Abstract:
:Multitargeting small regulatory RNAs (sRNAs) represent a potentially useful tool for metabolic engineering applications. Natural multitargeting sRNAs govern bacterial gene expression by binding to the translation initiation regions of protein-coding mRNAs through base pairing. We designed an Escherichia coli based genetic system to create and assay dual-acting retargeted-sRNA variants. The variants can be assayed for coordinate translational regulation of two alternate mRNA leaders fused to independent reporter genes. Accordingly, we began with the well-characterized E. coli native DsrA sRNA. The merits of using DsrA include its well-characterized separation of function into two independently folded stem-loop domains, wherein alterations at one stem do not necessarily abolish activity at the other stem. Expression of the sRNA and each reporter mRNA was independently controlled by small inducer molecules, allowing precise quantification of the regulatory effects of each sRNA:mRNA interaction in vivo with a microtiter plate assay. Using this system, we semirationally designed DsrA variants screened in E. coli for their ability to regulate key mRNA leader sequences from the Clostridium acetobutylicum n-butanol synthesis pathway. To coordinate intervention at two points in a metabolic pathway, we created bifunctional sRNA prototypes by combining sequences from two singly retargeted DsrA variants. This approach constitutes a platform for designing sRNAs to specifically target arbitrary mRNA transcript sequences, and thus provides a generalizable tool for retargeting and characterizing multitarget sRNAs for metabolic engineering.
journal_name
ACS Synth Bioljournal_title
ACS synthetic biologyauthors
Lahiry A,Stimple SD,Wood DW,Lease RAdoi
10.1021/acssynbio.6b00261subject
Has Abstractpub_date
2017-04-21 00:00:00pages
648-658issue
4issn
2161-5063journal_volume
6pub_type
杂志文章abstract::Mycoplasma hominis is a minimal human pathogen that is responsible for genital and neonatal infections. Despite many attempts, there is no efficient genetic tool to manipulate this bacterium, limiting most investigations of its pathogenicity and its uncommon energy metabolism that relies on arginine. The recent clonin...
journal_title:ACS synthetic biology
pub_type: 杂志文章
doi:10.1021/acssynbio.6b00379
更新日期:2017-05-19 00:00:00
abstract::Bacterial biofilms are three-dimensional networks of cells entangled in a self-generated extracellular polymeric matrix composed of proteins, lipids, polysaccharides, and nucleic acids. Biofilms can establish themselves on virtually any accessible surface and lead to varying impacts ranging from infectious diseases to...
journal_title:ACS synthetic biology
pub_type: 杂志文章
doi:10.1021/acssynbio.9b00192
更新日期:2019-07-19 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::Increasing protein expression levels is a key step in the commercial production of enzymes. Predicting promoter activity and translation initiation efficiency based solely on consensus sequences have so far met with mixed results. Here, we addressed this challenge using a "brute-force" approach by designing and synthe...
journal_title:ACS synthetic biology
pub_type: 杂志文章
doi:10.1021/acssynbio.8b00110
更新日期:2018-07-20 00:00:00
abstract::Characterization of genetic control elements is essential for the predictable engineering of synthetic biology systems. The current standard for in vivo characterization of control elements is through the use of fluorescent reporter proteins such as green fluorescent protein (GFP). Gene expression, however, involves n...
journal_title:ACS synthetic biology
pub_type: 杂志文章
doi:10.1021/sb400089c
更新日期:2014-03-21 00:00:00
abstract::Cell-free systems provide a versatile platform for the development of low-cost, easy-to-use sensors for diverse analytes. However, sensor affinity dictates response sensitivity, and improving binding affinity can be challenging. Here, we describe efforts to address this problem while developing a biosensor for vitamin...
