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Pier Ferdinando Casini nuovo PdR Ieri, #2. long. Visualizza Profilo Visualizza Messaggi Forum View Blog Entries Visualizza Articoli Member . Jul 13,  · Introduction. Natural products (NPs) produced by microbes are a major source of pharmacological agents and industrially useful compounds. With the spread of drug-resistant pathogens rendering widely used antibiotics ineffective, the discovery of new NPs has become an urgent necessity (Atanasov et al., ).The development of next-generation sequencing . Sito web istituzionale Istituto Comprensivo “Rossella Casini” - VIA SASSETTI, 1, SCANDICCI (FI).

Online casini information Article notes Copyright and License information Online casini. A f2p games switch for heterologous expression is to clone the target BGC into a suitable vector. This approach enables directional cloning in a ligase-free manner. Wenfang Wang 1 College of Life Sciences, Shanghai Normal University, Shanghai, China Find articles by Wenfang Wang. Ligation-independent cloning of PCR products LIC-PCR. No significant association was evidenced when specific types of cancer were analyzed.

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Direct Pathway Cloning DiPaC to unlock natural product biosynthetic potential. BMC Genom.

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Giornate di formazione per GENITORI e DOCENTI per online casini prevenzione di cyberbullismo e sexting: 18 e 21 marzo Il cssini Istituto ha aderito alla formazione promossa da AICS per favorire la comprensione del fenomeno del Cyberbullismo e del Online casini. As a result, each YAC clone must caasini carefully online casini to ensure that no DNA aktuelle lottozahlen mittwoch occur. Huang et al. Besides bacterial strains especially actinomycetesfungi are also prolific producers of NPs. They online casini online casini rapid and easy exchange of modules and boost the interoperability of bonus win spin no deposit and casino devices among different users Martinez-Garcia et al.

Cloning and heterologous expression of the grecocycline biosynthetic gene cluster. The metagenomics of soil. It was successfully used to isolate a New applications for phage integrases. Antibacterial and more info activities of orphan biosynthetic casiini clusters from atlantis II red sea brine pool.

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Methods Online casini. Cadasides, calcium-dependent acidic lipopeptides from the check this out metagenome that are active against multidrug-resistant bacteria. In the past decades, a large number of effective technologies for cloning NP BGCs have been established, which has greatly promoted drug discovery research. Guosong Zheng 1 College of Life Sciences, Shanghai Normal University, Shanghai, China Find articles by Guosong Zheng.

New applications for online casini integrases. Further, a highly sensitive novel method for the simultaneous separation and online casini of high-molecular-weight DNA fragments was established by optimizing the formulation of viscoelastic liquids and engineering a capillary system. Essilor di Del Vecchio fa causa a Jp Morgan.

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Casino hotel niagara falls Finally, the linear DNA assembly products were transformed into E. Meng et al.

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Received Apr 9; Accepted Jun Globalist Syndication 6 ore fa. Online casini Notizie. Broad-host-range expression reveals native and host regulatory elements that influence heterologous antibiotic production in Gram-negative bacteria. Therefore, pnline combination of different cloning approaches, and the establishment of novel, easy-to-use, highly efficient, and accurate cloning methods online casini a necessity.

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Online Gambling 2022 - Online Casino 2022 - Best 3 Online Casinos - Enhancement of neomycin production by online casini the entire biosynthetic gene cluster and feeding online casini precursors in Streptomyces fradiae CGMCC 4.

However, a major drawback is the problematic identification of naturally existing unique restriction enzyme recognition online casini on both sides of the slots casino 777 bay BGCs. They are multicopy plasmids in E. Selective isolation of large segments from individual microbial genomes and environmental DNA samples using transformation-associated recombination cloning in yeast. However, online casini described online casini, SSR-mediated cloning methods require the initial integration of specific sites into this web page chromosome in online casini. In vivo assembly approaches including: D phage-recombinase-mediated HR in E.

CNR 38 NR S. Publication types Leggi anche: Ceferin attacca la Superlega. Home Sport Calcio Lorenzo Casini nuovo presidente della Lega calcio Serie A. Dalla stessa sezione. So, enzymatic digestion is the most online casini used for DNA fragmentation of library construction. Currently, more and more microbial genome sequences are being published. For cloning small- to mid-sized BGCs, long-amplicon PCR could be used to amplify BGCs fragments, and then entire BGCs were obtained poker bonus DNA assembly Greunke et al.

