Development of a Pseudomonas putida cell-free protein synthesis platform for rapid screening of gene regulatory elements

Abstract

Cell-free protein synthesis (CFPS) systems enable the production of protein without the use of living, intact cells. An emerging area of interest is to use CFPS systems to characterize individual elements for genetic programs [e.g. promoters, ribosome binding sites (RBS)]. To enable this research area, robust CFPS systems must be developed from new chassis organisms. One such chassis is the Gram-negative Pseudomonas bacteria, which have been studied extensively for their diverse metabolism with promises in the field of bioremediation and biosynthesis. Here, we report the development and optimization of a high-yielding (198 ± 5.9 µg/ml) batch CFPS system from Pseudomonas putida ATCC 12633. Importantly, both circular and linear DNA templates can be applied directly to the CFPS reaction to program protein synthesis. Therefore, it is possible to prepare hundreds or even thousands of DNA templates without time-consuming cloning work. This opens the possibility to rapidly assess and validate genetic part performance in vitro before performing experiments in cells. To validate the P. putida CFPS system as a platform for prototyping genetic parts, we designed and constructed a library consisting of 15 different RBSs upstream of the reporter protein sfGFP, which covered an order of magnitude range in expression. Looking forward, our P. putida CFPS platform will not only expand the protein synthesis toolkit for synthetic biology but also serve as a platform in expediting the screening and prototyping of gene regulatory elements.

Keywords: Pseudomonas putida; TX-TL; cell-free protein synthesis; gene regulatory elements; prototyping; synthetic biology.

Figure 1.

Comparison of sfGFP yield with P. putida biomass harvested at different optical densities (OD₆₀₀). Reaction conditions: 33% (v/v) lysate content, 20 ng/µl plasmid template, 12 mM magnesium concentration and 26°C. Average sfGFP yield with error bars representing standard deviations of three independent experiments is shown.

Figure 2.

Cell-free protein synthesis optimization for P. putida enhances sfGFP expression yields. The CFPS reaction was optimized by surveying a range of (A) lysate content; (B) reaction temperatures; (C) plasmid concentrations; (D) magnesium concentrations. Initial reaction conditions before the optimization: 33% (v/v) lysate content, 20 ng/µl plasmid template, 12 mM magnesium concentration and 26°C. Average sfGFP yield with error bars representing standard deviations of three independent experiments is shown.

Figure 3.

sfGFP yield with various PCR templates and purification schemes compared to that of the pJL1-sfGFP plasmid. (A) sfGFP yield with different post-PCR purification. The purified PCR template used per reaction was normalized to the same molar concentration of the plasmid pJL1-sfGFP. (B) 15/340 bp upstream: space upstream of T7 promoter; all templates were purified by PCR clean-up. Average sfGFP yield with error bars representing standard deviations of three independent experiments is shown.

Figure 4.

Different levels of sfGFP expression with the RBS library highlights the ability of P. putida-based CFPS system for genetic part characterization. Rapid turn-over time for screening and characterization of DNA regulatory elements such as RBSs can be achieved with our P. putida CFPS system. Average sfGFP yield with error bars representing standard deviations of three independent experiments is shown.