms, and to learn to specifically modify these structures for applications in nanotechnology. Specifically we plan:
- To evaluate stages in silica cell wall formation in diatoms using visualization approaches to understand the underlying principles of structure formation.
- To identify and characterize genes and associated proteins involved in diatom cell wall formation.
- To develop tools for genetic manipulation of diatoms to enable alteration of structure for the purposes of identifying gene function and tailoring structure to suit particular nanotechnological applications.
Goal 1. Characterization of the stages in formation of diatom silica structures, especially of intermediates, provides insights into the control mechanisms and structural design principles, and enables the development of models for structure formation. We are applying high-resolution imaging techniques such as SEM, TEM, AFM, and fluorescence microscopy to understand the underlying principles of silica formation and assembly in diatoms. Comparative analysis of a variety of diatom species is providing insights into both conserved processes of formation that represent common themes in diatom silicification, and processes unique to a given species that generate species-specific differences in silica structure.
Goal 2. Biosilica structures in diatoms are formed by the coordinated interactions between organic components in a spatially and temporally controlled manner. These involve the controlled expression of genes and correct intracellular targeting of proteins. We are identifying genes and proteins involved in silicification using genomic and proteomic approaches. Development of synchronous culture procedures has enabled identification of a distinct pattern of changes in mRNA levels that is apparently diagnostic of cell wall synthesis genes, which has led to microarray probing of the entire genome for genes exhibiting a similar pattern.
Goal 3. Because diatom silica structures have a genetic basis, manipulation of genes involved in silicification will enable alteration of structure. This involves developing basic tools of genetic manipulation, such as controlled promoters and gene replacement and downregulation techniques. Genetic manipulation has a twofold purpose, 1) it facilitates understanding the function of individual genes, and 2) it will enable heritable and controlled changes to be made, to optimize structures for specific applications.
Prior Related Work
Hildebrand, et al. (1993) isolated the first clones derived from genes responding to the presence or absence of silicon in the diatom Cylindrotheca fusiformis. From these clones, Hildebrand et al. (1997, 1998) isolated and characterized genes encoding the proteins responsible for transporting silicic acid across the lipid bilayer cell membrane and into the diatom cell. These proteins (called Silicic acid Transporters or SITs) were the first shown to specifically recognize and interact with silicon. Additional work revealed that silicic acid uptake was tightly coupled to cell wall silicification and an apparent feedback mechanism controlled the uptake according to the demand for silica deposition (Hildebrand 2000, Hildebrand and Wetherbee 2003). Hildebrand was involved in the genome sequencing project for the diatom Thalassiosira pseudonana. His laboratory developed a synchronous growth procedure for T. pseudonana (Hildebrand et al. 2007), and used it to perform the first proteomic analysis of diatom cell wall proteins (Frigeri et al. 2006) and identify structural intermediates in cell wall formation (Hildebrand, et al., 2006).
The major MURI participants are:
Dr. Mark Hildebrand’s research interests include: i) the mechanisms of formation of diatom nanostructured silica, ii) diatom cellular silicon metabolism, iii) phytoplankton genomics and proteomics, iv) phytoplankton nutrient metabolism, and v) optical properties of biological compounds. His previous work includes cloning the first silicon responsive genes, and genes encoding silicic acid transporters in diatoms. The latter were the first genes shown to encode a protein that specifically interacted with silicon. He has also investigated the process of silicic acid transport in diatoms in conjunction with regulation of silicification. He was involved in genomic sequence analysis of the diatom Thalassiosira pseudonana.
In addition to the main MURI investigator for this thrust, collaborations with other MURI participants will be utilized to i) generate altered silica morphologies that may facilitate chemical conversion processes (K. H. Sandhage), and ii) experiment with confinement and controlled growth approaches to alter silica structure or enable patterning of living diatoms on substrates (D.J. Hansford).
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