## 4 Conclusions  
In this study, we report the fabrication of new antimicrobial protein-based polymers with different structural backbones (structural module), namely SELP-59-A, a silk-elastin-like protein able to form self-structured \(\beta\)-sheet structures, and A200, an elastin-like recombinant characterized by a more flexible conformation. The protein polymers were genetically engineered to include functional modules consisting of the amino acid sequences for antimicrobial peptides hepcidin, ABP-CM4 and BMAP18, as well as the recently isolated Synoeca-MP (Table S6). All constructs were successfully produced in _E. coli_, purified by simple non-chromatographic approaches and processed into free-standing films using water and formic acid as solvents. No statistically significant differences were observed in the antimicrobial activity of films produced with water or with formic acid, indicating the feasibility of using a water as mild and \"green\" alternative to more harsh chemicals. However, evaluation of the antimicrobial activity of the films indicates that the structural backbone plays a central role in antimicrobial performance suggesting that AMPs should be conformationally free to exert their antimicrobial effect. While the AMP-ELR films were highly effective in exerting an antimicrobial effect against _E. coli_ and _S. aureus_, we propose that the more rigid structure of the AMP-SELP materials restricts the ability of the AMPs to interact with bacterial cells and consequently results in a more limited performance. A strategy to overcome this limitation could be through the inclusion of flexible glycine/serine spacers in the genetic design to improve the conformational freedom of the AMPs [76; 77], thereby increasing their accessibility. [PERSON] et al. [36] demonstrated that the presence of flexible spacers and their length are determinant factors for antimicrobial activity since the presence of the spacer can enable peptide insertion into the cell. On the other hand, the antimicrobial activity of AMPs can be lost when immobilized on surfaces without the presence of a spacer [38; 78]. This supports our assumption that increased antibacterial activity can be achieved by introducing flexible spacers in the genetic design. Nevertheless, SELP-based antimicrobial biomaterials can be promising opportunity for novel materials, as SELPs combine the elasticity, resilience and solubility of elastin with the mechanical strength and versatility of processing of silk [5; 79]. Analysis of antimicrobial results against _S. aureus_ revealed a high variability because of its ability to form cell aggregates thus reducing the contact with the antimicrobial surface. This high variability could be reduced by lowering the number of cells in the inoculum, so as to ensure that the majority of bacteria are placed in contact with the film surface. As such, although the antimicrobial assays were performed based on ISO 22196, we suggest that results should be critically analyzed upon assessing the antimicrobial performance of surfaces while using cells with the ability to grow in aggregates. Overall, the findings of the present study represent an important contribution towards the rational design of genetically engineered protein polymers functionalized with antimicrobial peptides and provide cues for a next generation of antimicrobial materials. The following are available online at [[https://www.mdpi.com/article/10.3390/app11125352/s1](https://www.mdpi.com/article/10.3390/app11125352/s1)]([https://www.mdpi.com/article/10.3390/app11125352/s1](https://www.mdpi.com/article/10.3390/app11125352/s1)), Figure S1: SDS-PAGE (a) and Western blot (b) analysis of pure BMAP18-SELP (2), CM-SELP (3) and Hep-SELP (4) using anti-polyHistidine antibodies and pure SELP (1) as control. MWM-molecular weight marker (broad range unstained SDS-PAGE Standard from Bio-Rad) in kDa; Figure S2: Curve-fitted deconvoluted spectrum with band assignment for SELP-based films. The bands filled in green represent contributions of \(\beta\)-structures.