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
![Bringing Chlamy to iGEM](https://static.igem.org/mediawiki/2019/3/32/T--Humboldt_Berlin--chlamy2igem.png)
1.1 Golden Gate Modular Cloning for Chlamydomonas reinhardtii
1.2 Construction of a selection cassette
1.3 Expression analysis - testing our transgenic proteins
1.3.2 Testing secretion signals
1.3.3 PtxD - Phosphite Oxidoreductase
1.3.4 Cas9/sgRNA-mediated site-directed mutagenesis
1.3.5. Modeling photoautotrophic growth of Chlamy
2. Working on PET-Degradation as a proof-of-concept
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2.2 Transformation of PETase and MHETase into C. reinhardtii
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2.3 Testing the toxicity of TPA and EG for C. reinhardtii
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2.4 Selection cassette construction
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2.5 Measuring concentration of TPA and EG in medium (quantitative activity test)
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2.6 PnpB assay to test enzyme activity (quantitative test)
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2.7 Purification of PETase and MHETase from E. coli in order to characterize and compare enzyme activity
![Bringing Chlamy to iGEM](https://static.igem.org/mediawiki/2019/b/be/T--Humboldt_Berlin--microplastic_icon_ablauf.png)
2.1 Modeling PET degradation by C. reinhardtii using an optimized PETase
2.2 Transformation of PETase and MHETase into C. reinhardtii
2.3 Testing the toxicity of TPA and EG for C. reinhardtii
![Usage of a Bioreactor](https://static.igem.org/mediawiki/2019/f/fc/T--Humboldt_Berlin--Algae_cult.png)
As it is our goal to grow C. reinhardtii in a bioreactor in which it secretes PETase and MHETase we need to understand how it can deal with the produced degradation products terephthalic acid (TPA) and ethylene glycol (EG). Within this framework we measured the growth rates of several C. reinhardtii strains in a series of experiments. With the help of the Multi Cultivator MC 1000 we can test four different C. reinhardtii strains on these reagents to find out which one is the most suitable for further experiments and for transformation.