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Nucleic Acids Research 2005 33(9):e80; doi:10.1093/nar/gni077
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Published online 16 May 2005

© The Author 2005. Published by Oxford University Press. All rights reserved
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Methods Online

Triplet nucleotide removal at random positions in a target gene: the tolerance of TEM-1 ß-lactamase to an amino acid deletion

D. Dafydd Jones*

School of Biosciences, Biomedical Sciences Building, Cardiff University Cardiff CF10 3US, UK

*To whom correspondence should be addressed. Tel: +44 29 20874290; Fax: +44 29 20874116; Email: jonesdd{at}cardiff.ac.uk

Received February 15, 2005. Revised April 1, 2005. Accepted April 20, 2005.

The deletion of amino acids is one of the evolutionary mechanisms by which nature adapts the function of proteins. A simple method has been developed that mimics this event in vitro by introducing a deletion of exactly three nucleotides at random positions in a target gene. The method involved the engineering of the mini-Mu transposon to introduce a recognition sequence for the restriction enzyme MlyI. The new transposon, MuDel, was capable of efficient insertion into a target DNA sequence. To determine the efficacy of the method, the bla gene that encodes the TEM-1 ß-lactamase was used as the target and a small library containing 22 different sequence variants was created. Of these 22 variants, 8 were identified that conferred resistance to ampicillin on Escherichia coli. Each of the TEM-1 variants possessed a distinct ampicillin minimum inhibitory concentration, ranging from 500 to >10 000 µg/ml. Sequence analysis revealed that active TEM-1 variants contained deletions not just in loops but also helices, and included regions known to be involved in catalysis, antibiotic resistance and inhibitor binding. This new technology is transferable to most genes, permitting an extensive analysis of deletion mutations on protein function.


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