Gene Position More Strongly Influences Cell-Free Protein Expression from Operons than T7 Transcriptional Promoter Strength

Abstract

The cell-free transcription−translation of multiple proteins typically exploits genes placed behind strong transcriptional promoters that reside on separate pieces of DNA so that protein levels can be easily controlled by changing DNA template concentration. However, such systems are not amenable to the construction of artificial cells with a synthetic genome. Herein, we evaluated the activity of a series of T7 transcriptional promoters by monitoring the fluorescence arising from a genetically encoded Spinach aptamer. Subsequently the influences of transcriptional promoter strength on fluorescent protein synthesis from one, two, and three gene operons were assessed. It was found that transcriptional promoter strength was more effective at controlling RNA synthesis than protein synthesis in vitro with the PURE system. Conversely, the gene position within the operon strongly influenced protein synthesis but not RNA synthesis.

The PURE system for the cell-free synthesis of membrane proteins

“…To find the best proportion between the individual DNA fragments, it is recommended to perform preliminary experiments trying a variety of DNA concentrations and to quantify the synthesized protein products from each reaction. In contrast, it is generally difficult to control the protein product levels when using a polycistronic construct, although the order of the genes and the strength of the transcriptional promoter affect the productivity 25 . The excessive production of one component consumes the resources of the PURE system.…”

On Fine Stochastic Simulations of Liposome-Encapsulated PUREsystem™

“…E. coli extracts) also used in similar synthetic biology approaches. Such information is of great value for the most diverse applications, in both applied and basic research [31,32].…”

Communicating artificial cells

“…Recent screening of genetic constructs with different cell-free expression systems show that protein concentrations are easier to control by changing the strength of transcriptional promoters for E. coli RNA polymerase [38] rather than T7 RNA polymerase [39]. Protein yields can also be modulated by the genetic organization of the construct [39,40] and by the incorporation of degradation pathways [41]. Unfortunately, expression yield is significantly affected by many additional, poorly characterized factors making it difficult to get predictable behavior from newly constructed genetic sequences.…”

“…Unfortunately, expression yield is significantly affected by many additional, poorly characterized factors making it difficult to get predictable behavior from newly constructed genetic sequences. In terms of complexity, the PURE system has been used to express three gene operons [39] and the five subunits of E. coli RNA polymerase [42]. An E. coli cell extract was used to express a six gene transcriptional activation cascade, a five protein biosynthetic pathway, and the T7 genome, which encodes over 50 different proteins [32].…”