However, as the length of NP BGCs increases, the probability of mutations introduced by PCR also increases. Fortunately, the development of genome editing tool CRISPR-Cas online casini regularly interspaced short palindromic repeat-CRISPR-associated protein system has made online casini possible to isolate the exact sequence of target BGCs Lee et al. With the aid of Cas9 endonuclease, DNA segment of desired sizes can be obtained through generating the double strand breaks DSBs at specific sites within the genome guided by sgRNA. For example, bacterial cultures e. Finally, the plug was transferred into cleavage buffer containing Cas9 and corresponding sgRNA pairs, which were designed to target genome segments of different lengths 50, 75, kb. Clear DNA bands at the expected lengths were observed using pulsed-field gel electrophoresis PFGE assessment Jiang et al.

Recently, CISMR CRISPR-mediated isolation of specific megabase-sized regions of the genomewhich enables the isolation of contiguous megabase-sized segments of the mouse genome, has also been developed by improving in vitro CRISPR specificity with the aid of both Target Finder and ZiFIT Targeter software to design 17 base sgRNA other than traditional 20 base target sequences Bennett-Baker and Mueller, Further, a highly novel method for the simultaneous separation online casini concentration of high-molecular-weight DNA fragments was established by optimizing the formulation of viscoelastic liquids and engineering a capillary system.

It was successfully used to isolate a The quality of DNA fragments can be analyzed via fragment analyzer or horizontal agarose gel electrophoresis. DNA fragments of desired sizes can be separated and extracted through multiple rounds of PFGE with different ramped pulse times Clos and Zander-Dinse, Regardless of the cloning method used, sufficient amounts of DNA fragments are indispensable. Therefore, the preparation of high-quality and high-molecular-weight DNA fragments is recognized as a critical step in gene cluster cloning Sapojnikova et al. Given that most NP BGCs are of relatively large in length, appropriate vector systems capable of carrying the entire gene cluster as well as shuffling these genetic segments between different hosts are necessary.

Since the first generation of general cloning vectors was introduced ina variety of high-capacity vectors have been developed so far Bajpai, Despite the dazzling choice of commercial and other available vectors, cloning vector selection can be determined by several key criteria, such as the BGC size and GC content, vector copy number, host compatibility of different vectors, selection markers, and multiple cloning sites. Several types of high-capacity vectors are available for cloning large DNA fragments, including cosmid online casini artificial chromosomes, such online casini the fungal artificial chromosome FACyeast artificial chromosome YACbacterial artificial chromosome BACand P1 phage artificial chromosome PAC Monaco and Larin, ; Bajpai, ; Bok online casini al. Since the possibility for cloning large fragments in cosmid vectors was first confirmed inthey have been widely used for the construction of genomic libraries of various biological species, including Drosophila, mice, and humans.

The construction of cosmid library is relatively simple and has been widely applied for cloning various NP BGCs. However, as the cosmid library requires tedious nice big win casino slot machine was, it is necessary to combine high-throughput screening and sequencing methods. For example, the anisomycin BGC from Streptomyces hygrospinosus was identified using a bioactivity-guided high-throughput method for online casini library screening Zheng et al. Recently, CONKAT-seq co-occurrence network analysis of targeted sequences was used to uncover the potential of the rare BGCs from millions of cosmid clones harboring metagenomic Http:// inserts Libis et al.

Cosmid vectors can accommodate up to approximately 40 kb of DNA. They are multicopy plasmids in E. However, loss of inserted DNA sometimes occurs within cosmid clones, which may be indicative of sequences that are instability in E. In addition, fosmid has an inducible oriV replication start point online casini high copy propagation, if necessary. Recently, a fosmid library containing 10, clones of metagenomic DNA isolated from the ATII the Red Sea brine pool, Atlantis II Deep lower convective online casini LCL was visit web page screened, and the products of two putative NP BGCs were detected to exhibit antibacterial and anticancer effects Ziko et al.

Typically, a cosmid or fosmid vector can only accept relatively small BGCs up to 45 kbwhich greatly hampers their application in cloning large NP BGCs. YAC vectors contain two copies of yeast telomeres for chromosomal stability, a yeast centromere for segregation, a yeast ARS autonomously replicating sequence for replication, and appropriate markers for the selection of recombinant molecules Visit web page et al. YAC provides the largest DNA insert capacity among all cloning vector types. Exogenous DNA fragments with sizes up to several hundred kilobase pairs or even as much as 2 Mb can be cloned into YAC vectors Monaco and Larin, A cornerstone of the Human Genome Project HGP is the cloning of large chromosomal fragments using YAC vectors. However, problems are frequently observed during the use of YAC clones, including chimerics, deletions, and rearrangements.

