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 segregation of such a large plasmid. In addition, the use of a few site-specific recombination systems may facilitate cloning of large DNA segments. Here we show two strategies for constructing 1-megabase (1-Mb) secondary chromosomes by using new bacterial artificial chromosome (BAC) vectors. First, the 3-Mb genome of a genome-reduced E. coli strain was split into two chromosomes (2-Mb and 1-Mb), of which the smaller one has the origin of replication and the partitioning locus of the Vibrio tubiashii secondary chromosome. This chromosome fission method (Flp-POP cloning) works via flippase-mediated excision, which coincides with the reassembly of a split chloramphenicol resistance gene, allowing chloramphenicol selection. Next, we developed a new cloning method (oriT-POP cloning) and a fully equipped BAC vector (pMegaBAC1H) for developing a 1-Mb plasmid. Two 0.5-Mb genomic regions were sequentially transferred from two donor strains to a recipient strain via conjugation and captured by pMegaBAC1H in the recipient strain to produce a 1-Mb plasmid. This 1-Mb plasmid was transmissible to another E. coli strain via conjugation. Furthermore, these 1-Mb secondary chromosomes were amplifiable in vitro by using the reconstituted E. coli chromosome replication cycle reaction (RCR). These strategies and technologies would make popular E. coli cells a productive factory for designer chromosome engineering.
journal_name
ACS Synth Bioljournal_title
ACS synthetic biologyauthors
Mukai T,Yoneji T,Yamada K,Fujita H,Nara S,Su'etsugu Mdoi
10.1021/acssynbio.0c00008subject
Has Abstractpub_date
2020-06-19 00:00:00pages
1315-1327issue
6issn
2161-5063journal_volume
9pub_type
杂志文章abstract::Genetic switches in which the activity of T7 RNA polymerase (RNAP) is directly regulated by external signals are obtained with an engineering strategy of splitting the protein into fragments and using regulatory domains to modulate their reconstitutions. Robust switchable systems with excellent dark-off/light-on prope...
journal_title:ACS synthetic biology
pub_type: 杂志文章
doi:10.1021/acssynbio.6b00248
更新日期:2017-02-17 00:00:00
abstract::The availability of different host chassis will greatly expand the range of applications in synthetic biology. Members of the Acetobacteraceae family of Gram-negative bacteria form an attractive class of nonmodel microorganisms that can be exploited to produce industrial chemicals, food and beverage, and biomaterials....
journal_title:ACS synthetic biology
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abstract::Exploiting simple types of symmetry common to many natural protein oligomers as a starting point, several recent studies have succeeded in engineering complex self-assembling protein architectures reminiscent but distinct from those evolved in the natural world. Designing symmetric protein cages with a wide range of p...
journal_title:ACS synthetic biology
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journal_title:ACS synthetic biology
pub_type: 杂志文章
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更新日期:2014-09-19 00:00:00
abstract::We present the OpenPlant toolkit, a set of interlinked resources and techniques to develop Marchantia as testbed for bioengineering in plants. Marchantia is a liverwort, a simple plant with an open form of development that allows direct visualization of gene expression and dynamics of cellular growth in living tissues...
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abstract::While predictable design of a genetic circuit's output is a major goal of synthetic biology, it remains a significant challenge because DNA binding sites in the cell affect the concentration of available transcription factors (TF). To mitigate this problem, we propose to use a TF that results from the (reversible) pho...
journal_title:ACS synthetic biology
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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::Synthetic biology is today harnessing the design of novel and greener biosynthesis routes for the production of added-value chemicals and natural products. The design of novel pathways often requires a detailed selection of enzyme sequences to import into the chassis at each of the reaction steps. To address such desi...
journal_title:ACS synthetic biology
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更新日期:2016-06-17 00:00:00
abstract::Lactams are an important class of commodity chemicals used in the manufacture of nylons, with millions of tons produced every year. Biological production of lactams could be greatly improved by high-throughput sensors for lactam biosynthesis. To identify biosensors of lactams, we applied a chemoinformatic approach ins...
journal_title:ACS synthetic biology
pub_type: 杂志文章
doi:10.1021/acssynbio.6b00136
更新日期:2017-03-17 00:00:00
abstract::Melatonin is a commercially attractive tryptophan-derived hormone. Here we describe a bioprocess for the production of melatonin using Escherichia coli to high titers. The first engineered strain produced 0.13 g/L of melatonin from tryptophan under fed-batch fermentation conditions. A 4-fold improvement on melatonin t...
