How & why
Chlamy who?
Most projects at iGEM and in synthetic biology in general chose to work with bacterial chassis. However, a growing community of plant synthetic biologists have laid the focus increasingly in the utilization of microalgae such as Chlamydomonas reinhardtii as a photosynthetic expression platform (Jinkerson & Jonikas, 2015; Scaife et al., 2015). Being eukaryotic, this freshwater algae is able to perform post-translational modifications, allowing the expression of more complex proteins, while being easy and cost-efficient 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) enabling a wide range of possible combinations for individual applications.
Our project “Chlamylicious” has two goals: First we aim to establish C. reinhardtii in the iGEM competition as a photosynthetic chassis by proving a toolkit of basic parts and protocols and second, to demonstrate the advantage of an eco-friendly organism as platform to degrate PET plastic. To satisfy the need to reproducibly cultivate photoautotrophic organisms under controlled conditions, we built and optimised a Do-It-Yourself bioreactor. Modeling the algal growth during high protein expression under different conditions was also integrated in our efforts in optimizing cultivation.
Inclusion bodies
lack of eucaryotic posttranslational modification
Expensive cultivation
No motility
Post-translational modification
Photosynthesis
Enviromental-safe
Two expression compartments (nucleus & chloroplast)
Our Inspiration
The unicellular green alga C. reinhardtii has an impressive history. It is used since the 1950s as a model organism to not only elucidate the structure of flagellar and basic plant cellular processes, such as photosynthesis and light perception, but also in research regarding phototaxis, circadian rhythmicity, cell cycle and mating mechanisms (Elizabeth et al., 1989; E.H. Harris, 2009; ). However, C. reinhardtii has not only been used for fundamental research but is also a model for microalgal biotechnology and has become a photosynthetic expression platform to synthesize recombinant proteins. C. reinhardtii has been engineered to produce biofuels such as the biodiesel-precursor bisabolene (Wichmann et al., 2018). Chlamydomonas has also been used in therapeutic applications, having been modified to express an HIV antigen (Barahimipour et al., 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 steps in PET degradation (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 et al., 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 various 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 20th century (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 (1), who improved the activity levels of the PETase through direct mutagenesis. It was through their project that we started looking into improved versions of the PETase first discovered by Yoshida et al. We were intrigued by the effort Harvard BioDesign 2016 put into their project “Plastikback” (2), pushing us towards the direction of degrading microplastic in an aquatic environment secreting PETase into the enclosed system of a bioreactor. We also integrated the separation of both PET degradation products TPA and EG, useful precursors in the synthesis of new PET, in our bioreactor. The project of ASIJ Tokyo in 2016 (3) inspired our module design greatly. Their characterization of the expression strength of PETase using different promoters and the secretion tests conducted under different secretion signals struck us as good practices to optimize the expression of complex constructs. Therefore, while designing our transcriptional units and choosing possible parts we aimed for a high variety of interchangeable modules to compare their functions. The team from Tianjin in the year 2016 (4) combined photosynthetically active organisms and the degradation of PET, lending their reactor the capacity of fixating carbon dioxide using the energy from sunlight. The approaches by the team of ITB 2017 (5) aided us in our project design as well, inspiring us with their use of biofilms on plastics. Looking for microorganisms with the abillity of binding to the surface of PET, we came across flagellar adhesion of C. reinhardtii to surfaces, which is even light-switchable (Kreis et al., 2018). iGEM Team Yale´s (6) focus on improving functionality of the enzymes also greatly impacted our work.
Chlamydomonas as a model organism
We propose that by combining a photosynthetically active organism with at least the optimized PETase and the MHETase we can create a new way of recycling PET or even degrading PET completely to CO₂ and H₂O. The organism could then able to use the CO₂ obtained from the plastic as its carbon source. We immediately thought of Chlamydomonas reinhardtii as a chassis, as it proliferates quickly under energy-efficient conditions. Being not only able to conduct photosynthesis, fixing carbon dioxide and using it as its carbon source with the power of light, C. reinhardtii can also live off acetate as its carbon source.
easy to cultivate & phototrophic
