Functional whole-colony screening method to identify antimicrobial peptides

Journal of Microbiological Methods
E LoitIllimar Altosaar

Abstract

A high throughput method for screening cDNA libraries has been developed to identify putative antimicrobial peptides (AMPs). It is based on a rapid dye inclusion assay for assessing antagonism of bacterial viability. Colonies are grown on a membrane on a permissive medium until full colony size is reached. The membrane, supporting the array of colonies, is transferred onto an inductive medium containing a vital dye. Upon expression of any antagonizing peptides, the cell membrane becomes compromised allowing dye infusion to permit visual identification of deleterious peptides. Our approach was validated by screening a synthetic oligonucleotide library expressed in Escherichia coli. A random oligonucleotide library, containing inserts of up to 75 nucleotides in length was constructed and expressed in E. coli. From a potential pool of 100000 peptides, in a single round of screening, three were found to be antimicrobial: L1, L3, and L8. Peptide L1 was shown to have a concentration-dependent bactericidal effect against Gram-negative E. coli and moderate biostatic activity against the Gram-positive bacteria Listeria monocytogenes. L8 was found to have bacteriostatic, and possibly bactericidal effect against E. coli, Pseudomonas aerug...Continue Reading

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Citations

Mar 23, 2011·Annals of Clinical Microbiology and Antimicrobials·Satoshi UenoYusuke Kato
Feb 5, 2010·International Journal of Antimicrobial Agents·Evelin LoitIllimar Altosaar
Dec 2, 2009·Journal of Peptide Science : an Official Publication of the European Peptide Society·Xiaojing ShenCui Hu
Jun 16, 2021·Toxicon : Official Journal of the International Society on Toxinology·Yi-Chuan MengKen Tan

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