Synthetic biology (Oxford, England)最新文献

The diversity and flexibility of life offers a wide variety of molecules and systems useful for biosensing. A biosensor device should be robust, specific and reliable. Inorganic arsenic is a highly toxic water contaminant with worldwide distribution that poses a threat to public health. With the goal of developing an arsenic biosensor, we designed an incoherent feed-forward loop (I-FFL) genetic circuit to correlate its output pulse with the input signal in a relatively time-independent manner. The system was conceived exclusively based on the available BioBricks in the iGEM Registry of Standard Biological Parts. The expected behavior in silico was achieved; upon arsenic addition, the system generates a short-delayed reporter protein pulse that is dose dependent to the contaminant levels. This work is an example of the power and variety of the iGEM Registry of Standard Biological Parts, which can be reused in different sophisticated system designs like I-FFLs. Besides the scientific results, one of the main impacts of this synthetic biology project is the influence it had on team's members training and career choices which are summarized at the end of this article.

Quantifying the effect of vital resources on transcription (TX) and translation (TL) helps to understand the degree to which the concentration of each resource must be regulated for achieving homeostasis. Utilizing the synthetic TX-TL system, we study the impact of nucleotide triphosphates (NTPs) and magnesium (Mg²⁺) on gene expression. Recent observations of the counter-intuitive phenomenon of suppression of gene expression at high NTP concentrations have led to the speculation that such suppression is due to the consumption of resources by TX, hence leaving fewer resources for TL. In this work, we investigate an alternative hypothesis: direct suppression of the TL rate via stoichiometric mismatch in necessary reagents. We observe NTP-dependent suppression even in the early phase of gene expression, contradicting the resource-limitation argument. To further decouple the contributions of TX and TL, we performed gene expression experiments with purified messenger RNA (mRNA). Simultaneously monitoring mRNA and protein abundances allowed us to extract a time-dependent translation rate. Measuring TL rates for different Mg²⁺ and NTP concentrations, we observe a complex resource dependence. We demonstrate that TL is the rate-limiting process that is directly inhibited by high NTP concentrations. Additional Mg²⁺ can partially reverse this inhibition. In several experiments, we observe two maxima of the TL rate viewed as a function of both Mg²⁺ and NTP concentration, which can be explained in terms of an NTP-independent effect on the ribosome complex and an NTP-Mg²⁺ titration effect. The non-trivial compensatory effects of abundance of different vital resources signal the presence of complex regulatory mechanisms to achieve optimal gene expression.

Designer transcription activator-like effectors (dTALEs) are programmable transcription factors used to regulate user-defined promoters. The TALE DNA-binding domain is a tandem series of amino acid repeats that each bind one DNA base. Each repeat is 33-35 amino acids long. A residue in the center of each repeat is responsible for defining DNA base specificity and is referred to as the base specificying residue (BSR). Other repeat residues are termed non-BSRs and can contribute to TALE DNA affinity in a non-base-specific manner. Previous dTALE engineering efforts have focused on BSRs. Non-BSRs have received less attention, perhaps because there is almost no non-BSR sequence diversity in natural TALEs. However, more sequence diverse, TALE-like proteins are found in diverse bacterial clades. Here, we show that natural non-BSR sequence diversity of TALEs and TALE-likes can be used to modify DNA-binding strength in a new form of dTALE repeat array that we term variable sequence TALEs (VarSeTALEs). We generated VarSeTALE repeat modules through random assembly of repeat sequences from different origins, while holding BSR composition, and thus base preference, constant. We used two different VarSeTALE design approaches combing either whole repeats from different TALE-like sources (inter-repeat VarSeTALEs) or repeat subunits corresponding to secondary structural elements (intra-repeat VarSeTALEs). VarSeTALE proteins were assayed in bacteria, plant protoplasts and leaf tissues. In each case, VarSeTALEs activated or repressed promoters with a range of activities. Our results indicate that natural non-BSR diversity can be used to diversify the binding strengths of dTALE repeat arrays while keeping target sequences constant.

Because of the technological limitations of de novo DNA synthesis in (i) making constructs containing tandemly repeated DNA sequence units, (ii) making an unbiased DNA library containing DNA fragments with sequence multiplicity in a specific region of target genes, and (iii) replacing DNA fragments, development of efficient and reliable biochemical gene assembly methods is still anticipated. We succeeded in developing a biological standardized genetic parts that are flanked between a common upstream and downstream nucleotide sequences in an appropriate plasmid DNA vector (BioBrick)-based novel assembly method that can be used to assemble genes composed of 25 tandemly repeated BioBricks in the correct format in vitro. We named our new DNA part assembly system: 'Quick Gene Assembly (QGA)'. The time required for finishing a sequential fusion of five BioBricks is less than 24 h. We believe that the QGA method could be one of the best methods for 'gene construction based on engineering principles' at the present time, and is also a method suitable for automation in the near future.

