by Thomas H Segall‐Shapiro, Adam J Meyer, Andrew D Ellington, Eduardo D Sontag, Christopher A Voigt
"Synthetic genetic systems share resources with the host, including machinery for transcription and translation. Phage RNA polymerases (RNAPs) decouple transcription from the host and generate high expression. However, they can exhibit toxicity and lack accessory proteins (σ factors and activators) that enable switching between different promoters and modulation of activity. Here, we show that T7 RNAP (883 amino acids) can be divided into four fragments that have to be co‐expressed to function. The DNA‐binding loop is encoded in a C‐terminal 285‐aa ‘σ fragment’, and fragments with different specificity can direct the remaining 601‐aa ‘core fragment’ to different promoters. Using these parts, we have built a resource allocator that sets the core fragment concentration, which is then shared by multiple σ fragments. Adjusting the concentration of the core fragment sets the maximum transcriptional capacity available to a synthetic system. Further, positive and negative regulation is implemented using a 67‐aa N‐terminal ‘α fragment’ and a null (inactivated) σ fragment, respectively. The α fragment can be fused to recombinant proteins to make promoters responsive to their levels. These parts provide a toolbox to allocate transcriptional resources via different schemes, which we demonstrate by building a system which adjusts promoter activity to compensate for the difference in copy number of two plasmids."
by Yi Liu , Brian R. Fritz , Mark J. Anderson , Jennifer A. Schoborg , and Michael C. Jewett
"Complete cell-free synthesis of ribosomes could make possible minimal cell projects and the construction of variant ribosomes with new functions. Recently, we reported the development of an integrated synthesis, assembly, and translation (iSAT) method for in vitro construction of Escherichia coli ribosomes. iSAT allows simultaneous ribosomal RNA synthesis, ribosome assembly, and reporter protein expression as a measure of ribosome activity. Here we explore causes of iSAT reaction termination to improve efficiency and yields. We discovered that phosphoenolpyruvate (PEP), the secondary energy substrate, and nucleoside triphosphates (NTPs) were rapidly degraded during iSAT reactions. In turn, we observed a significant drop in the adenylate energy charge and termination of protein synthesis. Further, we identified that the accumulation of inorganic phosphate is inhibitory to iSAT. Fed-batch replenishment of PEP and magnesium glutamate (to offset the inhibitory effects of accumulating phosphate by repeated additions of PEP) prior to energy depletion prolonged the reaction duration 2-fold and increased yields by 75%. By adopting a semi-continuous method, where passive diffusion enables substrate replenishment and byproduct removal, we prolonged iSAT reaction duration 5-fold and increased superfolder green fluorescent protein yield 7-fold to 7.5 ± 0.7 μmol L-1. This protein yield is the highest ever reported for iSAT reactions. Our results underscore the critical role energy substrates play in iSAT and highlight the importance of understanding metabolic processes that influence substrate depletion for cell-free synthetic biology."
CITRIS is co-sponsoring an event in November that is designed to explore a new multi-leveled approach to speed up knowledge development and enter the fast track to optimal, large-scale and market-rele...
by Taton A, Unglaub F, Wright NE, Zeng WY, Paz-Yepez J, Brahamsha B, Palenik B, Peterson TC, Haerizadeh F, Golden SS, Golden JW
"Inspired by the developments of synthetic biology and the need for improved genetic tools to exploit cyanobacteria for the production of renewable bioproducts, we developed a versatile platform for the construction of broad-host-range vector systems. This platform includes the following features: (i) an efficient assembly strategy in which modules released from 3 to 4 donor plasmids or produced by polymerase chain reaction are assembled by isothermal assembly guided by short GC-rich overlap sequences. (ii) A growing library of molecular devices categorized in three major groups: (a) replication and chromosomal integration; (b) antibiotic resistance; (c) functional modules. These modules can be assembled in different combinations to construct a variety of autonomously replicating plasmids and suicide plasmids for gene knockout and knockin. (iii) A web service, the CYANO-VECTOR assembly portal, which was built to organize the various modules, facilitate the in silico construction of plasmids, and encourage the use of this system. This work also resulted in the construction of an improved broad-host-range replicon derived from RSF1010, which replicates in several phylogenetically distinct strains including a new experimental model strain Synechocystis sp. WHSyn, and the characterization of nine antibiotic cassettes, four reporter genes, four promoters, and a ribozyme-based insulator in several diverse cyanobacterial strains."
