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Engineering the esaR Promoter for Tunable Quorum Sensing-Dependent Gene Expression
Jasmine Shong † and Cynthia H. Collins
"Quorum sensing (QS) systems enable bacteria to coordinate their behavior as a function of local population density and are often used in synthetic systems that require cell–cell communication. We have engineered the esaR promoter, PesaR, which is repressed by the QS regulator EsaR. EsaR-dependent gene expression from PesaR is induced by 3-oxo-hexanoyl-homoserine lactone (3OC6HSL). Here, we report a set of modified PesaR promoters that contain a second EsaR binding site. We observed changes in gene expression levels, regulatory range, 3OC6HSL sensitivity, and the regulatory role of EsaR that are dependent on the position of the second binding site. Combining the new promoters with endogenous 3OC6HSL production led to QS-dependent systems that exhibit a range of expression levels and timing. These promoters represent a new set of tools for modulating QS-dependent gene expression and may be used to tune the regulation of multiple genes in response to a single QS signal."
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byBrynne C Stanton,Alec A K Nielsen,Alvin Tamsir,Kevin Clancy,Todd Peterson& Christopher A Voigt"Genetic circuits perform computational operations based on interactions between freely diffusing molecules within a cell. When transcription factors are combined to build a circuit, unintended interactions can disrupt its function. Here, we apply 'part mining' to build a library of 73 TetR-family repressors gleaned from prokaryotic genomes. The operators of a subset were determined using an in vitro method, and this information was used to build synthetic promoters. The promoters and repressors were screened for cross-reactions. Of these, 16 were identified that both strongly repress their cognate promoter (5- to 207-fold) and exhibit minimal interactions with other promoters. Each repressor-promoter pair was converted to a NOT gate and characterized. Used as a set of 16 NOT/NOR gates, there are >1054 circuits that could be built by changing the pattern of input and output promoters. This represents a large set of compatible gates that can be used to construct user-defined circuits."http://bit.ly/1aPORDL
"The cost and accuracy of genome sequencing have improved dramatically. George Church asks why so few people are opting to inspect their genome.
Synthetic biology -- unlike any research discipline that precedes it -- has the potential to bypass the less predictable process of evolution to usher in a new and dynamic way of working with living systems.
byKoeppl H, Hafner M, Lu J."With recent improvements of protocols for the assembly of transcriptional parts, synthetic biological devices can now more reliably be assembled according to a given design. The standardization of parts open up the way for in silico design tools that improve the construct and optimize devices with respect to given formal design specifications. The simplest such optimization is the selection of kinetic parameters and protein abundances such that the specified design constraints are robustly satisfied. In this work we address the problem of determining parameter values that fulfill specifications expressed in terms of a functional on the trajectories of a dynamical model. We solve this inverse problem by linearizing the forward operator that maps parameter sets to specifications, and then inverting it locally. This approach has two advantages over brute-force random sampling. First, the linearization approach allows us to map back intervals instead of points and second, every obtained value in the parameter region is satisfying the specifications by construction. The method is general and can hence be incorporated in a pipeline for the rational forward design of arbitrary devices in synthetic biology." http://bit.ly/18slJ5K
byNicolas Papon , Vincent Courdavault and Marc Clastre"Highlights
byMarkus J. Brçcker, Joanne M. L. Ho, George M. Church, Dieter Sçll, and Patrick ODonoghue"Selenocysteine (Sec) is naturally incorporated into proteins by recoding the stop codon UGA. Sec is not hardwired to UGA, as the Sec insertion machinery was found to be able to site-specifically incorporate Sec directed by 58 of the 64 codons. For 15 sense codons, complete conversion of the codon meaning from canonical amino acid (AA) to Sec was observed along with a tenfold increase in selenoprotein yield compared to Sec insertion at the three stop codons. This high-fidelity sense-codon recoding mechanism was demonstrated for Escherichia coli formate dehydrogenase and recombinant human thioredoxin reductase and confirmed by independent biochemical and biophysical methods. Although Sec insertion at UGA is known to compete against protein termination, it is surprising that the Sec machinery has the ability to outcompete abundant aminoacyl-tRNAs in decoding sense codons. The findings have implications for the process of translation and the information storage capacity of the biological cell."http://bit.ly/1964083
Cathal Garvey is the creator of the blog Indie Biotech, his personal endeavour to provide tools, materials and learning resources for biotechnology to indivi...
