Rainbow screening: Chromoproteins enable visualized molecular cloning

Molecular cloning facilitates the assembly of heterologous DNA fragments with vectors, resulting in the generation of plasmids that can steadily replicate in host cells. To efficiently and accurately screen out the expected plasmid candidates, various methods, such as blue-white screening, have been developed for visualization. However, these methods typically require additional genetic manipulations and costs. To simplify the process of visualized molecular cloning, here we report Rainbow Screening, a method that combines Gibson Assembly with chromoproteins to distinguish Escherichia coli (E. coli) colonies by naked eyes, eliminating the need for additional genetic manipulations or costs. To illustrate the design, we select both E. coli 16s rRNA and sfGFP expression module as two inserted fragments. Using Rainbow Screening, false positive colonies can be easily distinguished on LB-agar plates. Moreover, both the assembly efficiency and the construct accuracy can exceed 80%. We anticipate that Rainbow Screening will enrich the molecular cloning methodology and expand the application of chromoproteins in biotechnology and synthetic biology. Rainbow Screening is a visualized molecular cloning method that utilizes chromoproteins to distinguish Escherichia coli colonies by naked eyes, eliminating the need for additional genetic manipulation of host cells (e.g., lacZ modification for blue-white screening), and costs for substrates (e.g., X-gal and IPTG for blue-white screening) and/or equipment (e.g., UV transilluminator for fluorescence visualization) image