Synthesis of orthogonal transcription-translation networks

Abstract

Orthogonal, parallel and independent, systems are one key foundation for synthetic biology. The synthesis of orthogonal systems that are uncoupled from evolutionary constraints, and selectively abstracted from cellular regulation, is an emerging approach to making biology more amenable to engineering. Here, we combine orthogonal transcription by T7 RNA polymerase and translation by orthogonal ribosomes (O-ribosomes), creating an orthogonal gene expression pathway in Escherichia coli. We design and implement compact, orthogonal gene expression networks. In particular we focus on creating transcription-translation feed-forward loops (FFLs). The transcription-translation FFLs reported cannot be created by using the cells' gene expression machinery and introduce information-processing delays on the order of hours into gene expression. We refactor the rRNA operon, uncoupling the synthesis of the orthogonal 16S rRNA for the O-ribosome from the synthesis and processing of the rest of the rRNA operon, thereby defining a minimal module that can be added to the cell for O-ribosome production. The minimal O-ribosome permits the rational alteration of the delay in an orthogonal gene expression FFL. Overall this work demonstrates that system-level dynamic properties are amenable to rational manipulation and design in orthogonal systems. In the future this system may be further evolved and tuned to provide a spectrum of tailored dynamics in gene expression and investigate the effects of delays in cellular decision-making processes.

Fig. 1.

An orthogonal gene expression network. (A) Orthogonal gene expression is insulated from cellular gene expression. (B) The orthogonal AND function (Left) and an orthogonal transcription–translation FFL (Right).

Fig. 2.

The construction of an orthogonal AND function. (A) The logic of orthogonal gene expression (Left). A molecular strategy (Right) for the realization of orthogonal gene expression. P_lac, lac promoter; T7 RNAP, the gene encoding T7 RNAP; P_T7, T7 promoter; gfp, gene encoding the GFP; Ptrc, a hybrid of the tryptophan and lactose promoters; O-rDNA, encodes an orthogonal ribosomal RNA. (B) Sequences selected after 3 rounds of FACS sorting for O-ribosome-dependent translation of a T7 transcript. T7S2 was represented 3 times and T71503 was represented twice among the best clones for GFP expression. (C) Orthogonal transcription–translation shows AND logic. The expression construct pT7 O-rbs GST-GFP contains the regulatory sequences from T71504 (in B) upstream of a genetic fusion between GST and sfGFP. (Top) Expressed and purified GST-GFP. (Middle) A Northern blot to detect sfGFP mRNA. (Bottom) A loading control for total RNA.

Fig. 3.

Synthesis and characterization of an orthogonal transcription translation FFL. (A) (Left) Logic of the orthogonal transcription–translation FFL. (Right) A molecular strategy for the realization of an orthogonal transcription–translation FFL. Labels are as in Fig. 2. (B) The orthogonal transcription–translation FFL displays AND logic. pT7 O-rrnB is the rrnB operon that produces O-16S rRNA, 23S rRNA, and 5S rRNA. Cells were harvested 5 h after the addition of 1 mM IPTG. (C) The orthogonal genetic circuits constructed and examined for altered dynamics. Maroon, FFL; blue, simple AND; green, AND with constitutive O-rRNA production. (D) O-ribosome production causes an identical delay in the kinetics of orthogonal gene expression in the simple AND function and in the FFL. Percentage of maximal sfGFP expression produced from pT7 O-rbs GST-GFP as a function of time >60 h after the addition of 1 mM IPTG. Green solid circles, BL21 (DE3) (produce T7 RNAP from an IPTG inducible promoter), pSC101* O-ribosome, pT7 O-rbs GST-GFP. Blue solid circles, BL21 (DE3) pTrc RSF O-ribosome (produces O-rRNA from an IPTG inducible Trc promoter), pT7 O-rbs GST-GFP. Maroon solid circles, BL21 (DE3), pT7 RSF O-ribosome, pT7 O-rbs GST-GFP. In this and subsequent figures the error bars represent the SD of at least 3 independent trials and the growth of cells containing all combinations of constructs was comparable as judged by following the OD₆₀₀ (Figs. S4 and S5). Cells that lacked either T7 RNAP or the O-ribosome exhibited only backgorund fluorescence (Fig. S6).

Fig. 4.

Creation of a minimal O-ribosome. (A) Schematic of the truncations examined for the production of active O-ribosomes. (B) The activity of O-ribosomes produced from each truncation construct (constructs 1–10, filled bars) compared with the full-length operon (O-rrnB). Fluorescence was measured in cells containing pXR1 (a tetracycline-resistant p15A plasmid that directs GFP expression from a constitutive promoter and O-rbs). The empty bars show the expression of GFP produced when pXR1 is combined with wild-type ribosomes in the absence of O-ribosomes.

Fig. 5.

The FFLs with the minimal O-ribosome and the progenitor O-ribosome have identical topologies but mediate distinct delays. (A) The FFL using the progenitor O-ribosome (maroon) and the minimal O-ribosome (orange). (B) The delays in gene expression created by the orthogonal transcription translation networks. Orange solid circles, BL21 (DE3), pT7 RSF O-16S, pT7 O-rbs GST-GFP. The green blue and maroon curves are reproduced from Fig. 3 for comparison. The time taken to reach 50% of maximal expression, used to quantify the delay (22), is indicated.