Golden Gate Cloning - Loop and MoClo - part and primerdesign guidelines v1
Authors: Sparrow is 
Published: July 2024

This protocol is largely based on the original Loop and uLoop protocol dx.doi.org/10.17504/protocols.io.yxnfxmeby the corresponding authors. This protocol provides general guidelines for primer design for introducing parts into the Loop system of assembly. Golden Gate ligation/cloning/assembly, first developed by Engler et al., is a technique used to join DNA fragments in a specific order. It employs Type IIS restriction enzymes. These enzymes recognize a specific sequence and cut outside of the recognition sequence leaving overhangs. This has 2 implications: 1. Cut sites have directionality (i.e., the recognition sequence can be to the left or right of the cut site depending on the strand the sequence is in) 2. Cut sites' overhangs are not the same and will depend on the surrounding sequence to the recognition sequence. To simplify and optimize the Golden Gate assembly, multiple standardized systems have been developed to allow for easy interchange of parts and maximize efficiency of assembly. For example, BsaI enzyme cuts leaving a 4 base pair overhang. An overhang of 4 bases AAAT is less efficient than one of 4 bases ACGT. For standardized assembly, typical elements of DNA assemblies (e.g., promoter, coding sequence, terminator, etc.) are called "parts" and have defined 5' and 3' overhangs, so that no matter which promoter you choose, it can be ligated with a coding sequence because all promoters have the same overhangs. All of these elements are put in one pot, one step reaction alongside an acceptor vector, whose overhangs must match the first overhang of the parts (in order) (for example the 5' overhang of a promoter) and the last overhang of the parts (the 3' of the terminator). In acceptor vectors, the enzyme recognition sequences are diverging (cut sites facing away from each other) and are found inside the "insert" (typically a lacZ or other selection/screening gene) (in reality, the insert is defined by the presence of the cut sites), because we are interested in the backbone and not the actual insert. In donor vectors (which hold the parts/elements), the recognition sites are found convergently, because the insert is what we are interested in keeping in the final assembly. For any assembly, you will need any number of parts so long as all overhangs match up and can join together to form a plasmid. After digestion with the enzyme, we have a linear backbone, linear parts. If our reaction is correct, they will find each other in the solution, be joined if the overhangs match, and then be permanently ligated by ligase enzyme, as cut sites are removed, in the backbone of donor parts and insert of acceptor parts. For every final assembly, there is no longer any cut sites of that enzyme present in the vector, ensuring high efficiency and "no backsies". For more information read the original papers: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2574415/ Original Golden Gate https://pubs.acs.org/doi/10.1021/sb500366v Yeast MoClo kit https://nph.onlinelibrary.wiley.com/doi/full/10.1111/nph.15625 Loop https://nph.onlinelibrary.wiley.com/doi/full/10.1111/nph.13532 Plant MoClo https://currentprotocols.onlinelibrary.wiley.com/doi/full/10.1002/cpmb.115 Full description of Golden Gate assembly