byThomas E. Gorochowski , Eric van den Berg , Richard Kerkman , Johannes A Roubos , and Roel A.L. Bovenberg"Synthetic biology has developed numerous parts for the precise control of protein expression. However, relatively little is known about the burden these place on a host, or their reliability under varying environmental conditions. To address this, we made use of synthetic transcriptional and translational elements to create a combinatorial library of constructs that modulated expression strength of a green fluorescent protein. Combining this library with a microbioreactor platform, we were able to perform a detailed large-scale assessment of transient expression and growth characteristics of two Escherichia coli strains across several temperatures. This revealed significant differences in the robustness of both strains to differing types of protein expression, and a complex response of transcriptional and translational elements to differing temperatures. This study supports the development of reliable synthetic biological systems capable of working across different hosts and environmental contexts. Plasmids developed during this work have been made publicly available to act as a reference set for future research." http://bit.ly/18o4QJp
byHelen Shen"Scientists launch company to develop the therapeutic potential of gene-snipping enzymes.Instead of taking prescription pills to treat their ailments, patients may one day opt for genetic 'surgery' — using an innovative gene-editing technology to snip out harmful mutations and swap in healthy DNA.The system, called CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats), has exploded in popularity in the past year, with genetic engineers, neuroscientists and even plant biologists viewing it as a highly efficient and precise research tool. Now, the gene-editing system has spun out a biotechnology company that is attracting attention from investors as well.
Scientists have finally turned human stem cells into lung cells, and say the breakthrough paves the way for using a patient's own cells for lung transplant.
byZhang M, Wang F, Li S, Wang Y, Bai Y, Xu X."Transcription activator-like effectors (TALEs), first identified in Xanthomonas bacteria, are naturally occurring or artificially designed proteins that modulate gene transcription. These proteins recognize and bind DNA sequences based on a variable numbers of tandem repeats. Each repeat is comprised of a set of ∼34 conserved amino acids; within this conserved domain, there are usually two amino acids that distinguish one TALE from another. Interestingly, TALEs have revealed a simple cipher for the one-to-one recognition of proteins for DNA bases. Synthetic TALEs have been used to successfully target genes in a variety of species, including humans. Depending on the type of functional domain that is fused to the TALE of interest, these proteins can have diverse biological effects. For example, after binding DNA, TALEs fused to transcriptional activation domains can function as robust transcription factors (TALE-TFs), while fused to restriction endonucleases (TALENs) can cut DNA. Targeted genome editing, in theory, is capable of modifying any endogenous gene sequence of interest; this can be performed in cells or organisms, and may be applied to clinical gene-based therapies in the future. With current technologies, highly accurate, specific, and reliable gene editing cannot be achieved. Thus, recognition and binding mechanisms governing TALE biology are currently hot research areas. In this review, we summarize the major advances in TALE technology over the past several years with a focus on the interaction between TALEs and DNA, TALE design and construction, potential applications for this technology, and unique characteristics that make TALEs superior to zinc finger endonucleases." http://bit.ly/IErZ3z
byJared E. Toettcher, Orion D. Weinersend email, Wendell A. Lim"Highlights
RT @TeselaGen: A New Paper In Synthetic Biology Sheds “Light” On Future: http://t.co/82kugjtlbm #synbio #cyanobacteria #photosynthesis #sys…
"this is how I envisioned synthetic biology would look": comment on Murray paper on a designed concentration tracker http://t.co/80i5zZRNGN
bySun ZZ, Yeung E, Hayes CA, Noireaux V, Murray RM."Accelerating the pace of synthetic biology experiments requires new approaches for rapid prototyping of circuits from individual DNA regulatory elements. However, current testing standards require days to weeks due to cloning and in vivo transformation. In this work, we first characterized methods to protect linear DNA strands from exonuclease degradation in an Escherichia coli based transcription-translation cell-free system (TX-TL), as well as mechanisms of degradation. This enabled the use of linear DNA PCR products in TX-TL. We then compared expression levels and binding dynamics of different promoters on linear DNA and plasmid DNA. We also demonstrated assembly technology to rapidly build circuits entirely in vitro from separate parts. Using this strategy, we prototyped a four component genetic switch in under 8 h entirely in vitro. Rapid in vitro assembly has future applications for prototyping multiple component circuits if combined with predictive computational models." http://bit.ly/1bOWl07
byMaryJoe K Rice and Warren C Ruder"Synthetic biology is a new discipline that combines science and engineering approaches to precisely control biological networks. These signaling networks are especially important in fields such as biomedicine and biochemical engineering. Additionally, biological networks can also be critical to the production of naturally occurring biological nanomaterials, and as a result, synthetic biology holds tremendous potential in creating new materials. This review introduces the field of synthetic biology, discusses how biological systems naturally produce materials, and then presents examples and strategies for incorporating synthetic biology approaches in the development of new materials. In particular, strategies for using synthetic biology to produce both organic and inorganic nanomaterials are discussed. Ultimately, synthetic biology holds the potential to dramatically impact biological materials science with significant potential applications in medical systems."http://bit.ly/1kn5g8d
DNA sequencing seems to be an eternal theme for human due to the desire of ascertaining the nature of life.
