Part:BBa_K4633002

AmyL - signal peptide for Lactobucilli

Usage and Biology

Signal peptides play a crucial role in the secretion of proteins in bacteria, facilitating their transport across cellular membranes. The AmyL signal peptide, derived from Lactiplantibacillus plantarum S21 , a commonly used food-grade lactobacillus, is responsible for the secretion of α-amylase [1]. The amino acid sequence of this signal peptide was extracted from the research conducted by Tran, A. M. et al. [1] and subsequently reverse-translated to align with the genome of Lactococcus lactis using Benchling.

This signal peptide is secreted via the general secretion system, which is the predominant secretion system in gram-positive bacteria [2]. During the secretion process, the signal peptide undergoes cleavage by signal peptidase type I, with the cleavage site located two amino acids from the sequence's C-terminal end [2].

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Characterization and Measurements

To evaluate the efficiency of the AmyL signal peptide, we conducted experiments using Lactobacillus crispatus ATCC 33802 as the host organism. The AmyL signal peptide was incorporated into the pMG36E plasmid obtained from AddGene, positioned downstream of the constitutive promoter p32 present on the plasmid. As a reporter protein, we utilized Green Fluorescent Protein (GFP), encoded by BBa_E0040, with a linker.

To assess the performance of the AmyL signal peptide, we measured the levels of GFP expression both inside and outside bacterial cells and compared the results. Cell separation from the medium was achieved through centrifugation. In the supernatant, we quantified the extracellular GFP. Subsequently, we resuspended and lysed the cells to measure intracellular GFP levels. These measurements were conducted using a plate-reader with excitation at 488 nm and emission detection at 508 nm.

References

• [1] Tran, A. M. et al. Efficient Secretion and Recombinant Production of a Lactobacillal α-amylase in Lactiplantibacillus plantarum WCFS1: Analysis and Comparison of the Secretion Using Different Signal Peptides. Front. Microbiol. 12, 689413 (2021).
• [2] Anné, J., Economou, A. & Bernaerts, K. Protein secretion in gram-positive bacteria: From multiple pathways to biotechnology. Curr. Top. Microbiol. Immunol. 404, 267–308 (2017).