Biology subjects

Amit, R.

Publications and source records attributed to Amit, R..

6 recordsLinked to original sources

bioRxiv2018

Improved DNA based storage capacity and fidelity using composite DNA letters

DNA, with its remarkable density and long-term stability, is an appealing potential next generation data storage medium, most notably for long-term archiving. Megabyte scale DNA based storage was first reported in 2012. The Shannon information capacity of DNA was recently demonstrated, using fountain codes, to be [~]1.57 bit per synthesized position. However, synthesis and sequencing technologies process multiple nominally identical molecules in parallel, leading to significant information redundancies. We introduce composite DNA alphabets, using mixed DNA base types, to leverage this redundancy, enabling higher density. We develop encoding and decoding for composite DNA based storage, including error correction. Using current DNA synthesis technologies, we code 6.4 Megabyte data into composite DNA, achieving [~]25% increase in capacity as compared to literature. We further demonstrate, on smaller scales, how flexible synthesis leads to 2.7 fold increased capacity per synthesized position. Composite DNA can thus reduce costs for DNA based storage and can also serve in other applications.

synthetic biology↗

bioRxiv2017

Synthetic protein-sensing riboswitches

We study translational regulation by a 5 UTR sequence encoding the binding site of an RNA-binding protein (RBP) in bacteria, using a reporter assay and Selective 2-hydroxyl acylation analysed by primer extension sequencing (SHAPE-Seq). We tested constructs containing a single hairpin, based on the binding sites of the coat RBPs of bacteriophages GA, MS2, PP7, and Q{beta}, positioned in the 5 UTR of a reporter gene. With specifically-bound RBP present, either weak repression or up-regulation is observed, depending on the binding site and its flanking sequence. SHAPE-Seq data for a representative construct exhibiting up-regulation, indicates a partially-folded hairpin and non-reactive upstream and downstream flanking region, which we attribute to intermediate structures that apparently blocks translation. RBP binding stabilizes the fully-folded hairpin state and thus facilitates translation, suggesting that the up-regulating constructs are RBP-sensing riboswitches. This finding is further supported by lengthening the binding-site stem, which in turn destabilizes the translationally-inactive state, and abolishes the up-regulating behavior. Finally, we found that the combination of two binding sites, positioned in the 5 UTR and gene-header of the same transcript, can yield a cooperative regulatory response. Together, we show that the interaction of an RBP with its RNA target facilitates structural changes in the RNA, which is reflected by a controllable range of binding affinities and dose response behaviors. Thus, demonstrating that RNA-RBP interactions can provide a platform for constructing gene regulatory networks that are based on translational, rather than transcriptional, regulation.

synthetic biology↗

bioRxiv2017

An in vivo binding assay for RNA-binding proteins based on repression of a reporter gene

We employ a reporter assay and Selective 2'-hydroxyl acylation analysed by primer extension sequencing (SHAPE-seq) to study translational regulation by RNA-binding proteins, in bacteria. We designed 82 constructs, each with a single hairpin based on the binding sites of the RNA-binding coat proteins of phages MS2, PP7, GA, and Q{beta}, at various positions within the N-terminus of a reporter gene. In the absence of RNA-binding proteins, the translation level depends on hairpin location, and exhibits a three-nucleotide periodicity. For hairpin positions within the initiation region, we observe strong translational repression in the presence of its cognate RNA-binding protein. In vivo SHAPE-seq results for a representative construct indicate that the repression phenomenon correlates with a wide-swath of protection, including the hairpin and extending past the ribosome binding site. Consequently, our data suggest that the protection provided by the RBP-hairpin complex inhibits ribosomal initiation. Finally, utilizing the repression phenomenon for quantifying protein-RNA binding affinity in vivo, we both observe partially contrasting results to previous in vitro and in situ studies, and additionally, show that this method can be used in a high-throughput assay for a quantitative study of protein-RNA binding in vivo.

synthetic biology↗

bioRxiv2017

Demonstration of de novo chemotaxis in E. coli using a real-time, quantitative, and digital-like approach

