Open AccessAn efficient ORF selection system for DNA fragment libraries based on split beta-lactamase complementation
Author(s) -
Vaishali Verma,
Gopal Joshi,
Amita Gupta,
Vijay Chaudhary
Publication year - 2020
Publication title -
plos one
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.99
H-Index - 332
ISSN - 1932-6203
DOI - 10.1371/journal.pone.0235853
Subject(s) - open reading frame , biology , fragment (logic) , cloning (programming) , genetics , in vitro recombination , plasmid , genomic library , dna , genome , computational biology , cloning vector , molecular cloning , gene , microbiology and biotechnology , complementary dna , computer science , algorithm , peptide sequence , programming language
PCR-based amplification of annotated genes has allowed construction of expression clones at genome-scale using classical and recombination-based cloning technologies. However, genome-scale expression and purification of proteins for down-stream applications is often limited by challenges such as poor expression, low solubility, large size of multi-domain proteins, etc. Alternatively, DNA fragment libraries in expression vectors can serve as the source of protein fragments with each fragment encompassing a function of its whole protein counterpart. However, the random DNA fragmentation and cloning result in only 1 out of 18 clones being in the correct open-reading frame (ORF), thus, reducing the overall efficiency of the system. This necessitates the selection of correct ORF before expressing the protein fragments. This paper describes a highly efficient ORF selection system for DNA fragment libraries, which is based on split beta-lactamase protein fragment complementation. The system has been designed to allow seamless transfer of selected DNA fragment libraries into any downstream vector systems using a restriction enzyme-free cloning strategy. The strategy has been applied for the selection of ORF using model constructs to show near 100% selection of the clone encoding correct ORF. The system has been further validated by construction of an ORF-selected DNA fragment library of 30 genes of M . tuberculosis . Further, we have successfully demonstrated the cytosolic expression of ORF-selected protein fragments in E . coli .