Generation of MoClo Standard Parts Using Golden Gate Cloning.

Availability of efficient DNA assembly methods is a basic requirement for synthetic biology. A variety of modular cloning systems have been developed, based on Golden Gate cloning for DNA assembly, to enable users to assemble multigene 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 , coding sequence or terminator . Standard parts for the modular cloning system MoClo must be flanked by two BsaI restriction sites and should not contain internal sequences for two type IIS restriction sites, BsaI and BpiI, and optionally for a third type IIS enzyme, BsmBI. We provide here a detailed protocol for cloning of basic parts. This protocol requires the following steps (1) defining the type of basic part that needs to be cloned, (2) designing primers for amplification, (3) performing PCR amplification, (4) cloning of the fragments using Golden Gate cloning, and finally (5) sequencing of the part. For large 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.