Synthetic biology: advancing biological frontiers by building synthetic systems

Abstract

Advances in synthetic biology are contributing to diverse research areas, from basic biology to biomanufacturing and disease therapy. We discuss the theoretical foundation, applications, and potential of this emerging field.

Figure 1

Synthetic circuits that perform diverse functions can be coupled to achieve higher-order responses. (a) Interlinked positive and negative feedback loops of different strengths drive an oscillatory response. Arabinose-responsive transcriptional activator (AraC) expression positively modulates gene expression and results in a positive feedback loop, whereas the isopropyl-β-D-thio-galactoside (IPTG)-responsive inhibitor of the lac promoter (LacI) inhibits expression and generates a negative feedback loop. The small-molecule inducers arabinose and IPTG modulate the strength of these feedback loops [42]. GFP, green fluorescent protein. (b) A mammalian AND gate composed of RNA interference (RNAi) target sites evaluates small interfering (si)RNA inputs. Unique RNAi target sites are placed in the 3' UTR of two lacI genes, and LacI regulates the expression of a fluorescent reporter, resulting in an AND logic evaluator for the siRNA inputs m1 and m2 [52]. YFP, yellow fluorescent protein. (c) Quorum-sensing circuitry allows population control. Cell density is broadcast by the diffusible small molecule acyl-homoserine lactone (AHL), which is synthesized by the enzyme LuxI (X). As cell density and AHL concentration increase, LuxR (R), a transcriptional regulator, binds AHL and initiates expression of a 'killer' gene (encoding CcdB, a lethal protein that targets the DNA gyrase complex), ultimately reducing the steady-state cell density [58]. (d) Interlinking positive and negative feedback loops with communication circuitry enables oscillation synchronization across a population of cells. Expression of R positively regulates expression of X, R, GFP, and AiiA (A), an enzyme that degrades AHL. As A increases in concentration, it degrades AHL and negatively modulates protein expression levels [62]. (e) Combining logic processing with communication circuitry enables a synthetic biological edge detection system. The expression of X and the transcriptional repressor cI (Y) is turned ON in cells in the dark region, where Y represses the expression of the pigment-producing protein (pigment: β-galactosidase, an enzyme that cleaves a substrate to produce a black pigment). However, diffusion of AHL synthesized by cells in the dark region activates R in cells at the edge of the light region (where Y is turned OFF), thus turning ON expression of pigment only in cells along this edge [63].

Figure 2

Synthetic biological circuits can aid in understanding of biology, improve biomanufacturing productivity, and enable disease-targeted therapy. (a) The native circuit regulating competence in B. subtilis was compared with a synthetic circuit with similar dynamics to reveal architecture-specific variability in the duration of competence and consequent differences in the consistency of transformation efficiency over large ranges of DNA concentration [71]. (b) A synthetic protein scaffold was used to increase the biosynthesis of mevalonate from acetyl-CoA in E. coli. The scaffold consists of three protein-protein interaction domains (GBD, the GTPase binding domain from the actin polymerization switch N-WASP; SH3, the Src homology 3 domain from the adaptor protein CRK; and PDZ, the PSD95/DlgA/Zo-1 domain from the adaptor protein syntrophin) in various copy numbers connected by glycine-serine linkers. Pathway enzymes (AtoB, acetoacetyl-CoA thiolase; HMGS, hydroxymethylglutaryl-CoA synthase; HMGR, hydroxymethylglutaryl-CoA reductase) were each fused to the ligands of one interaction domain and recruited to the protein scaffold [39]. P_TET, tetracycline-inducible promoter; P_BAD, arabinose-inducible promoter. (c) A targeted therapeutic circuit was constructed by inserting an RNA aptamer near an alternatively spliced exon harboring a stop codon in a three-exon, two-intron minigene fused to herpes simplex virus thymidine kinase (HSV-TK). Binding of a disease marker protein to the aptamer results in exclusion of the alternative exon, expression of a suicide gene, and killing of diseased cells [35]. P_CMV, cytomegalovirus promoter.