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
![](https://static.igem.org/mediawiki/2019/1/16/T--Humboldt_Berlin--GrowthModeling.jpeg)
Fig. Overview of the components of a growth model.
In a first step to facilitate the ‚Design - Build - Test - Learn‘ cycle, we wanted to create a model that has the aim to give an overview of metabolic processes, genes and other parameters necessary for photosynthetic growth. We use the syntax defined in the Constraint Based Reconstruction Analysis (COBRA) Toolbox for Python (Ebrahim et al. 2013), already existing metabolic reconstructions of Chlamy (Imam et al 2015, Kliphuis et al. 2011), the fully sequenced genome (Merchant et. al 2007 ) and a recent -omics dataset (Strenkert et al. 2019) to define the components of the model. In a second step we combine these components in such a way that we are able to assess how they work together to give rise to growth of a Chlamy culture. The model should also provide the synthetic biologist with information useful for performing specific tasks in C. reinhardtii.
Unfortunately, we could not finish the model because there was not enough time. We still were able to use knowledge gathered along the way, though.
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
A C. reinhardtii which expresses and secretes the enzymes PETase and MHETase could pose as a solution for the problem of micro-plastic polluted water. Nevertheless, the viability of PET-degradation by C. reinhardtii at a larger scale is yet unknown. To assess the efficiency of PET-degradation by C. reinhardtii, a model of the expression, secretion and kinetics of the enzymes PETase and MHETase in C. reinhardtii was designed. The model can be seen here. here. The goal of the model is to simulate the degradation of PET while taking into account the parameters of enzyme kinetics, expression rate, secretion rate and cultivation density. By varying the parameters, an approximation on PET degradation under various conditions can be made to examine what the appropriate parameters are for an optimal PET-degradation. To achieve this, the kinetics of MHETase and the optimized PETase (I179F) were taken from literature (Palm et al., 2019) (Ma et al., 2019).
![PET degradation model](https://static.igem.org/mediawiki/2019/3/32/T--Humboldt_Berlin--modell_uebersicht.png)
2.2 Transformation of PETase and MHETase into C. reinhardtii
![Usage of a Bioreactor](https://static.igem.org/mediawiki/2019/f/fc/T--Humboldt_Berlin--Algae_cult.png)
Plastic degradation as intended by our project is to take place in the media of C. reinhardtii after expression and secretion of both the PETase and MHETase enzymes by the algae. To test the expression of PETase different constructs need to be implemented. We apply YFP as a fluorescent tag to optically screen our transformed clones for expression of the plasmid and therefore, PETase-production. Our other tag, the 3xHA-tag was intended for purification of the enzyme out of the C. reinhardtii cells. As secretion signals we chose the arylsulphatase 1 (ARS) and gametolysin (GLE) secretion signals, which were compared to each other. The serine-proline glycomodule (SP₂₀) is a secretion enhancer. While not only important for our goal to degrade plastics but also crucial in developing a toolkit for multi-purpose use, we test the following constructs:
PETase with secretion signal GLE and fluorescent tag YFP
PETase with secretion signal ARS, glycomodule SP₂₀ and YFP
PETase with HA purification tag and ARS
First, the clones of each transformation are tested for the uptake of the plasmids via colony PCR. Then, if bands of the expected length appear, the respective clones are continuously cultivated and algae cell suspensions are tested for fluorescence signals via fluorescence microscopy. If the secretion signals work, the medium around the cells emits fluorescence. This screening step is the most time-consuming. Once clones displaying fluorescence are detected, we try measuring the comparative fluorescences of the engineered clone and wildtype algae in a plate-reader. Constructs coding for the expression of MHETase are MHETase with YFP as a fluorescent tag, enabling screening for transformants and with an HA-tag for purification.
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.