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A Modular Assembly Platform for Rapid Generation of DNA Constructs.

ABSTRACT: Traditional cloning methods have limitations on the number of DNA fragments that can be simultaneously manipulated, which dramatically slows the pace of molecular assembly. Here we describe GMAP, a Gibson assembly-based modular assembly platform consisting of a collection of promoters and genes, which allows for one-step production of DNA constructs. GMAP facilitates rapid assembly of expression and viral constructs using modular genetic components, as well as increasingly complicated genetic tools using contextually relevant genomic elements. Our data demonstrate the applicability of GMAP toward the validation of synthetic promoters, identification of potent RNAi constructs, establishment of inducible lentiviral systems, tumor initiation in genetically engineered mouse models, and gene-targeting for the generation of knock-in mice. GMAP represents a recombinant DNA technology designed for widespread circulation and easy adaptation for other uses, such as synthetic biology, genetic screens, and CRISPR-Cas9.

SUBMITTER: Akama-Garren EH

PROVIDER: S-EPMC4757859 | biostudies-literature | 2016 Feb

REPOSITORIES: biostudies-literature

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A Modular Assembly Platform for Rapid Generation of DNA Constructs.

Akama-Garren Elliot H EH Joshi Nikhil S NS Tammela Tuomas T Chang Gregory P GP Wagner Bethany L BL Lee Da-Yae DY Rideout William M WM Papagiannakopoulos Thales T Xue Wen W Jacks Tyler T

Scientific reports 20160218

Traditional cloning methods have limitations on the number of DNA fragments that can be simultaneously manipulated, which dramatically slows the pace of molecular assembly. Here we describe GMAP, a Gibson assembly-based modular assembly platform consisting of a collection of promoters and genes, which allows for one-step production of DNA constructs. GMAP facilitates rapid assembly of expression and viral constructs using modular genetic components, as well as increasingly complicated genetic to ...[more]

PMID: 26887506

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Project description:Modulated molecular design-based intracellular self-assembly strategy has showed great potentiality in drug delivery, due to its assembling nature-resulted optimized drug biodistribution and metabolism. The modular designing concept endows the delivery system multiple functions, such as, selectivity and universality to improve the pharmacokinetics of loaded drugs. However, the accurate controlling of the self-assembling process in desired site to achieve optimal drug delivery is posed great challenges toward rational molecular design. Here, we fabricated a modulated drug-delivery system (MDS) through intracellular peptide self-assembly to realize effective drug delivery. MDS was designed based on modulated molecular designing strategy which contains five functional motifs and effectively transformed into fibrous nanostructures inside target cells by caspase3/7 hydrolysis directed in situ self-assembly. The experimental studies and molecular simulations were carried out to evaluate the successful construction and delivering efficacy of MDS. According to the experimental results and molecular simulation analysis, the percentage of solvent-exposed surface area of assembling modular (KLVFFAE), as well as its non-covalent interaction between four other modules synergeticly decide the solubility of molecules. The weak intramolecular forces of the peptide back bone, such as, hydrogen bond, as well as multivalent interactions of the side chains such as, salt bridge and hydrophobic interaction both contribute to the self-assembly of the molecules. The significant structural difference between delivering molecules optimize the system to adapt hydrophilic and hydrophobic drugs. Finally, the predicted drug delivery molecule specifically recognizes targeted cancer cell lines and self-assembles to form fibers intracellularly, resulting in prolonged drug retention and accumulation. The regular prediction and rational molecular design will benefit the further construction and optimization of modulated drug delivery platform.

Dataset Information

A Modular Assembly Platform for Rapid Generation of DNA Constructs.

Publications

A Modular Assembly Platform for Rapid Generation of DNA Constructs.

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