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

这项小企业创新研究（SBIR）第一阶段项目的广泛影响将是开发方法，以促进将大DNA片段高效、可重复地插入细菌载体、病毒和非病毒穿梭载体、原核和真核宿主细胞的染色体上的稳定位置，包括新的靶序列以及辅助和捐赠载体，这可能会影响合成生物学的许多领域。与野生型转座酶基因编码的产物相比，将进行定向进化实验，以恢复编码细菌转座酶变异的基因，这些基因改变了转座的特异性或提高了转座的效率。细菌靶位点的同源物将用于恢复编码变异转座酶的基因，这些基因应该在真核细胞中有效地起作用。修饰的辅助载体和供体载体也将与具有优化密码子偏好的启动子和基因一起构建，以促进在真核细胞中有效、直接地产生复合载体，并最终在宿主细胞染色体的一个或多个特定稳定位点上有效、可再生地产生含有大量DNA插入的细胞。该项目将利用细菌Tn7转座子系统的关键特性，在系统生物学的许多方面得到更广泛的利用。编码转座酶和辅助蛋白的基因将被诱变以改变特异性并提高插入事件在原核和真核细胞中的效率。与其他基因转移方法相比，该平台具有优势，因为它允许稳定、精确的插入事件，而不会在目标位点重新移动或产生索引/重排。以这种方式移动大片段DNA的能力将使合成生物学的许多领域受益。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

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.