Abstract：
:We study translation repression in bacteria by engineering a regulatory circuit that functions as a binding assay for RNA binding proteins (RBP) in vivo. We do so by inducing expression of a fluorescent protein-RBP chimera, together with encoding its binding site at various positions within the ribosomal initiation region (+11-13 nt from the AUG) of a reporter module. We show that when bound by their cognate RBPs, the phage coat proteins for PP7 (PCP) and Qβ (QCP), strong repression is observed for all hairpin positions within the initiation region. Yet, a sharp transition to no-effect is observed when positioned in the elongation region, at a single-nucleotide resolution. Employing in vivo Selective 2'-hydroxyl acylation analyzed by primer extension followed by sequencing (SHAPE-seq) for a representative construct, established that in the translationally active state the mRNA molecule is nonstructured, while in the repressed state a structured signature was detected. We then utilize this regulatory phenomena to quantify the binding affinity of the coat proteins of phages MS2, PP7, GA, and Qβ to 14 cognate and noncognate binding sites in vivo. Using our circuit, we demonstrate qualitative differences between in vitro to in vivo binding characteristics for various variants when comparing to past studies. Furthermore, by introducing a simple mutation to the loop region for the Qβ-wt site, MCP binding is abolished, creating the first high-affinity QCP site that is completely orthogonal to MCP. Consequently, we demonstrate that our hybrid transcriptional-post-transcriptional circuit can be utilized as a binding assay to quantify RNA-RBP interactions in vivo.
journal_name
ACS Synth Bioljournal_title
ACS synthetic biologyauthors
Katz N,Cohen R,Solomon O,Kaufmann B,Atar O,Yakhini Z,Goldberg S,Amit Rdoi
10.1021/acssynbio.8b00378subject
Has Abstractpub_date
2018-12-21 00:00:00pages
2765-2774issue
12issn
2161-5063journal_volume
7pub_type
杂志文章abstract：:Simple computation can be performed using the interactions between single-stranded molecules of DNA. These interactions are typically toehold-mediated strand displacement reactions in a well-mixed solution. We demonstrate that a DNA circuit with tethered reactants is a distributed system and show how it can be describ...
journal_title：ACS synthetic biology
pub_type： 杂志文章
doi：10.1021/acssynbio.5b00275
更新日期：2016-08-19 00:00:00
abstract：:Although Escherichia coli has been a popular tool for plasmid construction, this bacterium was believed to be "unsuitable" for constructing a large plasmid whose size exceeds 500 kilobases. We assumed that traditional plasmid vectors may lack some regulatory DNA elements required for the stable replication and segrega...
journal_title：ACS synthetic biology
pub_type： 杂志文章
doi：10.1021/acssynbio.0c00008
更新日期：2020-06-19 00:00:00
abstract：:The microbial ability to resist stressful environmental conditions and chemical inhibitors is of great industrial and medical interest. Much of the data related to mutation-based stress resistance, however, is scattered through the academic literature, making it difficult to apply systematic analyses to this wealth of...
journal_title：ACS synthetic biology
pub_type： 杂志文章
doi：10.1021/acssynbio.6b00150
更新日期：2016-12-16 00:00:00
abstract：:We recently developed a technique for rapidly and reversibly inhibiting protein function through light-inducible sequestration of proteins away from their normal sites of action. Here, we adapt this method for inducible inactivation of Bem1, a scaffold protein involved in budding yeast polarity. We find that acute inh...
journal_title：ACS synthetic biology
pub_type： 杂志文章
doi：10.1021/acssynbio.5b00053
更新日期：2015-10-16 00:00:00
abstract：:Staphylococci are prevalent skin-dwelling bacteria that are also leading causes of antibiotic-resistant infections. Viruses that infect and lyse these organisms (virulent staphylococcal phages) can be used as alternatives to conventional antibiotics and represent promising tools to eliminate or manipulate specific spe...
journal_title：ACS synthetic biology
pub_type： 杂志文章
doi：10.1021/acssynbio.7b00240
更新日期：2017-12-15 00:00:00
abstract：:To facilitate the construction of cell-free genetic devices, we evaluated the ability of 17 different fluorescent proteins to give easily detectable fluorescence signals in real-time from in vitro transcription-translation reactions with a minimal system consisting of T7 RNA polymerase and E. coli translation machiner...
journal_title：ACS synthetic biology
pub_type： 杂志文章
doi：10.1021/sb400003y
更新日期：2013-09-20 00:00:00
abstract：:Exploiting the ability of proteins to self-assemble into architectural templates may provide novel routes for the positioning of functional molecules in nanotechnology. Here we report the engineering of multicomponent protein templates composed of distinct monomers that assemble in repeating orders into a dynamic func...
journal_title：ACS synthetic biology
pub_type： 杂志文章
doi：10.1021/acssynbio.8b00241
更新日期：2018-10-19 00:00:00
abstract：:Engineered genetic systems are commonly unstable; if propagated, they evolve to reverse or modify engineered elements because the elements impair fitness. A goal of synthetic biology is thus to anticipate and avoid detrimental engineering, but little is yet known about which types of elements cause problems in differe...
