Home › Biochemistry › Antimicrobial Peptides Produced by Selective Pressure Incorporation of Non-canonical Amino Acids
Biochemistry JoVE (Open Access) Citable · DOI

Antimicrobial Peptides Produced by Selective Pressure Incorporation of Non-canonical Amino Acids

DOI: 10.3791/57551-v
What you'll learn
  • ✓ Incorporate non-canonical amino acids into recombinant nisin using selective pressure in E. coli
  • ✓ Assess antimicrobial peptide bioactivity via growth inhibition assays
  • ✓ Visualize peptide effects on bacterial cells using fluorescence microscopy
Protocol

The protocol presents the Escherichia coli-based selective pressure incorporation of non-canonical amino acids (ncAAs) into the lactococcal antimicrobial peptide nisin. Its properties can be changed during recombinant expression via substitution with desired ncAAs in defined growth media. Resulting changes in bioactivity are mapped by growth inhibition assays and fluorescence microscopy.

Difficulty
advanced
Total time
~3–5 days (protein expression 24–48 hrs, assays and microscopy 1–2 days)
Model organism
Escherichia coli; Lactococcus lactis (target peptide); Gram-positive test bacteria
Biosafety
BSL-1

Steps

1
Express recombinant nisin with proline analogs

Culture E. coli with selective pressure incorporation (SPI) of non-canonical amino acids into nisin in defined growth media. Induce protein expression and harvest cells at appropriate time points.

▶ 01:16
2
Perform antimicrobial activity growth inhibition assays

Test bioactivity of nisin variants by measuring growth inhibition of target bacteria in culture. Compare inhibition profiles across different ncAA-substituted peptides.

▶ 04:19
3
Prepare samples and acquire fluorescence microscopy images

Stain and image bacterial cells treated with recombinant nisin variants using fluorescence microscopy to visualize peptide localization and cellular effects.

▶ 07:37
4
Analyze fluorescence microscopy results on gram-positive cells

Evaluate and interpret microscopy data showing nisin effects on Gram-positive bacterial cell morphology and membrane perturbation.

▶ 09:02
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