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Abstract Development of strains for efficient production of chemicals and pharmaceuticals requires multiple rounds of genetic engineering. In this study, we describe construction and characterization of EasyClone vector set for baker's yeast Saccharomyces cerevisiae, which enables simultaneous expression of multiple genes with an option of recycling selection markers. The vectors combine the advantage of efficient uracil excision reaction-based cloning and Cre-LoxP-mediated marker recycling system. The episomal and integrative vector sets were tested by inserting genes encoding cyan, yellow, and red fluorescent proteins into separate vectors and analyzing for co-expression of proteins by flow cytometry. Cells expressing genes encoding for the three fluorescent proteins from three integrations exhibited a much higher level of simultaneous expression than cells producing fluorescent proteins encoded on episomal plasmids, where correspondingly 95% and 6% of the cells were within a fluorescence interval of Log10 mean ± 15% for all three colors. We demonstrate that selective markers can be simultaneously removed using Cre-mediated recombination and all the integrated heterologous genes remain in the chromosome and show unchanged expression levels. Hence, this system is suitable for metabolic engineering in yeast where multiple rounds of gene introduction and marker recycling can be carried out. EasyClone genetical toolbox allows faster development of yeast strains for biotechnological applications.

Thermostable DNA polymerases, such as Taq isolated from the thermophilic bacterium Thermus aquaticus , enable one-pot exponential DNA amplification known as polymerase chain reaction (PCR). However, properties other than thermostability - such as fidelity, processivity, and compatibility with modified nucleotides - are important in contemporary molecular biology applications. Here, we describe the engineering and characterization of a fusion between a DNA polymerase identified in the marine archaea Nanoarchaeum equitans and a DNA binding domain from the thermophile Sulfolobus solfataricus . The fusion creates a highly active enzyme, Neq2X7, capable of amplifying long and GC-rich DNA, unaffected by replacing dTTP with dUTP in PCR, and tolerant to various known PCR inhibitors. This makes it an attractive DNA polymerase for use, e.g., with uracil excision (USER) DNA assembly and for contamination-free diagnostics. Using a magnification via nucleotide imbalance fidelity assay, Neq2X7 was estimated to have an error rate lower than 2 ∙ 10 −5 bp −1 and an approximately 100x lower fidelity than the parental variant Neq2X, indicating a trade-off between fidelity and processivity – an observation that may be of importance for similarly engineered DNA polymerases. Neq2X7 is easy to produce for routine application in any molecular biology laboratory, and the expression plasmid is made freely available.

The Illumina DNA sequencing platform generates accurate but short reads, which can be used to produce accurate but fragmented genome assemblies. Pacific Biosciences and Oxford Nanopore Technologies DNA sequencing platforms generate long reads that can produce complete genome assemblies, but the sequencing is more expensive and error-prone. There is significant interest in combining data from these complementary sequencing technologies to generate more accurate \"hybrid\" assemblies. However, few tools exist that truly leverage the benefits of both types of data, namely the accuracy of short reads and the structural resolving power of long reads. Here we present Unicycler, a new tool for assembling bacterial genomes from a combination of short and long reads, which produces assemblies that are accurate, complete and cost-effective. Unicycler builds an initial assembly graph from short reads using the de novo assembler SPAdes and then simplifies the graph using information from short and long reads. Unicycler uses a novel semi-global aligner to align long reads to the assembly graph. Tests on both synthetic and real reads show Unicycler can assemble larger contigs with fewer misassemblies than other hybrid assemblers, even when long-read depth and accuracy are low. Unicycler is open source (GPLv3) and available at github.com/rrwick/Unicycler.

Engineering of the CRISPR/Cas9 system has opened a plethora of new opportunities for site-directed mutagenesis and targeted genome modification. Fundamental to this is a stretch of twenty nucleotides at the 5' end of a guide RNA that provides specificity to the bound Cas9 endonuclease. Since a sequence of twenty nucleotides can occur multiple times in a given genome and some mismatches seem to be accepted by the CRISPR/Cas9 complex, an efficient and reliable in silico selection and evaluation of the targeting site is key prerequisite for the experimental success. Here we present the CRISPR/Cas9 target online predictor (CCTop, http://crispr.cos.uni-heidelberg.de) to overcome limitations of already available tools. CCTop provides an intuitive user interface with reasonable default parameters that can easily be tuned by the user. From a given query sequence, CCTop identifies and ranks all candidate sgRNA target sites according to their off-target quality and displays full documentation. CCTop was experimentally validated for gene inactivation, non-homologous end-joining as well as homology directed repair. Thus, CCTop provides the bench biologist with a tool for the rapid and efficient identification of high quality target sites.

Quantum Cloud Computing is the technology which has the capability to shape the future of computing. In “Platform as a Service (PaaS)” type of cloud computing, the development environment is delivered as a service. In this paper, a multi-user broadcast protocol in network is developed with the mode of one master and N slaves together with a sequence of single photons. It can be applied to a multi-node network, in which a single photon sequence can be sent to all the slave nodes simultaneously. In broadcast communication networks, these single photons encode classical information directly through noisy quantum communication channels. The results show that this protocol can realize the secret key generation and sharing of multiple nodes. The protocol we propose is also proved to be unconditionally secure in theory, which indicates its feasibility in theoretical application.