journal_title:ACS synthetic biology
pub_type: 杂志文章
doi:10.1021/acssynbio.9b00429
更新日期:2020-02-21 00:00:00
abstract::In response to emergent antibiotic resistance, new strategies are needed to enhance the effectiveness of existing antibiotics. Here, we describe a phagemid-delivered, RNA-mediated system capable of directly knocking down antibiotic resistance phenotypes. Small regulatory RNAs (sRNAs) were designed to specifically inhi...
journal_title:ACS synthetic biology
pub_type: 杂志文章
doi:10.1021/sb500033d
更新日期:2014-12-19 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 streamlined approach toward the rapid fabrication of streptavidin-biotin-based protein microarrays was investigated. First, using our engineered versatile plasmid (pBADcM-tBirA) and an optimal coexpression strategy for biotin ligase and biotin acceptor peptide (BAP) chimeric recombinant protein, an autogeneration sy...
journal_title:ACS synthetic biology
pub_type: 杂志文章
doi:10.1021/acssynbio.0c00343
更新日期:2020-09-18 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::Fucosyl-oligosaccharides (FOSs) play physiologically important roles as prebiotics, neuronal growth factors, and inhibitors of enteropathogens. However, challenges in designed synthesis and mass production of FOSs hamper their industrial applications. Here, we report flexible biosynthetic routes to produce various FOS...
journal_title:ACS synthetic biology
pub_type: 杂志文章
doi:10.1021/acssynbio.8b00436
更新日期:2019-02-15 00:00:00
abstract::We developed a hybrid synthetic circuit that co-opts the genetic regulation of the native bacterial quorum sensing autoinducer-2 and imposes an extra external controller for maintaining tightly controlled gene expression. This dual-input genetic controller was mathematically modeled and, by design, can be operated in ...
journal_title:ACS synthetic biology
pub_type: 杂志文章
doi:10.1021/acssynbio.0c00179
更新日期:2020-10-16 00:00:00
abstract::A bacterial flavin transferase (ApbE) was recently employed for flavin mononucleotide (FMN) modification on the Na+-translocating NADH:quinone oxidoreductase C (NqrC) protein in the pathogenic Gram-negative bacterium Vibrio cholerae. We employed this unique post-translational modification in mammalian cells and found ...
journal_title:ACS synthetic biology
pub_type: 杂志文章
doi:10.1021/acssynbio.6b00284
更新日期:2017-04-21 00:00:00
abstract::The ability to rapidly design, build, and test prototypes is of key importance to every engineering discipline. DNA assembly often serves as a rate limiting step of the prototyping cycle for synthetic biology. Recently developed DNA assembly methods such as isothermal assembly and type IIS restriction enzyme systems t...
journal_title:ACS synthetic biology
pub_type: 杂志文章
doi:10.1021/acssynbio.8b00060
更新日期:2018-05-18 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::Quantification of biology's central dogma (transcription and translation) is pursued by a variety of methods. Direct, immediate, and ongoing quantification of these events is difficult to achieve. Common practice is to use fluorescent or luminescent proteins to report indirectly on prior cellular events, such as turni...
journal_title:ACS synthetic biology
pub_type: 杂志文章
doi:10.1021/acssynbio.8b00450
更新日期:2019-05-17 00:00:00
abstract::Materials synthesized by organisms, such as bones and wood, combine the ability to self-repair with remarkable mechanical properties. This multifunctionality arises from the presence of living cells within the material and hierarchical assembly of different components across nanometer to micron scales. While creating ...
journal_title:ACS synthetic biology
pub_type: 杂志文章
doi:10.1021/acssynbio.8b00448
更新日期:2019-01-18 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
abstract::Multidrug resistance (MDR) is a globally relevant problem that requires novel approaches. Two-component systems are a promising, yet untapped target for novel antibacterials. They are prevalent in bacteria and absent in mammals, and their activity can be modulated upon perception of various stimuli. Screening pre-exis...
