## 3 Frustule Modification: Techniques and Applications
### Frustule Genetic Modification
pseudonana_, of the first membrane protein located in SDVs, named Silicanin-1 (Sin1), highly conserved in all diatoms and that, in combination with long-chain polyamines, promote silica formation from silicic acid in vitro [124]. Later on, thanks to the advent of genome editing in diatoms, with the improvement of CRISPR/Cas9 precision technology, [PERSON] and collaborators generated Sin1 knockout mutants in _T. pseudonana_[118]. The phenotipical and mechanical performance of the frustule of the knockout mutants enable to confirm the previous function of the protein in promoting silica formation. Diatom shells are excellent natural porous materials for possible uses in nanotechnology such as drug delivery [22] and, with this aim, [PERSON] and co-workers [123] genetically modified _T. pseudonana_ to display an IgG-binding domain of protein G on the biosilica surface, enabling attachment of cell-targeting antibodies brought a genetic method termed living diatom silica immobilization (LiDSI). The method, introduced by [PERSON] and co-workers [127; 128], consists of the introduction of a recombinant gene encoding for an artificial protein formed by the desired enzyme or receptor protein fused to a protein of the organic matrix of the SVD (see Figure 11). In Ref. [123] the authors, for the simultaneous attachment of antibodies and hydrophobic drug molecules to diatom biosilica, used for the first time this genetic method to incorporate an immunoglobulin G (IgG)-binding domain of protein G19, named GB1, into the biosilica of the diatom _T. pseudonana_ in vivo, instead to the classical method with organic solvents and covalent cross-linking. Via recombinant DNA technology, the authors constructed a fusion gene GFP-GB1 that they introduced via the classical biolistic method into _Thalassiosira_ genome. GFP (Green Fluorescence Protein) tag was used to screen the trasformed cells. Then, they loaded the diatoms with chemotherapeutic drugs, demonstrating that diatom biosilica represent a novel, natural and nontocnologically tailored drug delivery system. Another approach linking genetic modification and frustule functionalization has been very recently proposed by [PERSON] and colleagues [PERSON] and colleagues [PERSON] (1975). It is already well known that there are two main classes of biomolecules involved in diatom biosilicification: phosphorylated proteins and long-chain polyamines. The first type of proteins are polypeptides rich in lysine amino acids and exhibiting post-translational modifications (PTMs) [PERSON] (1975). The authors demonstrated that, through PTMs, it is possible to control the structural features of the silica nanoparticles. In particular, they demonstrated that proteolytic cleavage of gene encoding for Silacin-1, Sil1, from C. _fusiformis_ releases multiple units. All these units can undergo to different PTMs processes such as phosphorylation and methilation. In particular, the authors proved that the PTMs of one specific peptides, named R5, impact on the silica precipitation process and consequently on shape, porosity, and surface of the frustule. The use of the modified peptides technique will enable the control of silica morphology, allowing to obtain nanostructures with controlled size and with many potential applications in nanotechnology [PERSON] (1975).