*Design criteria for synthetic riboswitches acting on transcription*
by Wachsmuth M, Domin G, Lorenz R, Serfling R, Findeiß S, Stadler PF, Mörl M.
"Riboswitches are RNA-based regulators of gene expression composed of a ligand-sensing aptamer domain followed by an overlapping expression platform. The regulation occurs at either the level of transcription (by formation of terminator or antiterminator structures) or translation (by presentation or sequestering of the ribosomal binding site). Due to a modular composition, these elements can be manipulated by combining different aptamers and expression platforms and therefore represent useful tools to regulate gene expression in synthetic biology. Using computationally designed theophylline-dependent riboswitches we show that 2 parameters, terminator hairpin stability and folding traps, have a major impact on the functionality of the designed constructs. These have to be considered very carefully during design phase. Furthermore, a combination of several copies of individual riboswitches leads to a much improved activation ratio between induced and uninduced gene activity and to a linear dose-dependent increase in reporter gene expression. Such serial arrangements of synthetic riboswitches closely resemble their natural counterparts and may form the basis for simple quantitative read out systems for the detection of specific target molecules in the cell."
"A method has been developed to produce and integrate single-stranded DNA into genomic locations in bacteria in response to exogenous signals. The system functions similarly to a cellular tape recorder by writing information into DNA and reading it at a later time. Much like other cellular memory platforms, its operation is based on DNA recombinase function. However, the scalability and recording capacity have been improved over previous designs. In addition, memory storage was reversible and could be recorded in response to analogue inputs, such as light exposure. This modular memory writing system is an important addition to the genomic editing toolbox available for synthetic biology."
Competing endogenous RNAs, which include mRNAs, transcribed pseudogenes, long noncoding RNAs (lncRNA), and circular RNA (circRNA), regulate other RNA transcripts by competing for shared microRNA
MicroRNA is a small non-coding RNA molecule containing about 22 nucleotides found in plants, animals, and some viruses, which functions in RNA silencing and post-transcriptional regulation of gene expression
Messenger RNA is a large family of RNA molecules that convey genetic information from DNA to the ribosome, where they specify the amino acid sequence of the protein products of gene expression
Pseudogenes are sections of a chromosome that are imperfect, dysfunctional copies of functional genes that have lost their protein-coding ability or are otherwise no longer expressed in the cell
Long noncoding RNA (lncRNA) comprises a large and diverse class of transcribed RNA molecules with a length of more than 200 nucleotides that do not encode proteins, and whose expression is developmentally regulated and that can be tissue- and cell-type specific
Circular RNA (circRNA) is a type of gene regulating noncoding RNA which, unlike the better-known linear RNA, forms a covalently closed continuous loop and that have not been shown to code for proteins
Small interfering RNA (siRNA), sometimes known as short interfering RNA or silencing RNA, is a class of double-stranded RNA molecules, 20-25 base pairs in length, that has many functions but is most notable in the RNA interference (RNAi) pathway where it interferes with the expression of specific genes with complementary nucleotide sequences..."
"DNA PRODUCTION IS becoming cheaper than ever, propelled down a Moore’s law curve by maturing technologies and cheaper reagents. This new biosynthetic industry allows researchers to order up a customized sequence for overnight delivery.
But many users don’t just want a chain of nucleotides, they want ready-to-use sequences that can be inserted into a cell to make a product of interest. Such DNA products, known as constructs, include two components – a vector that will be read by host machinery and initiate transcription, and an inserted gene that will generate the non-native biomolecule. Constructs can be thousands of bases in length, but once they’re uploaded to the cell, production should be good to go.
This niche is where Genscript is staking its claim. “We’re the world’s largest provider for construct based gene synthesis,” says Jeffrey Hung, a Genscript Vice President, “and a lot of our growth is coming from higher demand for biologics,” or medicinal biomolecules generated through a microbial host (as opposed to an exclusively chemical synthetic process). Most frequently, the company takes orders for non-native products to be expressed in a different organism, turning the unwitting target cell into a biofactory for recombinant proteins or antibodies. In many cases, biologics – the result of intentional expression of known biomolecules – are safer than uncharacterized but empirically promising small molecules taken from a cellular milieu. And using the cell as a production platform is an appealing prospect: organisms can tune behavior and metabolism to changing conditions, so small fluctuations in temperature or reactant concentration won’t doom a costly industrial process."
"Characteristics adapted from lizards, ivy and other natural materials could help to engineer everyday objects with remarkable properties.
made it so. Similarly, some scientists have gazed at geckos walking up walls and wondered whether humans could do the same. Now they can. In June 2014, a 100-kilogram man wearing a heavy pack climbed up a vertical sheet of glass using only a pair of hand-held paddles made from an advanced material inspired by geckos.
