## 3 Frustule Modification: Techniques and Applications
### Frustule Genetic Modification
The obtained results confirm the primary and already known role of these proteins in the silicon uptake from the environment into the cell but, unexpectedly, demonstrated further specialization of the SITs as sensors of cellular silicic acid levels that might elicit \begin{table}
\begin{tabular}{c c c c} \hline \hline
**Species** & **Genes or Related Proteins** & **Method** & **Reference** \\ \hline \multirow{8}{*}{T. pseudonana} & \multirow{4}{*}{Silicic acid transporters (SITs 1,2,3).} & Gateway technology for GFP fusion proteins. & [121] \\  & & RNAi and antisense knockdown. & [122] \\  & & Target mutations. & [122] \\  & Incorporated & LIDSI-based method & \\  & antibody-binding protein domains. & (live diatom silica immobilization). & [123] \\  & & & \\  & SAP1-2-3 (Silicalemma Associated Proteins). & Gateway technology for GFP fusion proteins. & [120] \\  & & RNAi and antisense knockdown. & [124] \\  & & Silacin-1 (Sin-1). & Cloning technology for GFP fusion proteins. & [124] \\  & & CRISPR/Cas9-based approach. & [118] \\  & & Silaffin-1 (Sill) and cingulin genes family & LIDSI-based method & \\  & & (CinY1, 2-3,-4 and CinW1, 2-,-3). & (live diatom silica immobilization). & [1] \\ \hline \multirow{4}{*}{_C. fusiformis_} & \multirow{4}{*}{Silicaffin-1 (Sill)} & _E. coli_ expression system for the & [125] \\  & & recombinant production of RS. & \\ \cline{1-1}  & & & \\ \cline{1-1}  & & & \\ \cline{1-1}  & & & \\ \cline{1-1}  & & & \\ \cline{1-1}  & & & \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Summary of the main model species used for frustule characterization with indication of the studied genes, encoded proteins and employed methods. and control cell wall formation and division processes. Afterwards, [PERSON] et al. [122], in order to shed light on the molecular mechanisms of silicic acid transport, developed a fluorescence method to characterize more in detail the kinetics parameters of the SITs and demonstrated, through protein mutagenesis approaches, that the conserved four GXQ amino acid motifs plays an essential role in SITs function. Transcriptome analysis in _T. pseudonana_ cells has enabled the identification of different genes potentially involved in silica formation [126]. In particular, the study identified a set of genes, named Silaffin Like Response Genes (SLRGs) and in 2017 [PERSON] and co-authors identified and characterized a subset of three related new proteins, conserved in other diatom species as well [120]. Using the same approach of [PERSON] and [PERSON] [121], the authors generated mutants expressing C-terminal GFP fusion proteins to determine their spatial sub-cellular localization. The results indicate an association of these proteins with the silicemma (the membrane surrounding the SDV), and, for this reason, they named the family Silicalemma Associated Proteins (SAPs 1, 2 and 3). To further investigate their role during cell wall formation, the authors, starting from the results of spatial localization, generated knock down lines by antisense RNA and RNAi methods for two out of three genes, TpSAP1 and TpSAP3. The knock down lines were molecularly analyzed and the frustule architecture phenotypically characterized. Interestingly, the valves of knock down lines showed altered pattern morphology compared to the control wild type cell. In detail, the mutants have an approximately two fold bigger cell volume and a mislocated pattern center, but in general exhibit the same biosilica morphology than the wild type [120]. The reported results represent one of the first evidence that demonstrates that the ability to manipulate and modulate the expression level of one of the genes involved in the biosilification process is sufficient to generate a consistent phenotypic change in silica structure. In 2017, [PERSON] and colleagues discovered and determined the spatial localization, in _T.