To synthesize and assemble the desired genetic elements, we applied the Type IIS “Golden Gate” cloning strategy (Engler et al., 2008). We used the Modular Cloning (MoClo) toolkit optimized for C. reinhardtii (Crozet et al., 2018), which follows the MoClo syntax of the plant synthetic biology community (Patron et al., 2015). Type IIS restriction enzymes cut outside their recognition sites, making them useful in this cloning method for consecutive assembly of fragments. Through the restriction, overhangs are formed which allow the fusion of said genetic fragments to complementary overhangs of the syntax and thereby determine the order of each in a transcriptional unit (Figure 1). These fusion sites allow for the assembly of several fragments in the right order in just one cloning reaction. The used MoClo-kit offers ten different options for the positioning inside a L1 plasmid which are defined by the parts’ functions.

Fig. 1. Universal MoClo fusion sites.

Within the MoClo syntax, there are three different cloning vectors, level 0, 1 and 2 (referred to as “L0”, “L1” and “L2”, respectively). L0 vectors carry one basic genetic fragment or part, L1 vectors are assembled fragments creating a transcriptional unit and L2 are multigenic constructs. Construction of an L0-part is done by flanking a gene of interest with the specific fusion site and the recognition site of BpiI by a PCR reaction. Upon digestion by BpiI it can be inserted into a previously digested L0-backbone. To then clone it into a L1-backbone, it is digested by BsaI, revealing the fusion sites for its assembly in a transcription unit. Lastly, a fusion of several transcription units (L1) into a L2 multigenic device is possible with the MoClo syntax.

As part of our contribution to a toolkit usable by future iGEM teams we design and construct not only the parts we intend to use on our goal of PET-degradation but several more, a L0-backbone and L1-backbone.

To ensure that all parts were designed correctly we cloned the PCR fragments into a L0 vector. To this end, we used a self-modified version of a L0 backbone containing RFP. After ligation, we transformed the L0 plasmids containing the parts into Escherichia coli and checked for white transformants. Only genes with the correct fusion and restriction sites could be inserted into the L0 backbone resulting in growth of white colonies, since the RFP gene was interrupted. For further verification, we checked the parts by DNA sequencing. We used the same control mechanisms for L1 assembly constructs.