The synthetic gecko skin on the paddles has plenty of company in the world of materials science. Researchers are increasingly looking towards plants and animals for ideas on how to design coatings and textures that imbue surfaces with special properties. The adhesive that ivy uses to cling to walls, for example, has inspired a material that might help damaged tissues to regenerate. Molecules taken from mussel adhesives could provide a way to target cancer cells. And the veins on nasturtium leaves have led to the development of a synthetic surface that could prevent rain from freezing on aeroplane wings or keep grimy fingerprints off smartphone screens. The trick is to take ideas sparked by nature — some of them long in development, others brand new — and make them practical and durable......"
"Compartmentalisation of cellular processes is fundamental to regulation of metabolism in Eukaryotic organisms and is primarily provided by membrane-bound organelles. These organelles are dynamic structures whose membrane barriers are continually shaped, remodelled and scaffolded by a rich variety of highly sophisticated protein complexes. Towards the goal of bottom-up assembly of compartmentalised protocells in synthetic biology, we believe it will be important to harness and reconstitute the membrane shaping and sculpting characteristics of natural cells. We review different in vitro membrane models and how biophysical investigations of minimal systems combined with appropriate theoretical modelling have been used to gain new insights into the intricate mechanisms of these membrane nanomachines, paying particular attention to proteins involved in membrane fusion, fission and cytoskeletal scaffolding processes. We argue that minimal machineries need to be developed and optimised for employment in artificial protocell systems rather than the complex environs of a living organism. Thus, well-characterised minimal components might be predictably combined into functional, compartmentalised protocellular materials that can be engineered for wide-ranging applications."
"The development of novel technologies in the late 19th and early 20th century lead to the creation of major new industries such as the petrochemical, automotive, aviation, and electronic. These industries have improved the lives of billions of people around the globe1 and propelled civilization forward. During the second half of the 20th century the digital revolution changed the world yet again, with the rise of personal computers and the internet. According to the UK Royal Academy of Engineering, we are on the cusp of another revolution—this one based on synthetic biology1. The applications of synthetic biology are broad, ranging from renewable energy production to agriculture. One exciting application that will have profound implications on human health is medicine. This paper will discuss the advent of synthetic biology and its medical applications."
by Masoumeh Emadpour, Daniel Karcher and Ralph Bock
"Riboswitches are RNA sensors that regulate gene expression in response to binding of small molecules. Although they conceptually represent simple on/off switches and, therefore, hold great promise for biotechnology and future synthetic biology applications, the induction of gene expression by natural riboswitches after ligand addition or removal is often only moderate and, consequently, the achievable expression levels are not very high. Here, we have designed an RNA amplification-based system that strongly improves the efficiency of riboswitches. We have successfully implemented the method in a biological system for which currently no efficient endogenous tools for inducible (trans)gene expression are available: the chloroplasts of higher plants. We further show that an HIV antigen whose constitutive expression from the chloroplast genome is deleterious to the plant can be inducibly expressed under the control of the RNA amplification-enhanced riboswitch (RAmpER) without causing a mutant phenotype, demonstrating the potential of the method for the production of proteins and metabolites that are toxic to the host cell."
"Synthetic small RNA transcriptional activators can regulate gene transcription in Escherichia coli.
'Learn from nature and copy what it does' is one of the guiding principles in the laboratory of Julius Lucks at Cornell University, but in their recent work, the researchers developed a strategy that seemingly expands what nature has to offer.
“We want to leverage our ability to model and measure RNA structures to make gene networks,” says Lucks. His team focuses on transcriptional control, and they aim to have RNA inputs control RNA outputs without involving proteins such as transcription factors. “The big conceptual advantage of RNA over proteins is that you can do design,” explains Lucks. “We know a lot more about RNA folding than we do about protein folding.”
The strategy of the Lucks team has been to observe RNA design principles in nature, characterize their structure and then apply these designs to the engineering of genetic circuits. The limitation is that whereas nature very efficiently uses small RNAs to repress transcription, there are to date no known instances of small RNAs alone activating transcription. “But,” says Lucks, “if you want to build networks, you need to turn things on as well as off.”
Melissa Takahashi, a graduate student in the lab, first focused on characterizing the function of a natural RNA transcriptional repressor mechanism: a special sequence upstream of a gene's coding region that can form RNA structures that allow or prevent progression of the RNA polymerase. These structures are switchable: in one case transcription is stopped by a transcriptional terminator RNA hairpin, and in the other case transcription is allowed by an antiterminator sequence that sequesters the terminator and prevents the formation of the blocking hairpin. Takahashi looked at the structural transitions needed in order to undergo the switch from active to inactive transcription; she then came up with a strategy to invert this repression mechanism into one that activates transcription by adding yet another layer of structural transitions using a small transcription activating RNA (STAR)...."
