Abstract
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.
Highlights
The majority of genetically engineered plants today are generated by integrating transgenes into the nuclear genome, engineering of the plastid genome has become a promising technology, both for basic science and applied plant biotechnology [1]
To further establish GoldenBraid as the modular cloning system overarching the full spectrum of plant genetic engineering, we demonstrate its reappropriation for plastid transformation
The across-the-board compatibility of the GoldenBraid system ensured boasts the potential for prospective establishment of an ever-expanding repository of reusable genetic components and bringing together multiple users within the plant scientific community
Summary
The majority of genetically engineered plants today are generated by integrating transgenes into the nuclear genome, engineering of the plastid genome has become a promising technology, both for basic science and applied plant biotechnology [1]. Their potential for successful genetic manipulation stems from the fact that plastids, as relicts of endosymbiotic cyanobacteria, still feature many characteristics of prokaryotes. Cells harbor a multitude of plastids, especially chloroplasts; these, in turn, carry multiple genome reprints This high (trans-) gene copy number per cell, coupled with the utilization of strong promoters, fosters significantly elevated expression rates resulting in unprecedented protein accumulation levels (e.g., 80% TSP for bacteriolysins [2]). This could be considered a built-in genetic containment feature, as the spread of transgenes by pollen is, largely excluded
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