How & why
![process sakaiensis](https://static.igem.org/mediawiki/2019/b/b4/T--Humboldt_Berlin--ablauf_sakaiensis.png)
Our Inspiration
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.
Chlamy who?
![chlamydomonas schaubild](https://static.igem.org/mediawiki/2019/0/0e/T--Humboldt_Berlin--chlamydomonas_schaubild_eng.png)
![Chlamydomonas](https://static.igem.org/mediawiki/2019/9/9e/T--Humboldt_Berlin--800px-Chlamydomonas6-1.png)
TODO Text
![chlamy sun co2 conversion](https://static.igem.org/mediawiki/2019/c/ce/T--Humboldt_Berlin--chlamy_sonne-compressor.png)
![e-coli illustration](https://static.igem.org/mediawiki/2019/4/49/T--Humboldt_Berlin--e_coli.png)
Inclusion bodies
lack of eucaryotic posttranslational modification
![eucaryotic cells illustration](https://static.igem.org/mediawiki/2019/b/bd/T--Humboldt_Berlin--eucaryotic_cells.png)
Low protein yields (yeast, cell lines)
Expensive cultivation
Handling problems
![chlamy illustration](https://static.igem.org/mediawiki/2019/2/23/T--Humboldt_Berlin--chlamy_nur_so.png)
rapid growth rates
Inexpensive & easy cultivation
Easy transgene insertion
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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.
![microplastic icon](https://static.igem.org/mediawiki/2019/b/bc/T--Humboldt_Berlin--microplastic_icon.png)
![chlamy organism](https://static.igem.org/mediawiki/2019/c/c5/T--Humboldt_Berlin--chlamy_organism.png)
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
one organism = single cell
well established as model organism
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