Furthermore, the isolation of YAC clones is challenging because of large sizes Ramsay, As a result, each YAC clone must be carefully analyzed to ensure that no DNA rearrangements occur. The first PAC vector pCYPAC1 combining the characteristics of P1-phage and F factor was online casini in Loannou et al. It can be efficiently transformed into E. Foreign DNA inserted in the PAC exhibits almost no chimerism or rearrangement. The PAC vector can carry DNA fragments of up to approximately kb. The recombinant PAC can stably exist as a single copy and propagate efficiently. Using these pESAC vectors, up to kb online casini of Actinomyces DNA can be cloned and introduced into genetically accessible Streptomyces lividans via protoplast transformation, stably maintaining the vector in an integrative form Sosio et al. Using PAC vector check this out a derivative of pESAC harboring an oriT site, which allows for conjugal transfer instead of time-consuming protoplast transformation, a genomic library of Streptomyces tsukubaensis was generated, and the entire The PAC library of Stretomyces sp.

PCS3-D2 was also constructed and analyzed in silico. Two clones containing and kb DNA inserts were identified to harbor Type I and Type III PKS polyketide synthase gene clusters, respectively Bayot Custodio and Alcantara, The positive rates of recombinant clones containing DNA inserts can be greatly improved by introducing the sacB or URA3 gene into PAC vectors as counter-screening markers, which can catalyze the production of toxicants in the presence of sucrose or 5-fluoroorotic acid 5-FOArespectively Noskov et al. Inthe first BAC vector pBACL was constructed based on the well-studied E. This BAC online casini has been reported to carry human genomic DNA fragments approaching kb Shizuya et al. Further, it enables the cloning of large-sized DNA fragments from complex genomic sources into bacteria, where they remain stable and are easily manipulated. Various BAC vectors, such as pStreptoBAC and pSBAC, have been extensively used for library construction with the purpose of cloning target large-sized NP BGCs Sosio et al.

These BAC vectors harbor two replication origins. One is ori that is essential for the initiation of single-copy replication in E. The other is oriVwhich can be induced to increase DNA yield. Thus far, when compared to YAC and PAC, the BAC vectors are more commonly employed for NP BGC cloning. When the genomic sequence information of BGCs is unknown e. Recently, using this online casini, several large NP BGCs, such as an aminopolyol polyketide BGC over kb, and a quinolidomicin BGC over kb, have been successfully cloned and heterologously expressed Zhang Online casini. However, due to the low positive rates, laborious screening is necessary Lin et al. Therefore, high-throughput screening methods have received considerable attention. Recently, the MAPLE Microfluidic automated plasmid library enrichment method, which combines BAC libraries with single-cell droplet microfluidic techniques for discovering functional biosynthetic pathways, was developed.

Using MAPLE, online casini type I PKS gene cluster from an Antarctic soil metagenome was isolated and sequenced Xu et al. In addition, when the genome sequence is available, the pSBAC vector can be inserted into online casini flanking regions of target BGCs within the chromosome in advance and the entire target BGCs can then be captured into pSBAC through specific your online slots casino opinion enzyme digestion and self-ligation. However, a major drawback is the problematic identification of naturally existing unique restriction enzyme recognition sites on both sides of the target BGCs. Therefore, artificial insertion of a specific DNA sequence into the online casini via homologous recombination HR is a prerequisite, limiting the application of this method in intractable strains.

Besides bacterial strains especially actinomycetesfungi online casini also prolific producers of NPs. However, despite the abundance of available fungal genome data that encode a large number of NP BGCs, the majority of them are silent in laboratory growth conditions and most fungi are online casini genetically amendable. Using FAC and metabolomic scoring MS56 recombinant FACs containing uncharacterized BGCs from article source fungal species were constructed and expressed in Aspergillus nidulans. Finally, 15 new metabolites were discovered and assigned with confidence to their BGCs Clevenger et al.

It could be anticipated that the development of FAC will facilitate NP research of fungi in the future. With the rapid development of synthetic biology, standardized and orthogonal vectors, which follow uniform and modular online casini, have been developed. They enable the rapid go here easy exchange of modules and boost the interoperability of genetic devices among different online casini Martinez-Garcia et al. However, within the field of specialized NP synthetic biology, even though there are multifarious vectors for large DNA online casini cloning, few such standard vectors have been constructed. It is well known that the size from a few kb to more than kb online casini NP BGCs, the please click for source GC content, and the repeat or similar sequence in the PKS or NRPS non-ribosomal peptide synthase genes can affect the choice of vectors for BGC cloning Aubry et al.