byRichard A. Stein"“Science is more than a body of knowledge, it’s a way of thinking,” remarked Carl Sagan, and probably his words were never more powerfully relevant than for portraying one of the newest biomedical felds, systems biology.A recent symposium inaugurating the depart- ment of systems biology at Columbia Univer- sity Medical Center comes at a very auspi- cious time, one in which biomedical sciences, chemistry, physics, engineering, bioinformat- ics, and computer sciences are converging to shape a vibrant new discipline.As Lee Goldman, M.D., M.P.H., execu- tive vice president for health and biomedical sciences at the Columbia University College of Physicians and Surgeons pointed out dur- ing the opening remarks, this new feld “rep- resents so much about our future.”Within a relatively short time, we have re- alized the possibility of sequencing and map- ping the genome of virtually any organism. Concomitantly, as increasingly sophisticated technologies allow whole-genome sequences to be completed within hours to days, navigat- ing the vast datasets has become the foremost challenge, opening a gap in our ability to un- derstand and interpret their significance....." http://bit.ly/18dDwmj
Metabolic modelling in the development of cell factories by synthetic biology
byPaula Tuulia Jouhten"Cell factories are commonly microbial organisms utilized for bioconversion of renewable resources to bulk or high value chemicals. Introduction of novel production pathways in chassis strains is the core of the development of cell factories by synthetic biology. Synthetic biology aims to create novel biological functions and systems not found in nature by combining biology with engineering. The workflow of the development of novel cell factories with synthetic biology is ideally linear which will be attainable with the quantitative engineering approach, high-quality predictive models, and libraries of well-characterized parts. Different types of metabolic models, mathematical representations of metabolism and its components, enzymes and metabolites, are useful in particular phases of the synthetic biology workflow. In this minireview, the role of metabolic modelling in synthetic biology will be discussed with a review of current status of compatible methods and models for the in silico design and quantitative evaluation of a cell factory."http://bit.ly/1gaBRk7
byYunzi Luo,Hua Huang,Jing Liang,Meng Wang,Lu Lu,Zengyi Shao,Ryan E. Cobb& Huimin Zhao"Polycyclic tetramate macrolactams (PTMs) are a widely distributed class of natural products with important biological activities. However, many of these PTMs have not been characterized. Here we apply a plug-and-play synthetic biology strategy to activate a cryptic PTM biosynthetic gene cluster SGR810-815 from Streptomyces griseus and discover three new PTMs. This gene cluster is highly conserved in phylogenetically diverse bacterial strains and contains an unusual hybrid polyketide synthase-nonribosomal peptide synthetase, which resembles iterative polyketide synthases known in fungi. To further characterize this gene cluster, we use the same synthetic biology approach to create a series of gene deletion constructs and elucidate the biosynthetic steps for the formation of the polycyclic system. The strategy we employ bypasses the traditional laborious processes to elicit gene cluster expression and should be generally applicable to many other silent or cryptic gene clusters for discovery and characterization of new natural products. http://bit.ly/IS5mcQcomment:*New method of DNA editing allows synthetic biologists to unlock secrets of a bacterial genome*byAnonymous"A group of University of Illinois researchers, led by Centennial Chair Professor of the Department of Chemical and Biomolecular Engineering Huimin Zhao, has demonstrated the use of an innovative DNA engineering technique to discover potentially valuable functions hidden within bacterial genomes. Their work was reported in a Nature Communications article on December 5, 2013.
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The ‘reading’ of DNA is a solved technological problem but what about ‘writing’ DNA? Could we program or reprogram biological systems and even generate new life forms? In this Friday Evening Discourse at the Ri, Paul Freemont explores how the powerful fusion of molecular biology, design and engineering could lead to a ‘Biotechnological Revolution’ and considers the implications of the extraordinary field of synthetic biology.
Scientists launch company to develop the therapeutic potential of gene-snipping enzymes
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