A Formalized Design Process for Bacterial Consortia That Perform Logic Computing

The concept of microbial consortia is of great attractiveness in synthetic biology. Despite of all its benefits, however, there are still problems remaining for large-scaled multicellular gene circuits, for example, how to reliably design and distribute the circuits in microbial consortia with limited number of well-behaved genetic modules and wiring quorum-sensing molecules. To manage such problem, here we propose a formalized design process: (i) determine the basic logic units (AND, OR and NOT gates) based on mathematical and biological considerations; (ii) establish rules to search and distribute simplest logic design; (iii) assemble assigned basic logic units in each logic operating cell; and (iv) fine-tune the circuiting interface between logic operators. We in silico analyzed gene circuits with inputs ranging from two to four, comparing our method with the pre-existing ones. Results showed that this formalized design process is more feasible concerning numbers of cells required. Furthermore, as a proof of principle, an Escherichia coli consortium that performs XOR function, a typical complex computing operation, was designed. The construction and characterization of logic operators is independent of “wiring” and provides predictive information for fine-tuning. This formalized design process provides guidance for the design of microbial consortia that perform distributed biological computation.     

Rapid construction of insulated genetic circuits via synthetic sequence-guided isothermal assembly

In vitro recombination methods have enabled one-step construction of large DNA sequences from multiple parts. Although synthetic biological circuits can in principle be assembled in the same fashion, they typically contain repeated sequence elements such as standard promoters and terminators that interfere with homologous recombination. Here we use a computational approach to design synthetic, biologically inactive unique nucleotide sequences (UNSes) that facilitate accurate ordered assembly. Importantly, our designed UNSes make it possible to assemble parts with repeated terminator and insulator sequences, and thereby create insulated functional genetic circuits in bacteria and mammalian cells. Using UNS-guided assembly to construct repeating promoter-gene-terminator parts, we systematically varied gene expression to optimize production of a deoxychromoviridans biosynthetic pathway in Escherichia coli. We then used this system to construct complex eukaryotic AND-logic gates for genomic integration into embryonic stem cells. Construction was performed by using a standardized series of UNS-bearing BioBrick-compatible vectors, which enable modular assembly and facilitate reuse of individual parts. UNS-guided isothermal assembly is broadly applicable to the construction and optimization of genetic circuits and particularly those requiring tight insulation, such as complex biosynthetic pathways, sensors, counters and logic gates.     

An efficient platform for genetic selection and screening of gene switches in Escherichia coli

Engineered gene switches and circuits that can sense various biochemical and physical signals, perform computation, and produce predictable outputs are expected to greatly advance our ability to program complex cellular behaviors. However, rational design of gene switches and circuits that function in living cells is challenging due to the complex intracellular milieu. Consequently, most successful designs of gene switches and circuits have relied, to some extent, on high-throughput screening and/or selection from combinatorial libraries of gene switch and circuit variants. In this study, we describe a generic and efficient platform for selection and screening of gene switches and circuits in Escherichia coli from large libraries. The single-gene dual selection marker tetA was translationally fused to green fluorescent protein (gfpuv) via a flexible peptide linker and used as a dual selection and screening marker for laboratory evolution of gene switches. Single-cycle (sequential positive and negative selections) enrichment efficiencies of >7000 were observed in mock selections of model libraries containing functional riboswitches in liquid culture. The technique was applied to optimize various parameters affecting the selection outcome, and to isolate novel thiamine pyrophosphate riboswitches from a complex library. Artificial riboswitches with excellent characteristics were isolated that exhibit up to 58-fold activation as measured by fluorescent reporter gene assay.     

A mechanistic model of the BLADE platform predicts performance characteristics of 256 different synthetic DNA recombination circuits

Boolean logic and arithmetic through DNA excision (BLADE) is a recently developed platform for implementing inducible and logical control over gene expression in mammalian cells, which has the potential to revolutionise cell engineering for therapeutic applications. This 2-input 2-output platform can implement 256 different logical circuits that exploit the specificity and stability of DNA recombination. Here, we develop the first mechanistic mathematical model of the 2-input BLADE platform based on Cre- and Flp-mediated DNA excision. After calibrating the model on experimental data from two circuits, we demonstrate close agreement between model outputs and data on the other 111 circuits that have so far been experimentally constructed using the 2-input BLADE platform. Model simulations of the remaining 143 circuits that have yet to be tested experimentally predict excellent performance of the 2-input BLADE platform across the range of possible circuits. Circuits from both the tested and untested subsets that perform less well consist of a disproportionally high number of STOP sequences. Model predictions suggested that circuit performance declines with a decrease in recombinase expression and new experimental data was generated that confirms this relationship.     

哺乳动物合成生物学在生物医学领域的研究进展

虽然合成生物学还处于早期研究阶段,但最近十年,该领域取得了非常显著的研究进展。合成生物学是以工程学思想为基础,通过人工设计、改造基因线路,从而赋予细胞或生物体新的功能,现已广泛应用于各个领域。随着人们对基因线路设计的深入研究,使得合成生物学研究走向临床应用成为可能。本文将围绕哺乳动物合成生物学在疾病治疗方面的研究进展,介绍基因线路的设计思路和方法、不同诱导因子调控的开环式基因线路以及用于疾病诊疗的闭环式基因环路在生物医学领域的应用。最后对合成生物学走向临床治疗的应用前景和挑战进行展望。