Thus, vectors that are flexible and adapted to various assembly methods are preferred. However, these vectors were mainly used for monocistronic gene expression Phelan et al. A set of 12 standardized and modular three different resistance markers and four orthogonal integration systems vectors based on model SEVA plasmids were designed to allow for the assembly of NP BGCs through various cloning methods in Streptomyces species Aubry et al. In these vectors, the FLP flippase recombination system was also incorporated for the recycling of antibiotic markers and for reducing unwanted homologous recombination when several vectors are used simultaneously Aubry et al.

It can be expected that through the modularization and orthogonalization of key vector elements, including orthogonal integration systems, online casini of replication, antibiotic selection markers, and a variety of cargoes with specific applications, a suitable vector can be online casini designed to efficiently assemble or clone large DNA fragments.

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It is worth to note that so far, many laboratories have designed and constructed a large number of multifarious vectors according to their own needs. To further promote NP research, laboratories should make their vectors freely available to other research groups. High fidelity, effective and seamless online casini of large DNA fragments and appropriate vectors is the pivotal step for obtaining entire NP Online casini for heterologous expression. With the rapid development of synthetic biology, various DNA cloning and assembly methods have been established and successfully utilized for cloning NP BGCs. Depending on the experimental setting, assembly methods can be divided into two categories: in vitro and in vivo Lotto uhrzeit assembly Juhas and Ajioka, ; Li et al.

In vitro cloning online casini assembly approaches include three main types: 1 restriction enzyme-mediated methods, e. The classic method for DNA assembly is via the use of enzymes for the cutting and ligation of DNA fragments and vectors. However, these will leave scars at the restriction site. To address this problem, type IIs restriction enzymes e. The DNA overhangs can be appropriately designed to guide the corresponding DNA fragments for ligation in a designated order. This method was named Golden Gate Figure 2Awhich reflects the concept of modular assembly Mitchell et al. Recently, it was employed for refactoring carotenoid biosynthetic pathways. Online casini particular, each biosynthetic gene equipped with different promoters and terminators was assembled, online casini in various expression cassettes.

A library containing 96 combinatorial refactored carotenoid pathways was then successfully generated by assembling these cassettes Ren et al. A similar method named MASTER methylation-assisted tailorable ends rational ligation based on MspJI, a specific type IIs endonuclease, was also developed for sequence-independent hierarchical DNA assembly. Using the MspJI-mediated method, the blue-colored antibiotic actinorhodin ACT BGC 29 kb from Streptomyces coelicolor was successfully assembled and expressed in a fast-growing Streptomyces sp. Chen et al. To online casini appropriate for Golden Gate cloning, special care should be taken to ensure that the type IIs restriction site this web page present in opposite orientation at the ends of the vector and DNA fragments but absent in internal sequences Marillonnet and Grutzner, However, type IIs enzymes are relatively rare, and thus few options check this out available.

Usually, internal type IIs restriction sites should be removed by silent mutations. In addition, the number of DNA fragments that can be simultaneously assembled is still limited Schmid-Burgk et online casini. The main cloning methods for BGC capturing. In vitro DNA assembly online casini including: A restriction enzyme-mediated digestion and ligation e. In vivo assembly approaches including: D phage-recombinase-mediated HR in E. F Site-specific integrase e. Although traditional restriction cutting and ligation methods are still widely used, their low efficiency and enzyme site-dependence do not meet the increasing demand for assembling large DNA fragments. Thus, recombination-based assembly online casini Figure 2B based on the existence of short homologous regions at the extremities of DNA fragments and vectors are attracting more attention.


These methods include ligation-independent cloning LIC Aslanidis and de Jong,sequence and ligation-independent cloning SLiC Li and Elledge,seamless ligation cloning extract SliCE Zhang Y. Online casini DNA assembly usually employs one to three enzymes in the in vitro reaction, wherein DNA polymerase, exonuclease, and ligase are the most commonly used. Hereafter, we provide a brief introduction to the above-mentioned assembly methods. It does not require the use of restriction enzymes and T4 DNA ligase. Based on this method, SLIC was developed, which can achieve the assembly of multiple DNA fragments in a single reaction by combining in vitro HR and single-strand annealing. Online casini is more efficient at very low DNA concentrations, especially in the presence of the HR protein RecA Li and Elledge, SLiCE is a highly cost-effective method, which utilizes cell extracts from E.

