## 3 Frustule Modification: Techniques and Applications
### Frustule Genetic Modification  
Diatom biosilica is a hybrid material which consists of amorphous silica and several organic compounds. The formation of the diatom frustule structure is coordinated and dependent by both silica polymerization in the SDV and self-assembly processes that involve different proteins and molecules [5; 9; 10]. In 1996, [PERSON] and co-workers identified and isolated the first protein from the frustule, that they called frustuline [108]. Over the past 25 years, through biochemical analyses and -omics studies, many other proteins and organic compounds involved in frustule formation and scaffold have been identified and partially characterized (e.g., long-chain polyamines, silaffins, silicidins, cingulins, and polysaccharides) [10]. Figure 10: SEM images of polyamine-rich scaffolds assembled by direct ink writing and reproducing the morphology of a _T. favus_ (**a**) and a _A. chrenbergii_ (**b**) diatom valve. Magnification views are reported in (**c,d**). Reproduced with permission from Ref. [105]. The process of frustule formation is under the control of a genetic, species specific, finely tuned mechanism which, at each division, is transmitted from the mother to the two derived daughter cells. The extraordinary precision and frequency with which this mechanism occurs has always fascinated and intrigued biologists and different models have been proposed. However, to date none of them is able to explain biosilica morphogenesis in its entirety [109]. Indeed, there are still many components of the process that should be clarified, such as how this higher-order structure is formed and what are the exact spatial and temporal mechanisms that are imparted on the process. The manipulation of the silification process and the alteration of the architecture of the frustule by genetic modification is challenging and can be an effective and powerful approach to address those questions [34]. In addition, understanding and modifying the process of biomineralization in diatoms might result in a powerful tool for a wide variety of nanotechnological applications [110]. Compared to other organisms, the genetic engineering of diatoms is still in its infancy. Genetic reporter systems to study gene expression and regulation and spatial localization of proteins [111], modulation of gene expression methods, overexpression [112], gene knockdown [113], knockout, tailored TALEN endonucleases [114] and the CRISPR/Cas9 system [115] are now available also for these microalgae, allowing genetic modifications and the generation of mutated strains [116]. These molecular tools allow to investigate the function and the role of the proteins involved in cell wall biogenesis and their properties [117; 118]. _Phaeodactylum tricornutum_ and _Thalassiosira pseudonana_ are the two molecular model species, since _P. tricornutum_ is characterized by fusiform cells with a cell wall poor in silica [119], while the centric _T. pseudonana_ has been developed as a model species to study the formation of silica cell wall [120]. More recently, _Cylindrotheca fusiformis_ is considered as well a model for silicification research [116]. In this section, we summarize up-to-date reports about the genetic engineering applied to diatom frustules, aimed at the characterization of the involved genes and the derived effects on the frustule formation, morphology, function and functionalization with implications for potential nanotechnological applications (see Table 1). One of the earliest examples of genetic engineering-based approach in the study of frustule biogenesis has been reported for _T. pseudonana_ by [PERSON] and [PERSON] in 2015 [121]. The authors, using Gateway technology for GFP fusion proteins and antisense RNA and RNA interference (RNAi) approaches, determined the spatial localization and the regulatory and signaling role for the individual silicon transporters genes (SITs).