Peptide synthesis and stock solution preparation

SPOT-synthesized peptides were prepared by Kinexus Inc. (Vancouver, BC. Canada) and were obtained as free molecules that had been released from the cellulose membrane by ammonia gas treatment³⁴. A library of experimental peptides derived from the parental sequence 1018 was used consisting of 96 single amino acids substitution variants (Supplementary Table ^S1), where each residue in 1018 was systematically replaced with nine amino acids (R, K, Q, G, A,W, V, L and I) that have previously been associated with antibiofilm potential in synthetic peptides. Each peptide spot (~100 nmol density, 60–70% purity) was dissolved in 200 μl of sterile water to generate a stock peptide solution. The concentration of a few SPOT-peptide stock solutions was determined by measuring the Trp absorbance at 280 nm on a NanoDrop instrument and calculated by Beer’s law using a Trp extinction coefficient of 5500 M⁻¹ cm⁻¹. This concentration (~250 µM) was then assumed to be representative of all the SPOT-peptide stock solutions. Synthetic 1018 at >95% purity was obtained from CPC Scientific (Sunnyvale, CA) while all other synthetic peptides (>95% purity) were obtained from Genscript (Piscataway, NJ). The C-terminal carboxyl groups of all >95% pure peptides were amidated to remove the negative charge. Stock solutions of >95% pure peptide were prepared to 2 mg/ml in sterile water then diluted to the appropriate working concentration on the day of the experiment.