Chlamy-HUB Collection
Chlamydomonas reinhardtii is a photoautotrophic organism, meaning that it needs few resources to grow optimally and fixates carbon dioxide (CO2) from our atmosphere. This makes it a perfect chassis to clear pollution from our environment, while holding potential for further protein synthesis applications.
As there are only few genetic parts for use in C. reinhardtii in the iGEM Registry, we have created the part collection „Chlamy-HUB Collection“ (Part number range: BBa_K2984000-BBa_K2984063). It contains parts optimized for use in Chlamydomonas that are easy-to-assemble and composite transcriptional units, ready for transformation. Each part fits the MoClo syntax for plants following the Golden Gate cloning standard and therefore the iGEM RFC[1000] Type IIS standard. Most of the level 0 parts were also designed to be compatible with the RFC[10] standard mainly used in the iGEM registry.
Utility
The Chlamy-HUB Collection features: one light-inducible and one constituent promoter, two secretion signals, a secretion-enhancer, the PETase enzyme, yellow and red fluorescent proteins, a self-cleaving peptide, purification tags, a metabolic marker for growth on phosphite, antibiotic selection markers for hygromycin, bleomycin and paromomycin and a terminator, all optimized for use in Chlamydomonas. These parts were chosen with a focus on maximizing protein expression, on using secretion modules to bring the active enzymes out of the organism and on detection specials. While designing the transcriptional units we followed a systematic approach, substituting each functional part by another. For example, the PETase constructs we created contain each of the markers and their secretion is regulated by both secretion signals, one by one.
Moreover, we designed and implemented our own backbones for MoClo cloning, specifically for use in Chlamydomonas. These vectors are smaller than the ones regularly employed in publications using the MoClo technique for an increased transformation efficiency. Their sequence has a high GC-content which fits the codon usage of Chlamydomonas. If transformed via the glass bead agitation or gene gun methods, they can still be used to conduct synthetic biology in other plant chassis.
To round up the Chlamy-HUB Collection we have put thought into the environmental aspects of our experimental design. We conducted cultivation tests of chlamy on regular tap water and water taken from different wastewater-treatment steps, where we discovered that they were unable to grow. Nevertheless, to avoid the survival of even a few algae these should only be cultivated in enclosed systems, like our self-designed photobioreactor. With this toolkit we hope to contribute to establishing chlamy as a green alternative to bacterial chassis for problem-solving with synthetic biology.
The Chlamy-HUB Collection contains parts that are easy-to-assemble and all composite transcriptional units are ready for transformation. Each part fits the MoClo syntax for plants following the Golden Gate cloning standard.