journal_title：ACS synthetic biology
pub_type： 杂志文章
doi：10.1021/sb300040v
更新日期：2012-09-21 00:00:00
abstract：:We have previously engineered green/red and red/far red photoreversible E. coli phytochrome and cyanobacteriochrome (CBCR) two-component systems (TCSs) and utilized them to program tailor-made gene expression signals for gene circuit characterization. Here, we transport the UV-violet/green photoreversible CBCR TCS Uir...
journal_title：ACS synthetic biology
pub_type： 杂志文章
doi：10.1021/acssynbio.6b00068
更新日期：2016-07-15 00:00:00
abstract：:A gene-directed chemical communication pathway between synthetic protocell signaling transmitters (lipid vesicles) and receivers (proteinosomes) was designed, built and tested using a bottom-up modular approach comprising small molecule transcriptional control, cell-free gene expression, porin-directed efflux, substra...
journal_title：ACS synthetic biology
pub_type： 信件
doi：10.1021/acssynbio.7b00306
更新日期：2018-02-16 00:00:00
abstract：:A major force shaping form and patterns in biology is based in the presence of amplification mechanisms able to generate ordered, large-scale spatial structures out of local interactions and random initial conditions. Turing patterns are one of the best known candidates for such ordering dynamics, and their existence ...
journal_title：ACS synthetic biology
pub_type： 杂志文章
doi：10.1021/acssynbio.5b00254
更新日期：2016-07-15 00:00:00
abstract：:Site directed RNA editing is an engineered tool for the posttranscriptional manipulation of RNA and proteins. Here, we demonstrate the inclusion of additional N- and C-terminal protein domains in an RNA editing-dependent manner to switch between protein isoforms in mammalian cell culture. By inclusion of localization ...
journal_title：ACS synthetic biology
pub_type： 杂志文章
doi：10.1021/acssynbio.7b00113
更新日期：2017-09-15 00:00:00
abstract：:The photoreceptor cryptochrome 2 (CRY2) has become a powerful optogenetic tool that allows light-inducible manipulation of various signaling pathways and cellular processes in mammalian cells with high spatiotemporal precision and ease of application. However, it has also been shown that the behavior of CRY2 under blu...
journal_title：ACS synthetic biology
pub_type： 杂志文章
doi：10.1021/acssynbio.5b00048
更新日期：2015-10-16 00:00:00
abstract：:Nanostructures formed by self-assembled peptides have been increasingly exploited as functional materials for a wide variety of applications, from biotechnology to energy. However, it is sometimes challenging to assemble free short peptides into functional supramolecular structures, since not all peptides have the abi...
journal_title：ACS synthetic biology
pub_type： 杂志文章
doi：10.1021/acssynbio.0c00415
更新日期：2020-12-18 00:00:00
abstract：:Transcription activator-like effectors (TALEs) are proteins secreted by Xanthomonas bacteria to aid the infection of plant species. TALEs assist infections by binding to specific DNA sequences and activating the expression of host genes. Recent results show that TALE proteins consist of a central repeat domain, which ...
journal_title：ACS synthetic biology
pub_type： 杂志文章,评审
doi：10.1021/sb400137b
更新日期：2014-10-17 00:00:00
abstract：:Cell-free systems, as part of the synthetic biology field, have become a critical platform in biological studies. However, there is a lack of research into developing a switch for a dynamical control of the transcriptional and translational process. The optogenetic tool has been widely proven as an ideal control switc...
journal_title：ACS synthetic biology
pub_type： 杂志文章
doi：10.1021/acssynbio.0c00211
更新日期：2020-08-21 00:00:00
abstract：:Scarless genetic manipulation of genomes is an essential tool for biological research. The restriction-modification (R-M) system is a defense system in bacteria that protects against invading genomes on the basis of its ability to distinguish foreign DNA from self DNA. Here, we designed an R-M system-mediated genome e...
journal_title：ACS synthetic biology
pub_type： 杂志文章
doi：10.1021/acssynbio.7b00254
更新日期：2018-01-19 00:00:00
abstract：:There is a growing interest in mediating information transfer between biology and electronics. By the addition of redox mediators to various samples and cells, one can both electronically obtain a redox "portrait" of a biological system and, conversely, program gene expression. Here, we have created a cell-based synth...
journal_title：ACS synthetic biology
pub_type： 杂志文章
doi：10.1021/acssynbio.9b00469
更新日期：2020-05-15 00:00:00
abstract：:Phenazine-1-carboxamide (PCN) produced by multifarious Pseudomonas strains represents a promising candidate as a new metabolite pesticide due to its broad-spectrum antifungal activity and capacity to induce systemic resistance in plants. The rice rhizosphere Pseudomonas strain PA1201 contains two reiterated gene clust...