Comment to: Creating small transcription activating RNAs
by James Chappell, Melissa K Takahashi & Julius B Lucks
"We expanded the mechanistic capability of small RNAs by creating an entirely synthetic mode of regulation: small transcription activating RNAs (STARs). Using two strategies, we engineered synthetic STAR regulators to disrupt the formation of an intrinsic transcription terminator placed upstream of a gene in Escherichia coli. This resulted in a group of four highly orthogonal STARs that had up to 94-fold activation. By systematically modifying sequence features of this group, we derived design principles for STAR function, which we then used to forward engineer a STAR that targets a terminator found in the Escherichia coli genome. Finally, we showed that STARs could be combined in tandem to create previously unattainable RNA-only transcriptional logic gates. STARs provide a new mechanism of regulation that will expand our ability to use small RNAs to construct synthetic gene networks that precisely control gene expression." http://bit.ly/1HjhZW7
What if you could design a house that would be encapsulated in a seed? Then to build that house you just had to plant the seed and add water. The Bio/Nano/Programmable Matter group at Autodesk Research is working to make this possible.
"OVER THE LAST several decades, DNA – the genetic material of life as we know it – has completed a remarkable scientific cycle. In 1953, it was a mysterious blur on an X-ray diffractogram. By the 1970s, it was possible to determine the sequence of short nucleotide chains. And now, a scientist can produce her own genetic code of choice with the click of a mouse.
What happens after the mouse click, after an order for a chain of DNA is sent, is an impressive series of events that represents one of the most mature, yet dynamic, sectors of the biotech industry. DNA synthesis companies range from scrappy start-ups to Cambridge-area behemoths, each touting a distinct set of tools that carves out a slice of the ever increasing pie.
For many groups, the human genome project – the $3 billion effort funded by the U.S. government – was an important launching point that both advanced DNA sequencing and synthesis technology and prompted important questions worthy of further scientific investigation. “We are a direct beneficiary of all the sequencing information that came out of the Project,” says Kevin Munnelly, CEO of Gen9, “and it’s all going to impact synthetic biology and our ability to write DNA.” Jerry Steele, the Director of Marketing for IDT, recalls that “the thing that really helped us take off was synthesizing the oligos for the human genome project. 10 or 15 years ago, it cost a few dollars per base to make oligos,” he recalls, “and now we’re down to a few cents.”
Several different industries are reaping the benefits, from agriculture to clean-tech to pharmaceuticals. Emily Leproust, CEO of Twist Bioscience, thinks the biochemical arms race between pathogens and pharmaceutical companies is worse than most people realize. With increasing antibiotic resistance and a diminished rate of new antibiotic discovery, “we’re going back to an era of pre-penicillin,” Leproust maintains, “and it will be a shock to people.” With affordable methods to produce alternative genes, regulatory structures, or even entire metabolic pathways now available, the range of possible products has grown exponentially. “Now we can make new candidates and new antibiotics that will enable us to start fighting back.”..."
by Zoltán Kis , Hugo Sant'Ana Pereira , Takayuki Homma , Ryan M. Pedrigi , Rob Krams
"In this review, we discuss new emerging medical applications of the rapidly evolving field of mammalian synthetic biology. We start with simple mammalian synthetic biological components and move towards more complex and therapy-oriented gene circuits. A comprehensive list of ON–OFF switches, categorized into transcriptional, post-transcriptional, translational and post-translational, is presented in the first sections. Subsequently, Boolean logic gates, synthetic mammalian oscillators and toggle switches will be described. Several synthetic gene networks are further reviewed in the medical applications section, including cancer therapy gene circuits, immuno-regulatory networks, among others. The final sections focus on the applicability of synthetic gene networks to drug discovery, drug delivery, receptor-activating gene circuits and mammalian biomanufacturing processes."
"Millions of years of evolution have made the biological world into a supremely effective materials-development laboratory. This Outlook examines the ways in which substances found in the natural world are inspiring imitations that might eventually endow humans with superhuman power..."
Genetically identical cells can have many variable properties. A study of correlations between cells in a lineage explains paradoxical inheritance laws, in which mother and daughter cells seem less similar than cousins. See Letter p.468
"Quorum-sensing networks enable bacteria to sense and respond to chemical signals produced by neighboring bacteria. They are widespread: over 100 morphologically and genetically distinct species of eubacteria are known to use quorum sensing to control gene expression. This diversity suggests the potential to use natural protein variants to engineer parallel, input-specific, cell–cell communication pathways. However, only three distinct signaling pathways, Lux, Las, and Rhl, have been adapted for and broadly used in engineered systems. The paucity of unique quorum-sensing systems and their propensity for crosstalk limits the usefulness of our current quorum-sensing toolkit. This review discusses the need for more signaling pathways, roadblocks to using multiple pathways in parallel, and strategies for expanding the quorum-sensing toolbox for synthetic biology."
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