Construction and Characterization of MoClo-Compatible Vectors for Modular Protein Expression in .

Cloning methods are fundamental to synthetic biology research. The capability to generate custom DNA constructs exhibiting predictable protein expression levels is crucial to the engineering of biology. Golden Gate cloning, a modular cloning (MoClo) technique, enables rapid and reliable one-pot assembly of genetic parts. In this study, we expand on the existing MoClo toolkits by constructing and characterizing compatible low- (p15A) and medium-copy (pBR322) destination vectors. Together with existing high-copy vectors, these backbones enable a protein expression range covering a 500-fold difference in normalized fluorescence output. We further characterize the expression- and burden profiles of each vector and demonstrate their use for the optimization of growth-coupled enzyme expression. The optimal expression of (encoding alcohol dehydrogenase) for ethanol-dependent growth of is determined using randomized Golden Gate Assembly, creating a diverse library of constructs with varying expression strengths and plasmid copy numbers. Through selective growth experiments, we show that relatively low expression levels of facilitated optimal growth using ethanol as the sole carbon source, demonstrating the importance of adding low-copy vectors to the MoClo vector repertoire. This study emphasizes the importance of varying vector copy numbers in selection experiments to balance expression levels and burden, ensuring accurate identification of optimal conditions for growth. The vectors developed in this work are publicly available via Addgene (catalog #217582-217609).