Living cells constantly process information from their living environment. It has recently been shown that a number of cell signaling mechanisms (e.g. G protein-coupled receptor and epidermal growth factor) can be interpreted as computing the logarithm of the ligand concentration. This suggests that logarithm is a fundamental computation primitive in cells. There is also an increasing interest in the synthetic biology community to implement analog computation and computing the logarithm is one such example. The aim of this article is to study how the computation of logarithm can be realized using chemical reaction networks (CRNs). CRNs cannot compute logarithm exactly. A standard method is to use power series or rational function approximation to compute logarithm approximately. Although CRNs can realize these polynomial or rational function computations in a straightforward manner, the issue is that in order to be able to compute logarithm accurately over a large input range, it is necessary to use high-order approximation that results in CRNs with a large number of reactions. This article proposes a novel method to compute logarithm accurately in CRNs while keeping the number of reactions in CRNs low. The proposed method can create CRNs that can compute logarithm to different levels of accuracy by adjusting two design parameters. In this article, we present the chemical reactions required to realize the CRNs for computing logarithm. The key contribution of this article is a novel method to create CRNs that can compute logarithm accurately over a wide input range using only a small number of chemical reactions.

To engineer cells for industrial-scale application, a deep understanding of how to design molecular control mechanisms to tightly maintain functional stability under various fluctuations is crucial. Absolute concentration robustness (ACR) is a category of robustness in reaction network models in which the steady-state concentration of a molecular species is guaranteed to be invariant even with perturbations in the other molecular species in the network. Here, we introduce a software tool, absolute concentration robustness explorer (ACRE), which efficiently explores combinatorial biochemical networks for the ACR property. ACRE has a user-friendly interface, and it can facilitate efficient analysis of key structural features that guarantee the presence and the absence of the ACR property from combinatorial networks. Such analysis is expected to be useful in synthetic biology as it can increase our understanding of how to design molecular mechanisms to tightly control the concentration of molecular species. ACRE is freely available at https://github.com/ramzan1990/ACRE.

Biosensors are valuable and versatile tools in synthetic biology that are used to modulate gene expression in response to a wide range of stimuli. Ligand responsive transcription factors are a class of biosensor that can be used to couple intracellular metabolite concentration with gene expression to enable dynamic regulation and high-throughput metabolite producer screening. We have established the Saccharomyces cerevisiae WAR1 transcriptional regulator and PDR12 promoter as an organic acid biosensor that can be used to detect varying levels of para-hydroxybenzoic acid (PHBA) production from the shikimate pathway and output green fluorescent protein (GFP) expression in response. The dynamic range of GFP expression in response to PHBA was dramatically increased by engineering positive-feedback expression of the WAR1 transcriptional regulator from its target PDR12 promoter. In addition, the noise in GFP expression at the population-level was controlled by normalising GFP fluorescence to constitutively expressed mCherry fluorescence within each cell. These biosensor modifications increased the high-throughput screening efficiency of yeast cells engineered to produce PHBA by 5,000-fold, enabling accurate fluorescence activated cell sorting isolation of producer cells that were mixed at a ratio of 1 in 10,000 with non-producers. Positive-feedback, ratiometric transcriptional regulator expression is likely applicable to many other transcription-factor/promoter pairs used in synthetic biology and metabolic engineering for both dynamic regulation and high-throughput screening applications.

Since the description, in 2000, of two artificial gene networks, synthetic biology has emerged as a new engineering discipline that catalyzes a change of culture in the life sciences. Recombinant DNA can now be fabricated rather than cloned. Instead of focusing on the development of ad-hoc assembly strategies, molecular biologists can outsource the fabrication of synthetic DNA molecules to a network of DNA foundries. Model-driven product development cycles that clearly identify design, build, and test phases are becoming as common in the life sciences as they have been in other engineering fields. A movement of citizen scientists with roots in community labs throughout the world is trying to democratize genetic engineering. It challenges the life science establishment just like visionaries in the 70s advocated that computing should be personal at a time when access to computers was mostly the privilege of government scientists. Synthetic biology is a cultural revolution that will have far reaching implications for the biotechnology industry. The work of synthetic biologists today prefigures a new generation of cyber-biological systems that may to lead to the 5^th industrial revolution. By catering to the scientific publishing needs of all members of a diverse community, Synthetic Biology hopes to do its part to support the development of this new engineering discipline, catalyze the culture changes it calls for, and foster the development of a new industry far into the twenty first century.