Quicker change - conventional vs. convenient mutant generation

Posted by Various Scientists

November 18, 2008

This success story was provided by Dr. B. Pohn & Dr. S. Feichtenhofer (Institute of Molecular Biotechnology, Graz University of Technology, Austria)

11 mutations and a deletion at one sweep
evolving mutant generation from conventional to convenient

To answer the question concerning the correlation between a structural feature of a wild-type enzyme and the activity towards a certain substrate we needed to excise seventeen amino acids of the sequence and introduce eleven mutations in the regions around the excision.

Conventional mutatant generation

Obviously with a standard mutation strategy this task would have been reached only by a multi-step process. At first we would have done the excision using an overlap PCR strategy. For the introduction of the site directed mutations in the second step either a rather long primer ensuring the binding in homologous regions of the gene in the neighborhood of the mutated region or several shorter primers would have been needed. To generate the mutated gene by ourselves the estimated costs only for the needed oligonucleotides would have been about € 190. Additionaly we would have needed polymerase, sequence analysis and labor time. The price Mr. Gene offerted us for the synthesised mutated gene in a plasmid provided with the cutting sites for cloning in our vector system was € 200.

The decision for the ordering of a synthetic gene at these costs was very easy to argue in our company. Two weeks after placing the order the synthesised gene and the sequencing data were delivered. We were very pleased with the service and the agreed price of Mr. Gene for that task. When we would have done the needed mutational changes by ourselves, neither we would have obtained the ready construct within two weeks, nor for that low costs.

Convenient mutant generation