Premium
Rapid in vivo evolution of a β‐lactamase using phagemids
Author(s) -
LongMcGie Jeffrey,
Liu Amy D.,
Schellenberger Volker
Publication year - 2000
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/(sici)1097-0290(20000405)68:1<121::aid-bit15>3.0.co;2-x
Subject(s) - biology , cefotaxime , mutant , directed evolution , population , mutagenesis , phagemid , escherichia coli , genetics , gene , microbiology and biotechnology , antibiotics , bacteriophage , demography , sociology
RNA viruses are capable of undergoing extremely rapid evolution due to their high rates of reproduction, small genome size, and a high frequency of spontaneous mutagenesis. Here we demonstrate that a virus‐like, evolutionary state can be created by propagating a phagemid population in a hypermutator strain of Escherichia coli in the presence of a helper phage. This enables one to subject individual phagemid‐encoded genes to rapid in vivo evolution. We applied this approach to TEM‐1 β‐lactamase which confers resistance to 0.05 mg/L of the antibiotic cefotaxime. After 3 weeks of in vivo evolution we were able to isolate a double mutant, E104K/G238S, of the enzyme which confers a 500‐fold increased level of resistance to cefotaxime compared to the starting enzyme. In two independent experiments we obtained a triple mutant, E104K/G238S/T263M, which confers a 1000‐fold increase in resistance compared to the wild type enzyme. The same three mutations have been previously observed in TEM‐4 β‐lactamase which was discovered in a highly cefotaxime‐resistant clinical isolate. The probability of randomly obtaining a β‐lactamase carrying three identical point mutations is less than 10 −10 . This indicates that phagemid evolution can rapidly reproduce evolution occurring in nature. © 2000 John Wiley & Sons, Inc. Biotechnol Bioeng 68: 121–125, 2000.