"As in other engineering fields, progress in synthetic biology depends on the exchange of parts and designs by researchers and will be most effective if designs adhere to common standards. A group of experimental and computational researchers from many different institutions in several countries, coordinated by Herbert Sauro from the University of Washington, describes such a data standard: the Synthetic Biology Open Language (SBOL). SBOL uses standard graphical notations—for example, for promoters, 5′ untranslated regions, coding s…"
Sami Ullah Jan, Burhan Ullah, Aimal Khan, Muhammad Asif Shahzad, Zeeshan Ali Yousaf, Atif Shafiqu1and Muhammad Ali Abbas
"Genomics and its related studies boosted explorations when applied in various dimensions of biology. The most common concept employed is to mix natural abilities of various living organisms or distant biological sources in the form of genes targeted for their products. With the advent of 21st century, this field gained a pace due to the attention by various scientific communities worldwide. Though, many hurdles still exist on its way but synthetic biology has led the basis for advanced outcomes by merging the potentials of genetic engineering and electronic techniques. This piece of literature reviews the research and development of synthetic biology accomplished since past in various life sciences with emphasis on pharmaceuticals, vaccines and biofuel development. The efforts of international scientific community and international organizations are also highlighted, who developed regulations and transmitted the importance to applied level. The production of biofuel, anti-microbial drugs, vaccines or other biological components with the help of genetic engineering technology was the first generation which after integration in synthetic biology has successfully transferred to a new generation. Along with the past, this paper also forecasts the future of synthetic biology in minimizing the limitations and problems faced in biological research with the help of synthetic biology. "
"A cornerstone of synthetic biology and biological engineering is achiev- ing regulatory control of genes of interest. Typically, this is attempted by placing binding sites for classic transcription factors upstream of genes. However, gene regulation is multilayered beyond transcription factor recruitment; thus, a new study has characterized how diverse chro- matin regulators might provide a flexible and powerful way to regulate different aspects of gene expression.
Chromatin states in eukaryotic cells are modulated in various ways — including by DNA methylation, histone modifications and nucleo- some remodelling — thus providing opportunities for ‘fine-tuning’ the regulation of gene expression. An emerging approach to assess the gene regulatory effects of specific chromatin regulator proteins is ‘epi- genome editing’, in which chromatin regulators are fused to sequence- specific DNA-binding proteins to allow their recruitment to a chosen locus. Such a strategy has so far characterized only a few chromatin- modifying enzymes. So, Keung et al. took a systematic approach by generating a library of 223 yeast chromatin regulators fused to zinc- finger (ZF) DNA-binding proteins, although the system is potentially also applicable to the transcription activator-like effector (TALE) and CRISPR–Cas genome targeting systems. ..."
comment to: ORIGINAL RESEARCH PAPER Keung, A. J. et al. Using targeted chromatin regulators to engineer combinatorial and spatial transcriptional regulation. Cell 158, 110–120 (2014)
by Dejana Jovicevic, Benjamin A. Blount andTom Ellis
"A team of US researchers recently reported the design, assembly and in vivo functionality of a synthetic chromosome III (SynIII) for the yeast Saccharomyces cerevisiae. The synthetic chromosome was assembled bottom-up from DNA oligomers by teams of students working over several years with researchers as the first part of an international synthetic yeast genome project. Embedded into the sequence of the synthetic chromosome are multiple design changes that include a novel in-built recombination scheme that can be induced to catalyse intra-chromosomal rearrangements in a variety of different conditions. This system, along with the other synthetic sequence changes, is intended to aid researchers develop a deeper understanding of how genomes function and find new ways to exploit yeast in future biotechnologies. The landmark of the first synthesised designer eukaryote chromosome, and the power of its massively parallel recombination system, provide new perspectives on the future of synthetic biology and genome research."
