Methods For Cloning Useful Extracellular Enzymes?

This manual explains how to create an expression construct to over-express a protein or a domain using an E.coli expression vector, which can be used for other systems. Cloning vectors in molecular biology have key features such as a suitable cloning site with restriction enzymes and a selectable marker. Restriction enzyme cloning, or “restriction cloning”, uses DNA restriction enzymes to cut a vector and insert at specific locations so they can be easily joined together by the enzyme DNA ligase to create recombinant DNA.

Researchers often take advantage of heterologous expression systems by cloning genes into artificial vectors designed to operate within easily accessible environments. An initial modeling approach was applied to analyze how a single, nonmotile, free-living, heterotrophic bacterial cell may optimize the deployment of its extracellular enzymes.

Molecular cloning is made possible by co-opting cellular enzymes normally involved in DNA replication, recombination, and repair. In vitro selections for catalytic activity have been designed for the isolation of genes encoding enzymes from libraries of proteins displayed on filamentous surfaces.

Functional cloning is based on identification of the protein that is altered in a disorder, with subsequent sequencing of the protein and design of cDNA probes. Restriction enzyme cloning involves making multiple copies of a DNA fragment to enable it to be more easily studied and manipulated.

In summary, this manual provides an overview of how to create expression constructs for protein expression using various cloning vectors. It also discusses the importance of Ca2+ for enzymatic activity and the use of restriction enzyme cloning for functional cloning.