journal_title:ACS synthetic biology
pub_type: 杂志文章
doi:10.1021/acssynbio.6b00288
更新日期:2017-07-21 00:00:00
abstract::As a product of a multistep enzymatic reaction, accumulation of poly(3-hydroxybutyrate) (PHB) in Escherichia coli (E. coli) can be achieved by overexpression of the PHB synthesis pathway from a native producer involving three genes phbC, phbA, and phbB. Pathway optimization by adjusting expression levels of the three ...
journal_title:ACS synthetic biology
pub_type: 杂志文章
doi:10.1021/acssynbio.6b00083
更新日期:2016-11-18 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::The versatility of Ca2+ signals allows it to regulate diverse cellular processes such as migration, apoptosis, motility and exocytosis. In some receptors (e.g., VEGFR2), Ca2+ signals are generated upon binding their ligand(s) (e.g., VEGF-A). Here, we employed a design strategy to engineer proteins that generate a Ca2+...
journal_title:ACS synthetic biology
pub_type: 杂志文章
doi:10.1021/acssynbio.6b00310
更新日期:2017-03-17 00:00:00
abstract::The Sc2.0 project is perhaps the largest synthetic biology project in the public domain, and ultimately aims to construct a new version of the humble brewer's yeast, Saccharomyces cerevisiae. Each year, the Sc2.0 consortium gather to discuss progress in this ambitious project and highlight new developments at the fore...
journal_title:ACS synthetic biology
pub_type: 杂志文章,评审
doi:10.1021/acssynbio.6b00227
更新日期:2016-09-16 00:00:00
abstract::The cell contents are encapsulated within a compartment, the volume of which is a fundamental physical parameter that may affect intracompartmental reactions. However, there have been few studies to elucidate whether and how volume changes alone can affect the reaction kinetics. It is difficult to address these questi...
journal_title:ACS synthetic biology
pub_type: 杂志文章
doi:10.1021/sb300041z
更新日期:2012-09-21 00:00:00
abstract::The deeply red-colored natural compound prodigiosin is a representative of the prodiginine alkaloid family, which possesses bioactivities as antimicrobial, antitumor, and antimalarial agents. Various bacteria including the opportunistic human pathogen Serratia marcescens and different members of the Streptomycetaceae ...
journal_title:ACS synthetic biology
pub_type: 杂志文章
doi:10.1021/acssynbio.7b00099
更新日期:2017-09-15 00:00:00
abstract::While inducing agents are often used to redirect resources from growth and proliferation toward product outputs, they can be prohibitively expensive on the industrial scale. Previously, we developed an autonomously guided protein production system based on the rewiring of E. coli's native quorum sensing (QS) signal tr...
journal_title:ACS synthetic biology
pub_type: 杂志文章
doi:10.1021/acssynbio.5b00261
更新日期:2016-09-16 00:00:00
abstract::Improved plants are necessary to meet human needs. Agrobacterium-mediated transformation is the most common method used to rewire plant capabilities. For plant gene delivery, DNA constructs are assembled into binary T-DNA vectors that rely on broad host range origins for bacterial replication. Here we present pLX vect...
journal_title:ACS synthetic biology
pub_type: 杂志文章
doi:10.1021/acssynbio.6b00354
更新日期:2017-10-20 00:00:00
abstract::Directed evolution requires the creation of genetic diversity and subsequent screening or selection for improved variants. For DNA mutagenesis, conventional site-directed methods implicitly utilize the Boolean AND operator (creating all mutations simultaneously), producing a combinatorial explosion in the number of ge...
journal_title:ACS synthetic biology
pub_type: 杂志文章
doi:10.1021/acssynbio.9b00063
更新日期:2019-06-21 00:00:00
abstract::The structural complexity of nonribosomal peptides (NRPs) impeding economic chemical synthesis and poor cultivability of source organisms limits the development of bioprocesses for novel bioactive compounds. Since nonribosomal peptide synthetases (NRPSs) assemble NRPs from simple amino acid building blocks, heterologo...
journal_title:ACS synthetic biology
pub_type: 杂志文章
doi:10.1021/sb400082j
更新日期:2014-07-18 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