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

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 microg/mL luciferase in batch reactions and 60 ± 4 microg/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.