To synthesize and assemble all the needed gene constructs to let Chlamydomonas express the PETase and MHETase together with different promotors, terminators, secretion signals, tags and selection markers cloning was done using the Golden Gate Modular Cloning (referred to hereafter as “MoClo”) toolkit for C. reinhardtii (Crozet et al., 2018). The Chlamydomonas MoClo kit is standardized to fit the syntax of the plant synthetic biology community (Patron et al., 2015).

MoClo is an assembly method using type IIS restriction sites first introduced by (Weber, Engler, Gruetzner, Werner, & Marillonnet, 2011). Type IIS restriction enzymes (BpiI; BsaI) cleave outside of their recognition site leaving a four base pair overhang also called a fusion site (Engler, Kandzia, & Marillonnet, 2008). Placing those restriction sites before the 5’ beginning and the 3’ end of a desired DNA fragment in inverse orientation will allow ligation of DNA fragments with compatible fusion sites to be correctly assembled (Weber et al., 2011). Type IIS restriction sites can be constructed to build different overhangs making an assembly of multiple fragments possible (Weber et al., 2011).

Fig. 1 Overview of the hierarchical and modular cloning system (Weber et al., 2011). (A) Level 0 plasmid modules containing cloned and sequenced promoters (P), 5′ untranslated regions (U), coding sequences (CDS) and terminators (T). Because of the standardization of the toolkit the desired transcription units can be assembled from selected level 0 plasmids in a one-step digestion and ligation reaction into a level 1 vector. (B) Scheme of a Level 0 (L0) and L1 module. The gene part of a L0 module is flanked by compatible fusion sites providing the correct assembly of these modules when cloned into a L1 destination vector. The fusion sites are four nucleotides long (1-4 and 5-8) and flanked by a BsaI recognition sites.

The MoClo toolkit for Chlamydomonas consists of three different cloning vectors called level 0, 1 and 2. They are used in consecutive assembly steps. Level 0 (referred to hereafter as “L0”) destination vectors contain a selection marker gene such as lacZ or RFP surrounded by two cloning sites (BpiI; BsaI). It is possible to insert gene parts into L0 plasmids such as promotors, coding sequences or UTRs with specific fusion sites and surrounded by BsaI restriction sites (Fig. 1 B). The specific fusion sites and BsaI restriction sites make it possible to correctly assemble those L0 modules onto a next plasmid level 1 (referred to hereafter as “L1”) in one step generating a transcriptional unit (Fig. 1, Tab. 1).

Each assembly is performed in a single reaction mix with the desired insert, the destination vector, DNA ligase and one type IIS restriction enzyme. Correctly assembled L1 modules can then be transformed into C. reinhardtii.

Each L0 module has designated nucleotides as their fusion site determining their cloning position in a L1. The kit offers ten different options for positioning in a L1 plasmid. Those cloning positions for each gene part are defined by its function and altogether its position in a L1 plasmid (Tab. 1).