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Library construction and evaluation for site saturation mutagenesis

Paper ID Volume ID Publish Year Pages File Format Full-Text
17181 42649 2013 8 PDF Available
Title
Library construction and evaluation for site saturation mutagenesis
Abstract

We developed a method for creating and evaluating site-saturation libraries that consistently yields an average of 27.4 ± 3.0 codons of the 32 possible within a pool of 95 transformants. This was verified by sequencing 95 members from 11 independent libraries within the gene encoding alkene reductase OYE 2.6 from Pichia stipitis. Correct PCR primer design as well as a variety of factors that increase transformation efficiency were critical contributors to the method's overall success. We also developed a quantitative analysis of library quality (Q-values) that defines library degeneracy. Q-values can be calculated from standard fluorescence sequencing data (capillary electropherograms) and the degeneracy predicted from an early stage of library construction (pooled plasmids from the initial transformation) closely matched that observed after ca. 1000 library members were sequenced. Based on this experience, we suggest that this analysis can be a useful guide when applying our optimized protocol to new systems, allowing one to focus only on good-quality libraries and reject substandard libraries at an early stage. This advantage is particularly important when lower-throughput screening techniques such as chiral-phase GC must be employed to identify protein variants with desirable properties, e.g., altered stereoselectivities or when multiple codons are targeted for simultaneous randomization.

► A site-saturation mutagenesis protocol yielding 27.4 ± 3.0/32 codons in a 95-member library was developed. ► Critical parameters impacting library quality were uncovered and optimized. ► A quantitative method correlating early-stage DNA sequencing data with library quality is described.

Keywords
Mutagenesis; Site-saturation; Codon libraries; DNA sequencing
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Library construction and evaluation for site saturation mutagenesis
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Publisher
Database: Elsevier - ScienceDirect
Journal: Enzyme and Microbial Technology - Volume 53, Issue 1, 10 June 2013, Pages 70–77
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us