Synthetic Biology approaches in algae
Engineering Chlamydomonas reinhardtii using synthetic biology (SynBio) represents a promising approach for protein synthesis. Following the Design-Build-Test cycle as good conduct to establish synthetic biological approaches, our project aims at providing experience and resources needed to increase the utility of algae as a tool for SynBio. We used the algae’s qualities as a photoautotrophic, sustainable chassis to try and degrade PET-plastic, while fixating carbon dioxide. We designed and implemented parts and vectors following the Golden Gate standard for easy assembly and cloning into the green alga, thus increasing the number of possible tools for alga SynBio in the iGEM Registry. Our Chlamy-HUB Collection features enzymes, fluorescence tags, secretion signals, antibiotic selection or metabolic markers, purification tags, a terminator and promoters for strong expression. Although we were unsuccessful in measuring plastic degradation, the Chlamy-HUB Collection proved to be useful for protein expression, demonstrated by fluorescence of YFP in the engineered algae. Our parts are codon-optimized for C. reinhardtii and the backbones we created are smaller than those conventionally used for MoClo, enabling future users to easily transform and achieve high protein yields. By introducing a self-cleaving peptide within our parts collection we enable future users to increase the yield of their transgenic protein. Adding to the experimental data we collected, we have shared all protocols we usedto engineer the green algae. Furthermore, we present the OpenPBR, a DIY-photobioreactor to monitor growth of any phototrophic organism under comparative conditions. Its automated measurements give users the chance to gain reproducible data, while controlling different parameters. This way, teams could ramp up protein expression by keeping the growth conditions in the optimum range for their cultivated algae.
ChlamyHUB sheds light on the many advantages protein synthesis by C. reinhardtii has, while remaining a sustainable SynBio platform.
Modeling and Design Iterations
As part of our way towards enabling C. reinhardtii to degrade PET, we modeled a biological system to assess the efficiency of PET-degradation by an algae culture. One of the lessons learned from this model was that protein expression had to be increased further. In consequence, we chose a light-inducible promoter, PsaD, which regulates the expression of an abundant chloroplast protein and added the SP20 module for enhanced protein secretion to our parts resources. Additionally, we chose the UVM4 strain of Chlamydomonas for our transformations, which allows efficient expression of transgenes. A second model was developed to simulate light-limited growth of Chlamydomonas, leading us to conclusions on how to design the measurements of optical density by the OpenPBR.
Overall, we hope our project will shed light on the many advantages protein synthesis by C. reinhardtii has, while remaining a sustainable SynBio platform. The standardization of genetic parts and protocols will have a high impact on their usefulness for future applications and holds potential for reproducible experimental setups. Finally, by supplying instructions on building the OpenPBR and publishing our models we are providing other researchers with an infrastructure to work on algal SynBio.
