A Modular Cloning Toolbox for the Generation of Chloroplast Transformation Vectors

Plastid transformation is a powerful tool for basic research, but also for the generation of stable genetically engineered plants producing recombinant proteins at high levels or for metabolic engineering purposes. However, due to the genetic makeup of plastids and the distinct features of the transformation process, vector design, and the use of specific genetic elements, a large set of basic transformation vectors is required, making cloning a tedious and time-consuming effort. Here, we describe the adoption of standardized modular cloning (GoldenBraid) to the design and assembly of the full spectrum of plastid transformation vectors. The modular design of genetic elements allows straightforward and time-efficient build-up of transcriptional units as well as construction of vectors targeting any homologous recombination site of choice. In a three-level assembly process, we established a vector fostering gene expression and formation of griffithsin, a potential viral entry inhibitor and HIV prophylactic, in the plastids of tobacco. Successful transformation as well as transcript and protein production could be shown. In concert with the aforesaid endeavor, a set of modules facilitating plastid transformation was generated, thus augmenting the GoldenBraid toolbox. In short, the work presented in this study enables efficient application of synthetic biology methods to plastid transformation in plants.