Part Collection

Part Collection overview

The Golden Gate Assembly was used to create the plasmids, which were later transformed into P. pastoris. The system is based on type IIs restriction enzymes. These cut the DNA outside of their recognition site and create DNA overhangs called fusion sites, which can consist of any nucleotides (shown in figure 4). As a result, seamless junctions can be created by designing compatible overhangs. The restriction and ligation event of the desired fragments result in the loss of the original restriction sites. Therefore, it is possible to perform the reaction in a one pot restriction – ligation reaction.

A hierarchical cloning system was used which contains three levels of complexity, which are shown in figure 5. Level 1 called BB1 contains the basic donor plasmids containing genetic building blocks like promotors, genes of interests and terminators. At Level 2 called BB2 a promotor, a gene of interest and a terminator are combined to a transcription unit. The level with the highest complexity is level 3 by combining different transcription units to a multigene construct. At level 3 called BB3 up to 7 BB2 can be combined.