SBIR Phase I: Directed evolution of site-specific bacterial transposase genes to alter specificity and efficiency of insertion of large DNA segments into restorable gene fusions

点击翻译按钮获取中文摘要

The broader impact of this Small Business Innovation Research (SBIR) Phase I project will be to develop methods to facilitate the efficient, reproducible insertion of large DNA segments into stable locations on bacterial vectors, viral and non-viral shuttle vectors, and the chromosomes of prokaryotic and eukaryotic host cells comprising novel target sequences plus helper and donor vectors that could impact many areas of synthetic biology. Directed evolution experiments will be carried out to recover genes encoding bacterial transposase variants that have altered specificity or increased efficiency of transposition, compared to those recovered by products encoded by the wild-type transposase genes. Homologues of the bacterial target site will be used to recover genes encoding variant transposases that should function efficiently in eukaryotic cells. Modified helper and donor vectors will also be constructed with promoters and genes having optimized codon preferences to facilitate the efficient, direct generation of composite vectors harbored in eukaryotic cells, and eventually, the efficient, reproducible generation of cells harboring large DNA insertions at one or more specific stable sites within a host cell chromosome.The proposed project will exploit the key properties of the bacterial Tn7 transposon system for much broader utilization in many aspects of systems biology. Genes encoding transposases and accessory proteins will be mutagenized to alter the specificity and enhance the efficiency of insertion events in both prokaryotic and eukaryotic cells. This platform could have advantages over other gene transfer approaches by allowing stable, precise insertion events without the subsequent remobilization or the creation of indels/rearrangements at the target site. The ability to move large segments of DNA in such a manner would benefit many fields of synthetic biology.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.