journal_title：ACS synthetic biology
pub_type： 杂志文章
doi：10.1021/acssynbio.0c00161
更新日期：2020-07-17 00:00:00
abstract：:2-Pyrone-4,6-dicarboxylic acid (PDC) is a pseudoaromatic dicarboxylic acid and is a promising biobased building block chemical that can be used to make diverse polyesters with novel functionalities. In this study, Escherichia coli was metabolically engineered to produce PDC from glucose. First, an efficient biosynthet...
journal_title：ACS synthetic biology
pub_type： 杂志文章
doi：10.1021/acssynbio.8b00281
更新日期：2018-09-21 00:00:00
abstract：:Microorganisms often use specific autoinducers other than common metabolites for quorum sensing (QS). Herein, we demonstrated that Escherichia coli produced sulfide (H2S, HS-, and S2-) with the concentrations proportionally correlated to its cell density. We then designed synthetic gene circuits that used H2S as an au...
journal_title：ACS synthetic biology
pub_type： 杂志文章
doi：10.1021/acssynbio.9b00210
更新日期：2019-09-20 00:00:00
abstract：:Movement of molecules across membranes in response to a stimulus is a key component of cellular programming. Here, we characterize and manipulate the response of a protein-based piston capable of puncturing membranes in a pH-dependent manner. Our protein actuator consists of modified R bodies found in a bacterial endo...
journal_title：ACS synthetic biology
pub_type： 信件
doi：10.1021/acssynbio.5b00237
更新日期：2016-04-15 00:00:00
abstract：:Medium-chain fatty acids (MCFAs) are key intermediates in the synthesis of medium-chain chemicals including α-olefins and dicarboxylic acids. In bacteria, microbial production of MCFAs is limited by the activity and product profile of fatty acyl-ACP thioesterases. Here, we engineer a heterologous bacterial medium-chai...
journal_title：ACS synthetic biology
pub_type： 杂志文章
doi：10.1021/acssynbio.7b00334
更新日期：2018-05-18 00:00:00
abstract：:Synthetic biology relies on the manufacture of large and complex DNA constructs from libraries of genetic parts. Golden Gate and other Type IIS restriction enzyme-dependent DNA assembly methods enable rapid construction of genes and operons through one-pot, multifragment assembly, with the ordering of parts determined...
journal_title：ACS synthetic biology
pub_type： 杂志文章
doi：10.1021/acssynbio.8b00333
更新日期：2018-11-16 00:00:00
abstract：:Agricultural soils are often cocontaminated with multiple pesticides. Unfortunately, microorganisms isolated from natural environments do not possess the ability to simultaneously degrade different classes of pesticides. Currently, we can use the approaches of synthetic biology to create a strain endowed with various ...
journal_title：ACS synthetic biology
pub_type： 杂志文章
doi：10.1021/acssynbio.6b00025
更新日期：2016-05-20 00:00:00
abstract：:Cell-free gene expression systems are emerging as an important platform for a diverse range of synthetic biology and biotechnology applications, including production of robust field-ready biosensors. Here, we combine programmed cellular autolysis with a freeze-thaw or freeze-dry cycle to create a practical, reproducib...
journal_title：ACS synthetic biology
pub_type： 杂志文章
doi：10.1021/acssynbio.7b00253
更新日期：2017-12-15 00:00:00
abstract：:Type I modular polyketide synthases (PKSs) are polymerases that utilize acyl-CoAs as substrates. Each polyketide elongation reaction is catalyzed by a set of protein domains called a module. Each module usually contains an acyltransferase (AT) domain, which determines the specific acyl-CoA incorporated into each conde...
journal_title：ACS synthetic biology
pub_type： 杂志文章
doi：10.1021/acssynbio.6b00176
更新日期：2017-01-20 00:00:00
abstract：:Heterochiral DNA strand-displacement reactions enable sequence-specific interfacing of oligonucleotide enantiomers, making it possible to interface native d-nucleic acids with molecular circuits built using nuclease-resistant l-DNA. To date, all heterochiral reactions have relied on peptide nucleic acid (PNA), which p...
journal_title：ACS synthetic biology
pub_type： 杂志文章
doi：10.1021/acssynbio.9b00335
更新日期：2019-12-20 00:00:00
abstract：:Author: Recently, increasing research in siderophores has been dedicated to their possible medical applications in diagnostics and therapeutics for human pathogenic infections. Fusarinine C (FsC) is a natural hydroxamate siderophore that harbors three amino groups, which allow the easy chemical modification of FsC for...
journal_title：ACS synthetic biology
pub_type： 杂志文章
doi：10.1021/acssynbio.9b00026
更新日期：2019-08-16 00:00:00
abstract：:Episodic gene expression, with periods of high expression separated by periods of no expression, is a pervasive biological phenomenon. This bursty pattern of expression draws from a finite reservoir of expression machinery in a highly time variant way, i.e., requiring no resources most of the time but drawing heavily ...
journal_title：ACS synthetic biology
pub_type： 杂志文章
doi：10.1021/acssynbio.6b00189
更新日期：2017-02-17 00:00:00