"Split T7 RNA polymerase provides new avenues for creating synthetic gene circuits that are decoupled from host regulatory processes—but how many times can this enzyme be split, yet retain function? New research by Voigt and colleagues (Segall‐Shapiro et al, 2014) indicates that it may be more than you think."
"We demonstrate how logical operations can be implemented in ensembles of protoplasmic tubes of acellular slime mold Physarum polycephalum. The tactile response of the protoplasmic tubes is used to actuate analogs of two- and four-input logical gates and memory devices. The slime mold tube logical gates display results of logical operations by blocking flow in mechanically stimulated tube fragments and redirecting the flow to output tube fragments. We demonstrate how XOR and NOR gates are constructed. We also exemplify circuits of hybrid gates and a memory device. The slime mold based gates are non-electronic, simple and inexpensive, and several gates can be realized simultaneously at sites where protoplasmic tubes merge."
by Catherine Jefferson, Filippa Lentzos and Claire Marris
"Synthetic biology, a field that aims to ‘make biology easier to engineer’, is routinely described as leading to an increase in the ‘dual use’ threat, i.e. the potential for the same piece of scientific research to be ‘used’ for peaceful purposes or ‘misused’ for warfare or terrorism. Fears have been expressed that the ‘de-skilling’ of biology, combined with online access to the genomic DNA sequences of pathogenic organisms and the reduction in price for DNA synthesis, will make biology increasingly accessible to people operating outside well-equipped professional research laboratories, including people with malevolent intentions. The emergence of DIY biology communities and of the student iGEM competition has come to epitomize this supposed trend towards greater ease of access and the associated potential threat from rogue actors. In this article, we identify 5 ‘myths’ that permeate discussions about synthetic biology and biosecurity, and argue that they embody misleading assumptions about both synthetic biology and bioterrorism. We demonstrate how these myths are challenged by more realistic understandings of the scientific research currently being conducted in both professional and DIY laboratories, and by an analysis of historical cases of bioterrorism. We show that the importance of tacit knowledge is commonly overlooked in the dominant narrative: the focus is on access to biological materials and digital information, rather than on human practices and institutional dimensions. As a result, public discourse on synthetic biology and biosecurity tends to portray speculative scenarios about the future as realities in the present or the near future, when this is not warranted. We suggest that these ‘myths’ play an important role in defining synthetic biology as a ‘promissory’ field of research and as an ‘emerging technology’ in need of governance."
"The method dubbed Assembly of Designed Oligonucleotides (ADO) is a powerful tool in synthetic biology to create combinatorial DNA libraries for gene, protein, metabolic, and genome engineering. In directed evolution of proteins, ADO benefits from using reduced amino acid alphabets for saturation mutagenesis and/or DNA shuffling, but all 20 canonical amino acids can be also used as building blocks. ADO is performed in a two-step reaction. The first involves a primer-free, polymerase cycling assembly or overlap extension PCR step using carefully designed overlapping oligonucleotides. The second step is a PCR amplification using the outer primers, resulting in a high-quality and bias-free double-stranded DNA library that can be assembled with other gene fragments and/or cloned into a suitable plasmid subsequently. The protocol can be performed in a few hours. In theory, neither the length of the DNA library nor the number of DNA changes has any limits. Furthermore, with the costs of synthetic DNA dropping every year, after an initial investment is made in the oligonucleotides, these can be exchanged for alternative ones with different sequences at any point in the process, fully exploiting the potential of creating highly diverse combinatorial libraries. In the example chosen here, we show the construction of a high-quality combinatorial ADO library targeting sixteen different codons simultaneously with nonredundant degenerate codons encoding various reduced alphabets of four amino acids along the heme region of the monooxygenase P450-BM3."
"Saccharomyces cerevisiae can serve as a key production platform for biofuels, nutraceuticals, industrial compounds, and therapeutic proteins. Over the recent years, synthetic biology tools and libraries have expanded in yeast to provide newfound control over regulation and synthetic circuits. This review provides an update on the status of the synthetic biology toolbox in yeast for use as a cell factory. Specifically, we discuss the impact of plasmid selection and composition, promoter, terminator, transcription factor, and aptamer selection. In doing so, we highlight documented interactions between these components, current states of development, and applications that demonstrate the utility of these parts with a particular focus on synthetic gene expression control."