Journal: Biomolecules
Article Title: A Rapid, High-Throughput Method for the Construction of Mutagenesis Libraries
doi: 10.3390/biom15111511
Figure Lengend Snippet: Library design and intermediate plasmid construction strategy. ( A ) Schematic of oligonucleotide library design. The full-length PSMD10 gene was divided into 10 sublibraries (P1–P10). Taking sublibrary P4 as an example, each sublibrary’s oligonucleotides contain 19 bp constant primer-binding regions (blue segments) at both ends, enabling specific amplification of the corresponding variable region. The central region is the variable segment (yellow), where each position is systematically mutated to an amber stop codon (TAG). Each sublibrary’s oligonucleotide pool consists of 24 unique sequences that share identical primer-binding regions and carry a single TAG mutation at a specific site within the variable region. ( B ) Intermediate plasmid assembly strategy. Using sublibrary P4 as an example, the PSMD10 gene was divided into constant and variable regions, with the variable region corresponding to each sublibrary. The intermediate plasmid backbone contains predefined BsmBI restriction sites flanking the vacant variable region. During sublibrary construction, all other regions remain unchanged, and only the corresponding variable region sequences are synthesized and inserted into the intermediate plasmid via BsmBI-mediated assembly (indicated by thick black arrows).
Article Snippet: During the construction of the original plasmid, the vector pQTEV- PSMD10 (#31332, Addgene, Cambridge, MA, USA) was used as the template to amplify the target sequence using the following primers: EF1α-PSMD10-F: 5′-AGGTAGCTGATATCGAATTCCTGGGGATCCACCGGTCCACGGGGGTGTGTCTAAC-3′ and EF1α-PSMD10-R: 5′-AGGTCAGGCTCGCTGAATTCCCCAATGTCAAGC-3′.
Techniques: Plasmid Preparation, Binding Assay, Amplification, Mutagenesis, Synthesized
