Sequential Permutation Libraries
(# of codons x 20 variants)
successive site-saturation mutagenesis
The Problem
Frequently, proteins need to be optimized by directed evolution without specific knowledge about the molecule. Random mutagenesis libraries of such genes create only a tiny fraction of all possible variants, rendering succesful screenings unlikely or suboptimal.
The Solution
A systematic approach to improve a protein's performance is sequential permutation. It resembles an alanine scan but replaces each amino acid by all 20 possible amino acids simultaneously. For each codon, a small site-saturated library is constructed, which can be delivered as a pool or separated for all 19 substitution variants. This strategy scans the whole (or part of the) protein to create a map of neutral, beneficial and detrimental amino acids for each position.
Deliverables
All gene variants are subcloned into the customer's vector and delivered as 10 µg of plasmid DNA.
Depending on the details of the subsequent screening assay, site-saturated constructs can be delivered in two different formats for each codon:
- mixed (small library containing all 20 variants)
- separate (each of the 19 substitution variants will be delivered separately)
Quality control
A mixed site-saturated library will be bulk sequenced for each codon.
Separated variants will be 100 % sequence verified each.
Fill out our online quotation form to get a quote for your specific project.
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