Expression optimization and synthetic gene networks in cell-free systems

Abstract

Synthetic biology offers great promise to a variety of applications through the forward engineering of biological function. Most efforts in this field have focused on employing living cells, yet cell-free approaches offer simpler and more flexible contexts. Here, we evaluate cell-free regulatory systems based on T7 promoter-driven expression by characterizing variants of TetR and LacI repressible T7 promoters in a cell-free context and examining sequence elements that determine expression efficiency. Using the resulting constructs, we then explore different approaches for composing regulatory systems, leading to the implementation of inducible negative feedback in Escherichia coli extracts and in the minimal PURE system, which consists of purified proteins necessary for transcription and translation. Despite the fact that negative feedback motifs are common and essential to many natural and engineered systems, this simple building block has not previously been implemented in a cell-free context. As a final step, we then demonstrate that the feedback systems developed using our cell-free approach can be implemented in live E. coli as well, illustrating the potential for using cell-free expression to fast track the development of live cell systems in synthetic biology. Our quantitative cell-free component characterizations and demonstration of negative feedback embody important steps on the path to harnessing biological function in a bottom-up fashion.

Figure 1.

Genetic components characterized and role of cell-free systems in synthetic biology. (A) T7 Tet system. The repressor TetR binds the operator TetO and represses GFP expression. The inducer aTc may be added to relieve repression. (B) T7 Lac system. The repressor LacI binds the operator LacO and represses GFP expression. The inducer IPTG may be added to relieve repression. (C) Tet negative feedback system. A TetR repressible T7 promoter expresses TetR and GFP. A similar system can be assembled using a LacI repressible T7 promoter. (D) Most efforts in synthetic biology have focused on assembling biological components to form systems that are introduced into living cells for applications such as chemical synthesis, drug production, biosensors and energy production. This procedure of engineering biological systems can also be carried out in a cell-free context. Cell-free systems may be directly used in applications. Alternatively, initial deployment of synthetic systems in a cell-free context can help to fast track the development of live cell synthetic systems.

Figure 2.

Promoter sequences and dosage responses. (A) Sequences of TetR repressible promoters T7tet13 and T7tet19 and LacI repressible promoters T7lacO1 and T7lacOID, (B) Fluorescence after 10 h of expression from the T7, T7tet13 and T7tet19 promoters using constructs pT7, pT7tet and pT7tet2. (C) Dosage responses of T7, T7tet13 and T7tet19 constructs to purified TetR. (D) Fluorescence after 10 h of expression from the T7, T7lacO1 and T7lacOID promoters using constructs pKSGFP, pT7lacO1 and pT7lacOID. Error bars in (B) and (D) depict standard deviation of triplicate measurements. Note that the T7 controls in (B) and (D) correspond to plasmids (pT7 and pKSGFP, respectively) with different backbones, ribosome binding sites, GFP variants and terminators as described in Table 1.

Figure 3.

Optimization results. (A) Upstream sequences. Underlined base pairs are ribosome binding sites, and start codons are shown in italics. The 3′ end of the 16S rRNA sequence is shown above each ribosome binding site. (B) Effect of these different RBS sequences on EGFP production as measured by fluorescence after 10 h of expression. (C) Effect of transcriptional terminator on EGFP yield using plasmids pUCT7tet and pUCT7tet-T7term. pUCT7tet has the vsv terminator, while pUCT7tet-T7term has a T7 terminator. These plasmids are otherwise identical. (D) Comparison of T7 and T7tet13 promoter expression after incorporation of g10RBS and the T7 terminator into a pBluescript backbone. Error bars depict standard deviation of triplicate measurements.

Figure 4.

Plasmids and results for exploring different system composition approaches. (A) Constitutive T7 construct pKSGFP, and bicistronic constructs pCtltetKS, placI-GFP and pGFP-lacI. (B) Fluorescence after 10 h of EGFP expression from these constructs. (C) To investigate multi-plasmid systems, separate plasmids containing either GFP (pKSGFP) or lacI (pT7lacI) were constructed. (D) Results for co-expression of pKSGFP and pT7lacI for different percentages of pKSGFP by molar concentration. Error bars depict standard deviation of triplicate measurements.

Figure 5.

Negative feedback system and results. (A) Negative feedback constructs pNFB-T7tet and pNFBtetKS. pNFB-T7tet has the ZE21 RBS for tetR and GFP along with the T1 terminator, while pNFBtetKS has the g10 RBS for tetR and GFP along with the T7 terminator. (B) Responses of pNFB-T7tet (red triangles) and pNFBtetKS (green squares) to different aTc concentrations. (C) Response dynamics of pNFB-T7tet with no aTc (open red triangles), pNFB-T7tet with 3300 ng/ml aTc (closed red triangles), pNFBtetKS with no aTc (open green squares), pNFBtetKS with 3300 ng/ml aTc (closed green squares). (D) Response dynamics of pNFBtetKS to different aTc concentrations in the PURE system. (E) Responses of E. coli BL21-AI harboring pNFBtetKS to inducer aTc.