Establishing a High-Yield Chloroplast Cell-Free System for Prototyping Genetic Parts

Abstract

Plastid engineering offers the potential to carry multigene traits in plants; however, it requires reliable genetic parts to balance expression. The difficulty of chloroplast transformation and slow plant growth makes it challenging to build plants just to characterize genetic parts. To address these limitations, we developed a high-yield cell-free system from Nicotiana tabacum chloroplast extracts for prototyping genetic parts. Our cell-free system uses combined transcription and translation driven by T7 RNA polymerase and works with plasmid or linear template DNA. To develop our system, we optimized lysis, extract preparation procedures (e.g., runoff reaction, centrifugation, and dialysis), and the physiochemical reaction conditions. Our cell-free system can synthesize 34 ± 1 μg/mL luciferase in batch reactions and 60 ± 4 μg/mL in semicontinuous reactions. We apply our batch reaction system to test a library of 103 ribosome binding site (RBS) variants and rank them based on cell-free gene expression. We observe a 1300-fold dynamic range of luciferase expression when normalized by maximum mRNA expression, as assessed by the malachite green aptamer. We also find that the observed normalized gene expression in chloroplast extracts and the predictions made by the RBS Calculator are correlated. We anticipate that chloroplast cell-free systems will increase the speed and reliability of building genetic systems in plant chloroplasts for diverse applications.

Chloroplast Cell-Free Systems from Different Plant Species as a Rapid Prototyping Platform

“…Our study successfully demonstrated the development of CFE systems from the chloroplasts of spinach, wheat, and poplar, demonstrating that a previously developed protocol for tobacco is versatile across species. We also showcased the potential for native transcription and the characterization and prototyping of regulatory sequences at both transcription and translation levels.…”

“…Our objective was to develop a high-yielding CFE system, derived from wheat (a monocot) as well as spinach and poplar trees (dicots). We adapted the workflow for generating highly active cell-free transcription and translation systems from tobacco chloroplasts (Figure S1). In contrast to cell-free expression systems from bacteria, yeast, and mammalian cells, ,,,,, preparation of chloroplast extracts requires separation of an organelle with its own bacteria-like translation machinery from other organelles and subcellular and cytoplasmic components of the plant cell.…”

“…The isolation and lysis procedure was adapted from Clark et al All steps of the chloroplast isolation procedure were carried out at 4 °C or on ice. Harvested plant material was divided into 50–100 g batches, and leaves of wheat and poplar were cut into 3 cm stripes prior to processing in a blender (8011ES, Waring).…”

“…We focused on testing regulatory components involved in post-transcriptional and translation control, as these stages are the primary ways for the regulation of gene expression in chloroplasts . Our work builds off a recently established protocol for generating a highly active tobacco chloroplast-based cell-free transcription and translation system, which demonstrated the feasibility of using CFE systems for the characterization of plant-based ribosome-binding sites …”