Chemotaxis is the movement of an organism in response to an external chemical stimulus. This system enables bacteria to sense their immediate environment and adapt to changes in its chemical composition. Bacterial chemotaxis is mediated by chemoreceptors, membrane proteins that bind an effector and transduce the signal to the downstream proteins. From a synthetic biology perspective, the natural chemotactic repertoire is of little use since bacterial chemoreceptors have evolved to sense specific ligands that either benefit or harm the cell. Here we demonstrate that using a combined computational design approach together with a quantitative, real-time, and digital detection approach, we can rapidly design, manufacture, and characterize a synthetic chemoreceptor in E. coli for histamine (a ligand for which there are no known chemoreceptors). First, we employed a computational protocol that uses the Rosetta bioinformatics software together with high threshold filters to design mutational variants to the native Tar ligand binding domain that target histamine. Second, we tested different ligand-chemoreceptors pairs with a novel chemotaxis assay, based on optical reflectance interferometry of porous silicon (PSi) optical transducers, enabling label-free quantification of chemotaxis by monitoring real-time changes in the optical readout (expressed as the effective optical thickness, EOT). We found that different ligands can be characterized by an individual set of fingerprints in our assay. Namely, a binary, digital-like response in EOT change (i.e. positive or negative) that differentiates between attractants and repellants, the amplitude of change of EOT response, and the rate by which steady state in EOT change is reached. Using this assay, we were able to positively identify and characterize a single mutational chemoreceptor variant for histamine that mediated chemotaxis comparably to the natural Tar-aspartate system. Our results demonstrate the possibility of not only expanding the natural chemotaxis repertoire, but also provide a new quantitative assay by which to characterize the efficacy of the chemotactic response.

synthetic biology↗

bioRxiv2016

Short CT-rich motifs can trigger context-specific silencing of gene expression in bacteria

We use an oligonucleotide library of over 10000 variants together with a synthetic biology approach to identify an insulation mechanism encoded within a subset of {sigma}54 promoters. Insulation manifests itself as dramatically reduced protein expression for a downstream gene that may be expressed by transcriptional read-through. The insulation we observe is strongly associated with the presence of short CT-rich motifs (3-5 bp), positioned within 25 bp upstream of the Shine-Dalgarno (SD) motif of the silenced gene. We hypothesize that insulation is effected by binding of the RBS to the upstream CT-rich motif. We provide evidence to support this hypothesis using mutations to the CT-rich motif and gene expression measurements on multiple sequence variants. Modelling is also consistent with this hypothesis. We show that the strength of the silencing, effected by insulation, depends on the location and number of CT-rich motifs encoded within the promoters. Finally, we show that in E.coli these insulator sequences are preferentially encoded within {sigma}54 promoters as compared to other promoter types, suggesting a regulatory role for these sequences in natural contexts. Our findings suggest that context-related regulatory effects may often be due to sequence-specific interactions encoded sparsely by short motifs that are not easily detected by lower throughput studies. Such short sequence-specific phenomena can be uncovered with a focused OL design that filters out the sequence noise, as exemplified herein.

systems biology↗

bioRxiv2016

A looping-based model for quenching repression

We model the regulatory role of proteins bound to looped DNA using a simulation in which dsDNA is represented as a self-avoiding chain, and proteins as spherical protrusions. We simu-late long self-avoiding chains using a sequential importance sampling Monte-Carlo algorithm, and compute the probabilities for chain looping with and without a protrusion. We find that a protrusion near one of the chains termini reduces the probability of looping, even for chains much longer than the protrusion-chain-terminus distance. This effect increases with protrusion size, and decreases with protrusion-terminus distance. The reduced probability of looping can be explained via an eclipse-like model, which provides a novel inhibitory mechanism. We test the eclipse model on two possible transcription-factor occupancy states of the eve 3/7 enhancer, and show that it provides a possible explanation for the experimentally-observed eve stripe 3 and 7 expression patterns.\n\nThe authors declare no conflict of interests

biophysics↗