Gene cloning is an important step in investigating gene structure and function. To verify gene sequence, Sanger sequencing is used, which may produce several overlapping sequencing files that need to be merged before alignment to the target gene sequence is performed. Previously, we reported the Python program to Merge Sanger sequences ( https://peerj.com/articles/11354/ ), which ran in command line and relied heavily on EMBOSS suite. In this updated version of the program, we have made several remarkable improvements. It provides a graphical user interface (GUI) written with tkinter, which is convenient and stable. It does not require users to rename the input sequences before performing merging. With regard to the implementation, the updated version utilizes Python function (Align.PairwiseAligner) to align adjacent sequences, which is more flexible (can adjust program parameter i.e. , the number of first-time consecutive matching bases). The new version of the program makes merging Sanger sequences much more convenient and facilitates gene study.

Everyone today actively uses online social networks to get in touch with their friends, for career opportunities, and business also. Some of the most popular social networks are Facebook, Instagram, LinkedIn, and Twitter. But the question is how much their authentication systems are built in a secured way. The authentication systems mostly depend on the user's general details such as name, photo, and location. In such systems, mischievous persons can easily create fake profiles by cloning the user’s identity to abuse the user’s information. Using fake profiles they can misuse original user’s photos, contacts, and videos. This kind of mischievous person can be identified by the use of privacy detection mechanisms. So, this research emphasizes some data mining techniques for protecting the original user’s information and to identify the malicious accounts in social network sites. By the assessment of 3PS (Publically Privacy Protection System), this work employs the malicious account detection method in OSN depending upon the mischievous person’s uncountable shared posts in a day and latest activity and behaviors. Examining the network similarity and comparison of attributes threshold values referred to the original user’s profile can be used to identify the malicious accounts. For this E_SVM-NN classifier is used based on the feature reduction techniques. This work involves in creating OSN accounts for experiments and investigates the latest updates, posts, comments, photos, and performing online search etc. which are used to evaluate the effectiveness and significance of the proposed work in contrast to the previous works.

Plant synthetic biology aims to apply engineering principles to plant genetic design. One strategic requirement of plant synthetic biology is the adoption of common standardized technologies that facilitate the construction of increasingly complex multigene structures at the DNA level while enabling the exchange of genetic building blocks among plant bioengineers. Here, we describe GoldenBraid 2.0 (GB2.0), a comprehensive technological framework that aims to foster the exchange of standard DNA parts for plant synthetic biology. GB2.0 relies on the use of type IIS restriction enzymes for DNA assembly and proposes a modular cloning schema with positional notation that resembles the grammar of natural languages. Apart from providing an optimized cloning strategy that generates fully exchangeable genetic elements for multigene engineering, the GB2.0 toolkit offers an evergrowing open collection of DNA parts, including a group of functionally tested, premade genetic modules to build frequently used modules like constitutive and inducible expression cassettes, endogenous gene silencing and protein-protein interaction tools, etc. Use of the GB2.0 framework is facilitated by a number of Web resources that include a publicly available database, tutorials, and a software package that provides in silico simulations and laboratory protocols for GB2.0 part domestication and multigene engineering. In short, GB2.0 provides a framework to exchange both information and physical DNA elements among bioengineers to help implement plant synthetic biology projects.

Sulfatases cleave sulfate groups from various molecules and constitute a biologically and industrially important group of enzymes. However, the number of sulfatases whose substrate has been characterized is limited in comparison to the huge diversity of sulfated compounds, yielding functional annotations of sulfatases particularly prone to flaws and misinterpretations. In the context of the explosion of genomic data, a classification system allowing a better prediction of substrate specificity and for setting the limit of functional annotations is urgently needed for sulfatases. Here, after an overview on the diversity of sulfated compounds and on the known sulfatases, we propose a classification database, SulfAtlas (http://abims.sb-roscoff.fr/sulfatlas/), based on sequence homology and composed of four families of sulfatases. The formylglycine-dependent sulfatases, which constitute the largest family, are also divided by phylogenetic approach into 73 subfamilies, each subfamily corresponding to either a known specificity or to an uncharacterized substrate. SulfAtlas summarizes information about the different families of sulfatases. Within a family a web page displays the list of its subfamilies (when they exist) and the list of EC numbers. The family or subfamily page shows some descriptors and a table with all the UniProt accession numbers linked to the databases UniProt, ExplorEnz, and PDB.

Summary Oligonucleotide (oligo)‐fluorescence in situ hybridization (FISH) has rapidly becoming the new generation of FISH technique in plant molecular cytogenetics research. Genome‐scale identification of single‐copy oligos is the foundation of successful oligo‐FISH experiments. Here, we introduce Chorus2, a software that is developed specifically for oligo selection. We demonstrate that Chorus2 is highly effective to remove all repetitive elements in selection of single‐copy oligos, which is critical for the development of successful FISH probes. Chorus2 is more effective than Chorus, the original version of the pipeline, and OligoMiner for repeat removal. Chorus2 allows to select oligos that are conserved among related species, which extends the usage of oligo‐FISH probes among phylogenetically related plant species. We also implemented a new function in Chorus2 that allows development of FISH probes from plant species without an assembled genome. We anticipate that Chorus2 can be used in plants as well as in mammalian and other non‐plant species. Chorus2 will broadly facilitate the design of FISH probes for various types of application in molecular cytogenetics research.