In vivo assembly to switch fluorescent protein expression in Escherichia coli: a simple laboratory class to introduce molecular cloning.

Molecular cloning is a fundamental technique in life science research. However, learning to perform traditional restriction-ligation-based methods is time-consuming and prone to failure, discouraging new students. Here, we describe a compact, two- to three-session undergraduate practical class using In vivo Assembly, a simple, homology-based method that takes advantage of the native recombination machinery of Escherichia coli. Students swap a green fluorescent protein coding sequence with a red fluorescent protein coding sequence in a bacterial expression vector using PCR-amplified DNA fragments with short homology arms, followed by DpnI restriction enzyme digestion to remove template DNA, and then bacterial transformation. Success is directly visible by red versus green fluorescent bacterial colonies, providing rapid feedback and high student engagement. This low-cost protocol introduces key cloning concepts (PCR, homology design, template removal, bacterial transformation, and recombination) with reduced hands-on time and fewer failure points than classic approaches, making it ideal for effective teaching in resource-limited settings.