As previously explained on our Description page it is our aim to establish C. reinhardtii in the iGEM competition. To reach this goal we created a tool kit of various functional parts and multi-use constructs that future iGEM teams can use and optimize.
So, what is our focus?
1. Establishing C. reinhardtii as a platform in the competition
2. Working on the PET-degradation as a proof of concept
3. Building a bioreactor, in which we can cultivate C. reinhardtii and test its growth rates under different conditions
1. Establishing Chlamy in the iGEM competition
Golden Gate Modular Cloning
for Chlamydomonas reinhardtii
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 (BpiI; BsaI) cleave outside of their recognition site leaving a four base pair overhang also called a fusion site (Engler, Kandzia, & Marillonnet, 2008). These fusion sites are determined by the used syntax. Placing restriction- and fusion sites in front of the 5’ beginning and after the 3’ end of a desired DNA fragment in an inverse orientation allows the ligation of DNA fragments with compatible fusion sites (Weber et al., 2011). Thus, the required order of sequences is defined and it is possible to assemble multiple fragments at the same time (Weber et al., 2011).
Fig. 1
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 restriction sites; the inner being BpiI, the outer BsaI. It is possible to insert gene parts into L0 plasmids such as promoters, coding sequences or UTRs with specific fusion sites and surrounded by BpiI restriction sites. The construct is a “level 0 part”, which is flanked by the syntax-specific fusion sites and a BsaI recognition site. The designated fusion sites (shown in green) determine the modules’ cloning position in a Level 1 (“L1”) plasmid. The used MoClo-kit offers ten different options for the positioning inside a L1 plasmid which are defined by the parts’ function.
When using these fusion sites, it is crucial to maintain the reading frame of the coding sequence, since a four base insertion can change the triplet code. Since the fusion site B3 contains the start codon ATG, the ATG within the original coding sequence needs to be inserted into this fusion site or has to be deleted. In any case, since the overhang contains four bases, it is crucial that after every element, which is part of the transcribed unit, two additional bases are integrated after the gene sequence to avoid a frame-shift, a shift in the reading frame of the following parts. To keep the native function of the protein, the selection of additional bases is not completely arbitrary and non-disturbing amino acids like alanine should be selected when possible. The stop codon TAA is not a part of any fusion site and must be attached to the end of the last part of the transcriptional unit (e.g. B5) or in front of the terminator (e.g. B5/C1, B6/C1) when creating a primer.
This makes it possible to correctly assemble those L0 parts onto the next level 1 plasmid (referred to hereafter as “L1”) in one step generating a transcriptional unit. Each assembly of L1 or L2 is performed in a single reaction mixed with the desired insert, the destination vector, DNA ligase and the needed type IIS restriction enzyme.
