How & why
Chlamy who?
For various biosynthetic experimental designs, chosen chassis have been bacterial, with the disadvantage of lacking post-translational modifications. A growing community of plant synthetic biologists have however laid the focus increasingly in the utilization of freshwater alga Chlamydomonas reinhardtii as a biosynthetic expression platform (Jinkerson & Jonikas, 2015; Scaife et al., 2015). Being eukaryotic, this microalga is able to perform post-translational modifications, allowing the expression of more complex proteins, while being easy to cultivate and to handle (Merchant et al., 2007). A variety of transformation methods, including but not limited to biolistic transformation, glass bead agitation and electroporation are well-established for this model organism (Boynton et al., 1988). Its ability to grow photoautotrophically makes it an ideal chassis to tackle a variety of complex problems in an environmentally-friendly way. For the iGEM competition 2019 we have developed a toolkit for C. reinhardtii containing a variety of functional parts and multi-use constructs in the MoClo syntax, based on the Golden Gate cloning method. This design aids the work of synthetic biologists, providing a method for an easy one-step, one-pot assembly (Weber et al., 2011) posing vast possible combinations for individual use-cases. The aim of our project “Chlamylicious” is two-fold: establishing C. reinhardtii in the iGEM competition as a biosynthetic chassis and proving the usefulness of our toolkit of parts and constructs, while working on the degradation of PET plastic. To satisfy the need for a Do-It-Yourself tool to reproducibly cultivate photoautotrophic organisms at lab-scale under controlled conditions, we built and optimised a bioreactor. Modeling the algal growth during expression of a high-copy plasmid under different conditions was also integrated in our efforts of optimizing cultivation.
Inclusion bodies
lack of eucaryotic posttranslational modification
Low protein yields (yeast, cell lines)
Expensive cultivation
Handling problems
rapid growth rates
Inexpensive & easy cultivation
Easy transgene insertion
Our Inspiration
The unicellular and genetically modifiable green alga C. reinhardtii has an impressive history. It is used since the 1950s as a model organism to not only elucidate basic plant cellular processes, such as photosynthesis and light perception, but also in research regarding phototaxis, circadian rhythmicity, cell cycle and mating mechanisms (E.H. Harris, 2009; Elizabeth H Harris, Stern, & Witman, 1989). The now best characterized microalga has since then not only been used for fundamental research but also industrial biotechnology, as the algal species has become an expression platform to synthesize recombinant proteins. C. reinhardtii has been engineered to produce biofuels such as the biodiesel-precursor bisabolene (Wichmann, Baier, Wentnagel, Lauersen, & Kruse, 2018). Chlamydomonas has also been used in therapeutic applications, having been modified to express an HIV antigen (Barahimipour, Neupert, & Bock, 2016). In 2016, the bacterium Ideonella sakaiensis that is able to use polyethylene terephthalate (PET) as a primary carbon and energy source was discovered (Yoshida et al., 2016). This bacterium secretes two different hydrolases that perform the first two PET degradation steps (Yoshida et al., 2016). The first hydrolase, PETase, breaks down PET to mono(2-hydroxyethyl) terephtalic acid (MHET). The second hydrolase, the MHETase, then digests MHET to terephthalic acid (TPA) and ethylene glycole (EG). The bacterium itself grows optimally within a pH range of 7-7,5 and a temperature of 30-37°C (Tanasupawat, Takehana, Yoshida, Hiraga, & Oda, 2016). It was also demonstrated that it cannot grow anaerobically and that it has a GC-rich genome (70,4%) (Tanasupawat et al., 2016; Yoshida et al., 2016). In 2018, the PETase was characterized and engineered to improve its performance (Austin et al., 2018). Astonished by the possibility to degrade one of the most commonly used plastics, we were inspired to try to integrate the PETase and MHETase Enzymes into C. reinhardtii in order to illustrate the capabilities of our favorite algae using a toolkit of varied genetic parts. One of the issues of the gravest impact on our generation is the environmental pollution. Especially the production of synthetic polymers, like plastic, has increased considerably since the 20thcentury (Andrady, 2011). Plastic is the most frequent material collected in studies on the surface of the Ocean (Law et al., 2010) and is also observed on the seafloor (Galgani et al., 2000). Even though the exact implication of microplastic to organisms is still unknown, the appearance of microplastic inside a wide variety of marine organisms is significant (Murray & Cowie, 2011).
We know that we are not after something completely new. But we want to do this right. So we chose a different organism and tried to tackle obstacles other teams failed to solve.
The iGEM projects that inspired us
Degrading microplastic is not a new idea when it comes to iGEM projects. Similarly inspired by the works of Yoshida and his colleagues (Yoshida et al., 2016) a multitude of different teams have worked on comparable topics. We know that we are not after something completely new. But we wanted to do this right. So we chose a different organism and tried to tackle obstacles other teams failed to solve. Our work was inspired by TJUSLS project on PETase 2016 (1), we are intrigued by the effort Harvard BioDesign 2016 put into their project “Plastikback” (2) and the project of ASIJ Tokyo in 2016 struck the same nerve (3). The approaches by the Teams of Tianjin 2016 (4) and ITB 2017 (5) have gravely encouraged our project as well.
Chlamydomonas as a model organism
We propose that by combining a photosynthesis active organism with at least the optimized PETase and the MHETase we can create a new way of recycling PET or even degrade PET completely to CO2 and H2O. The organism is then able to use the CO2 coming from the plastic as carbon source. We immediately thought of Chlamydomonas reinhardtii as an organism as it grows fast under energy-efficient conditions.
easy to cultivate & phototrophic
