Generation of MoClo Standard Parts Using Golden Gate Cloning

Availability of efficient DNA assemblyDNA assemblies methods is a basic requirement for synthetic biologySynthetic biology. A variety of modular cloningModular cloning systems have been developed, based on Golden Gate Golden GatecloningCloning for DNA assemblyDNA assemblies, to enable users to assemble multigene constructsMultigene constructs from libraries of standard parts using a series of successive one-pot assembly reactions. Standard parts contain the DNA sequence coding for a genetic element of interest such as a promoter Promoters , coding sequenceCoding sequences or terminator Terminators . Standard parts for the modular cloningModular cloning system MoCloMoClo must be flanked by two BsaIBsaI restriction sites and should not contain internal sequences for two type IIS restriction sites, BsaIBsaI and BpiIBpiI, and optionally for a third type IIS enzyme, BsmBIBsmBI. We provide here a detailed protocol for cloningCloning of basic parts. This protocol requires the following steps (1) defining the type of basic Biopart Assembly Standard for Idempotent Cloning (BASIC) part that needs to be cloned, (2) designing primers for amplification, (3) performing PCR amplification, (4) cloningCloning of the fragments using Golden Gate Golden GatecloningCloning, and finally (5) sequencing of the part. For large basic Biopart Assembly Standard for Idempotent Cloning (BASIC) parts, it is preferable to first clone subparts as intermediate level −1 constructs. These subparts are sequenced individually and are then further assembled to make the final level 0 module.