*2.2. Construction of Lacticaseicin 30 Variant Peptides Carrying N-Terminal Part (N-Ter Lacticaseicin 30), or the Central and C-Terminal Parts (C-Ter Lacticaseicin 30) and Their Expression in E. coli Cells*

All oligonucleotides used in this work are listed in the supplementary materials (Table S2). The molecular cloning and other standard techniques were used thereof to perform genetic construction of lacticaseicin 30 and variants. N-ter-lacticaseicin 30, C-ter lacticaseicin 30 and N-ter-H1 lacticaseicin 30 plasmids were described by Sambrook et al. [32]. The *orf030*, *orf030-nter*, *orf030-cter* and *orf030-nter-h1* were amplified by PCR using pT7-6his-030 plasmid as template and F-*Bam*HI-030 and R-030-*Hind*III (for *orf030*), F-*Bam*HI-030 and R-Nter\_030-*Hind*III (for *orf030-nter*), F-*Bam*HI-Cter\_030 and R-030-*Hind*III (for *orf030-cter*) and F-*Bam*HI-030 and R-Nter-H1\_030-*Hind*III (for *orf030-nter-h1*) primers. Then, each PCR product was cloned between the *Bam*HI and *Hind*III sites of the pET-32b(+) plasmid.

Phusion High-Fidelity DNA Polymerase, restriction endonucleases and T4 ligase were obtained from ThermoFisher Scientific (Thermo Scientific, Waltham, MA, USA) and used in accordance with the manufacturer's instructions. Plasmids and PCR products were purified using NucleoSpin kits (Macherey-Nagel, Düren, Germany) and the final plasmid constructions were verified by PCR and sequencing (Eurofins, Ebersberg, Germany). The resulting sequences were analyzed using the SnapGenes tool (GSL Biotech LLC, San Diego, CA, USA).
