Efficient De Novo Assembly of 100 kb-Scale Human Functional Immunoglobulin Heavy Variable (IGHV) Gene Fragments In Vitro

Synthetic biology provides a powerful approach to functional studies of viral and microbial genomes. However, in vitro, efficient and scarless DNA manipulation on large and complex human genomes remains an inevitable challenge. Here, we de novo design and successfully assemble human functional immunoglobulin heavy variable (IGHV) gene fragments up to hundred-kilobase (kb)-sized, using an iterative in vitro assembly via <i>Escherichia coli</i> (<i>E. coli</i>) based on Gibson isothermal assembly. We describe an efficient method for "scarless" (without leaving any non-native sequences) engineering of the assembled ordered functional IGHV gene fragments, which contain complex and highly repetitive regions. Our method provides a suitable way to construct bacterial artificial chromosomes (BACs) (30-100 kb) with common materials, easy manipulations, and low cost. The construction of ordered functional IGHV gene BACs expands the synthetic biologist's chassis repertoire. It is essential for the adaptive immune response and constructing immunity humanized animal models.