journal_title:ACS synthetic biology
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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::The Design-Build-Test-Learn (DBTL) cycle, facilitated by exponentially improving capabilities in synthetic biology, is an increasingly adopted metabolic engineering framework that represents a more systematic and efficient approach to strain development than historical efforts in biofuels and biobased products. Here, ...
journal_title:ACS synthetic biology
pub_type: 杂志文章
doi:10.1021/acssynbio.9b00020
更新日期:2019-06-21 00:00:00
abstract::Recent advances in the design and construction of synthetic multicelled systems in E. coli and S. cerevisiae suggest that it may be possible to implement sophisticated distributed algorithms with these relatively simple organisms. However, existing design frameworks for synthetic biology do not account for the unique ...
journal_title:ACS synthetic biology
pub_type: 杂志文章
doi:10.1021/sb300034m
更新日期:2012-08-17 00:00:00
abstract::Robust and precise ratio control of heterogeneous phenotypes within an isogenic population is an essential task, especially in the development and differentiation of a large number of cells such as bacteria, sensory receptors, and blood cells. However, the mechanisms of such ratio control are poorly understood. Here, ...
journal_title:ACS synthetic biology
pub_type: 杂志文章
doi:10.1021/acssynbio.9b00030
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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::Traditional enzyme characterization methods are low-throughput and therefore limit engineering efforts in synthetic biology and biotechnology. Here, we propose a DNA-linked enzyme-coupled assay (DLEnCA) to monitor enzyme reactions in a high-throughput manner. Throughput is improved by removing the need for protein pur...
journal_title:ACS synthetic biology
pub_type: 杂志文章
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更新日期:2015-07-17 00:00:00
abstract::A common approach to design genetic circuits is to compose gene expression cassettes together. While appealing, this modular approach is challenged by the fact that expression of each gene depends on the availability of transcriptional/translational resources, which is in turn determined by the presence of other genes...
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doi:10.1021/acssynbio.6b00361
更新日期:2017-07-21 00:00:00
abstract::Optogenetic control of endogenous signaling can be an important tool for probing cell behavior. Using the photoresponse of the LOV2 domain of Avena sativa phototropin 1, we developed analogues of kinase inhibitors whose activity is light dependent. Inhibitory peptides were appended to the Jα helix, where they potently...
journal_title:ACS synthetic biology
pub_type: 信件
doi:10.1021/sb5001356
更新日期:2014-11-21 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::The Synthetic Biology Open Language (SBOL) is an emerging synthetic biology data exchange standard, designed primarily for unambiguous and efficient machine-to-machine communication. However, manual editing of SBOL is generally difficult for nontrivial designs. Here, we describe ShortBOL, a lightweight SBOL scripting ...
journal_title:ACS synthetic biology
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doi:10.1021/acssynbio.9b00470
更新日期:2020-04-17 00:00:00
abstract::DNA replication is one of the central functions of the cell. The complexity of modern DNA replication systems raises a question: is it possible to achieve a simpler continuous isothermal DNA replication using fewer proteins? Here, we searched such replication using an evolutionary approach. Through a long-term serial ...
journal_title:ACS synthetic biology
pub_type: 杂志文章
doi:10.1021/acssynbio.0c00137
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abstract::Eukaryotic transcriptional factors (TFs) typically recognize short genomic sequences alone or together with other proteins to modulate gene expression. Mapping of TF-DNA interactions in the genome is crucial for understanding the gene regulatory programs in cells. While chromatin immunoprecipitation followed by sequen...
journal_title:ACS synthetic biology
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doi:10.1021/acssynbio.6b00358
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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
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更新日期:2017-03-17 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
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journal_title:ACS synthetic biology
pub_type: 信件
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更新日期:2018-01-19 00:00:00
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journal_title:ACS synthetic biology
pub_type: 杂志文章
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更新日期:2012-09-21 00:00:00
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journal_title:ACS synthetic biology
pub_type: 杂志文章
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更新日期:2017-05-19 00:00:00
abstract::Combinatorial metabolic engineering has been widely established for the development of efficient microbial cell factories to produce the products of interest by precisely regulating the expression levels of multiple genes simultaneously. Here, we report a novel multifunctional CRISPR system that enables simultaneous g...
journal_title:ACS synthetic biology
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doi:10.1021/acssynbio.0c00218
更新日期:2020-09-18 00:00:00
abstract::Engineered bacteria promise to revolutionize diagnostics and therapeutics, yet many applications are precluded by the limited number of detectable signals. Here we present a general framework to engineer synthetic receptors enabling bacterial cells to respond to novel ligands. These receptors are activated via ligand-...
journal_title:ACS synthetic biology
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