Department of Pathology, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106, United States.
ACS Synth Biol. 2023 Nov 17;12(11):3352-3365. doi: 10.1021/acssynbio.3c00355. Epub 2023 Nov 3.
The Bxb1 bacteriophage serine DNA recombinase is an efficient tool for engineering recombinant DNA into the genomes of cultured cells. Generally, a single engineered "landing pad" site is introduced into the cell genome, permitting the integration of transgenic circuits or libraries of transgene variants. While sufficient for many studies, the extent of genetic manipulation possible with a single recombinase site is limiting and insufficient for more complex cell-based assays. Here, we harnessed two orthogonal Bxb1 recombinase sites to enable alternative avenues for using mammalian synthetic biology to characterize transgenic protein variants. By designing plasmids flanked by a second pair of auxiliary recombination sites, we demonstrate that we can avoid the genomic integration of undesirable bacterial DNA elements using the same starting cells engineered for whole-plasmid integration. We also created "double landing pad" cells simultaneously harboring two orthogonal Bxb1 recombinase sites at separate genomic loci, allowing complex cell-based genetic assays. Integration of a genetically encoded calcium indicator allowed for the real-time monitoring of intracellular calcium signaling dynamics, including kinetic perturbations that occur upon overexpression of the wild-type or variant version of the calcium signaling relay protein STIM1. A panel of missense mutants of the HIV-1 accessory protein Vif was paired with various paralogs within the human Apobec3 innate immune protein family to identify combinations capable or incapable of interacting within cells. These cells allow transgenic protein variant libraries to be readily paired with assay-specific protein partners or biosensors, enabling new functional readouts for large-scale genetic assays for protein function.
Bxb1 噬菌体丝氨酸 DNA 重组酶是将重组 DNA 工程化导入培养细胞基因组的有效工具。通常，将单个工程化的“着陆垫”位点引入细胞基因组中，允许转基因电路或转基因变体文库的整合。虽然对于许多研究来说已经足够了，但单个重组酶位点的遗传操作程度是有限的，不足以进行更复杂的基于细胞的测定。在这里，我们利用两个正交的 Bxb1 重组酶位点，为利用哺乳动物合成生物学来表征转基因蛋白变体提供了替代途径。通过设计侧翼带有第二对辅助重组位点的质粒，我们证明我们可以避免使用相同的起始细胞进行全质粒整合时整合不想要的细菌 DNA 元件。我们还同时创建了“双着陆垫”细胞，这些细胞在不同的基因组位点上同时含有两个正交的 Bxb1 重组酶位点，允许进行复杂的基于细胞的遗传测定。遗传编码钙指示剂的整合允许实时监测细胞内钙信号动力学，包括在野生型或变体钙信号中继蛋白 STIM1 过表达时发生的动力学干扰。一组 HIV-1 辅助蛋白 Vif 的错义突变体与人类 Apobec3 先天免疫蛋白家族中的各种同源物配对，以鉴定能够或不能在细胞内相互作用的组合。这些细胞允许转基因蛋白变体文库与特定于测定的蛋白伴侣或生物传感器轻松配对，为大规模的蛋白功能遗传测定提供新的功能读数。
