10 research outputs found
The USER cloning standard
This BioBricks Foundation Request for Comments (BBF RFC) provides information about the design of uracil-containing primers used for USER cloning and USER fusion
deFUME: Dynamic exploration of functional metagenomic sequencing data
BACKGROUND: Functional metagenomic selections represent a powerful technique that is widely applied for identification of novel genes from complex metagenomic sources. However, whereas hundreds to thousands of clones can be easily generated and sequenced over a few days of experiments, analyzing the data is time consuming and constitutes a major bottleneck for experimental researchers in the field. FINDINGS: Here we present the deFUME web server, an easy-to-use web-based interface for processing, annotation and visualization of functional metagenomics sequencing data, tailored to meet the requirements of non-bioinformaticians. The web-server integrates multiple analysis steps into one single workflow: read assembly, open reading frame prediction, and annotation with BLAST, InterPro and GO classifiers. Analysis results are visualized in an online dynamic web-interface. CONCLUSION: The deFUME webserver provides a fast track from raw sequence to a comprehensive visual data overview that facilitates effortless inspection of gene function, clustering and distribution. The webserver is available at cbs.dtu.dk/services/deFUME/and the source code is distributed at github.com/EvdH0/deFUME. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s13104-015-1281-y) contains supplementary material, which is available to authorized users
PHUSER (Primer Help for USER): a novel tool for USER fusion primer design
Uracil-Specific Exision Reagent (USER) fusion is a recently developed technique that allows for assembly of multiple DNA fragments in a few simple steps. However, designing primers for USER fusion is both tedious and time consuming. Here, we present the Primer Help for USER (PHUSER) software, a novel tool for designing primers specifically for USER fusion and USER cloning applications. We also present proof-of-concept experimental validation of its functionality. PHUSER offers quick and easy design of PCR optimized primers ensuring directionally correct fusion of fragments into a plasmid containing a customizable USER cassette. Designing primers using PHUSER ensures that the primers have similar annealing temperature (Tm), which is essential for efficient PCR. PHUSER also avoids identical overhangs, thereby ensuring correct order of assembly of DNA fragments. All possible primers are individually analysed in terms of GC content, presence of GC clamp at 3â˛-end, the risk of primer dimer formation, the risk of intra-primer complementarity (secondary structures) and the presence of polyN stretches. Furthermore, PHUSER offers the option to insert linkers between DNA fragments, as well as highly flexible cassette options. PHUSER is publicly available at http://www.cbs.dtu.dk/services/phuser/
Molecular Buffers Permit Sensitivity Tuning and Inversion of Riboswitch Signals
Predictable integration
of foreign biological signals and parts
remains a key challenge in the systematic engineering of synthetic
cellular actuations, and general methods to improve signal transduction
and sensitivity are needed. To address this problem we modeled and
built a molecular signal buffer network in <i>Saccharomyces cerevisiae</i> inspired by chemical pH buffer systems. The molecular buffer system
context-insulates a riboswitch enabling synthetic control of colony
formation and modular signal manipulations. The riboswitch signal
is relayed to a transcriptional activation domain of a split transcription
factor, while interacting DNA-binding domains mediate the transduction
of signal and form an interacting molecular buffer. The molecular
buffer system enables modular signal inversion through integration
with repressor modules. Further, tuning of input sensitivity was achieved
through perturbation of the buffer pair ratio guided by a mathematical
model. Such buffered signal tuning networks will be useful for domestication
of RNA-based sensors enabling tunable outputs and library-wide selections
for drug discovery and metabolic engineering
Improved biotin, thiamine, and lipoic acid biosynthesis by engineering the global regulator IscR
Software-Supported USER Cloning Strategies for Site-Directed Mutagenesis and DNA Assembly
USER cloning is a fast and versatile
method for engineering of
plasmid DNA. We have developed a user friendly Web server tool that
automates the design of optimal PCR primers for several distinct USER
cloning-based applications. Our Web server, named AMUSER (<u>A</u>utomated DNA <u>M</u>odifications with <u>USER</u> cloning), facilitates DNA assembly and introduction
of virtually any type of site-directed mutagenesis by designing optimal
PCR primers for the desired genetic changes. To demonstrate the utility,
we designed primers for a simultaneous two-position site-directed
mutagenesis of green fluorescent protein (GFP) to yellow fluorescent
protein (YFP), which in a single step reaction resulted in a 94% cloning
efficiency. AMUSER also supports degenerate nucleotide primers, single
insert combinatorial assembly, and flexible parameters for PCR amplification.
AMUSER is freely available online at http://www.cbs.dtu.dk/services/AMUSER/