This method provides an effective strategy for directional seamless DNA cloning from BAC or complex genomes Zhang Online casini. The Gibson assembly method uses three commercial enzymes T5 exonuclease, Phusion DNA polymerase, and Taq DNA ligase for the assembly of DNA fragments with short homologous ends in vitro. See more, Gibson assembly cannot be efficiently employed for super lucky casino mod apk assembly of DNA fragments with high GC content due to high vector self-ligation background. Recently, a modified Gibson assembly method was developed by adding a pair of universal terminal overhangs online casini high AT content 21 bp to the ends of the BAC vector, greatly reducing vector self-ligation Li L.

Using this method, a 67 kb online casini II PII BGC from Streptomyces pristinaespiralis was hierarchically assembled from 15 PCR-amplified fragments Li L. In addition, a T5 exonuclease-mediated DNA assembly TEDA method was established, in which homologous ends were treated with T5 exonuclease alone. After annealing, the reaction sample was transformed into E. The results indicated that the cloning efficiency of TEDA was higher than that of the traditional Gibson assembly tool Xia et al. The development of CRISPR technology has greatly promoted recombination-mediated DNA assembly.

It should be noted these recombination-based methods e. Unlike Cas9 which introduces double-strand breaks DSBs and produces blunt ends, Cpf1 Cas12a cleaves target DNA and produces sticky overhangs, which makes Cpf1 an alternative tool for DNA assembly in vitro Lei et al. Recently, a method named CCTL Cpf1-assisted Cutting and Taq DNA ligase mediated Ligation was developed, with the 8-nt sticky end produced by Cpf1 cleavage for ligation instead of homologous sequences in recombination-based methods, which therefore make the CCTL is suitable for cloning complicated DNA sequences Lei et al.

However, the requirement of specific PAM limits its application. To address this limitation, the PAM specificity of Cas12a was expanded via specific structure-guided mutagenesis and two engineered Cas12a variant EP15 and EP16 were obtained, which increased the targeting range by fourfold. Based on this modified Cas12a, the iCOPE improved Cas12a-assisted one-pot DNA Editing method was developed, which can avoid many of the DNA sequence constraints Wang et al. In addition to the methods described above, various in vitro novel DNA assembly methods for BGCs cloning have been designed and established. These include the assembly of fragment ends after PCR Click here cloning Zeng et read more. Enzyme-mediated DNA assembly online casini are efficient and straightforward. As mentioned above, Online casini Gate assembly is robust and suitable for assembling over 15 DNA fragments with high efficiency and fidelity.

However, due to limited commercially available Type IIs endonucleases, it is not always possible to find suitable restriction enzymes that avoid the naturally occurring Type IIs sites within BGCs Liang et al. Therefore, additional efforts are needed to modify the sequences of BGCs in order to eliminate the undesired cut sites.

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Gibson assembly is versatile, but its efficiency and fidelity drop sharply when the number of fragments is more than four. Furthermore, essential components such as promoters, ribosomal binding sites, and terminators are notoriously difficult for Gibson assembly because of their secondary structures Liang et al. Enzyme-independent DNA online casini methods can realize DNA assembly without enzymes, which saves costs and is especially suitable for high-throughput settings. These approaches mainly include enzyme-free cloning EFCpolymerase incomplete primer extension PIPEand Twin primer non-enzymatic DNA assembly TPA.

A highly efficient EFC procedure for DNA assembly was previously established, utilizing tailed PCR primer sets to generate complementary staggered overhangs on both fragments and vectors via a denaturation-hybridization reaction Tillett and Neilan, This approach enables directional cloning in a ligase-free manner. Therefore, it is not constrained by the requirement for appropriate enzyme sites. However, this method is mainly used for the assembly of two DNA fragments, and its efficiency is low. TPA cloning is also seamless and sequence-independent, and its performance rivals even the best in vitro assembly methods.

Although these enzyme-free cloning tools provide a number of advantages over other cloning strategies, they still have limitations. For example, these methods usually require a number of specially designed primers, and the assembly capability as well online casini fidelity drop sharply with increasing fragment size Online casini and Neilan, ; Yuan et al. In vitro assembly methods provide flexible cloning of DNA fragments, wherein the DNA fragments can be produced through multiple rounds of PCR or direct chemical synthesis. However, random mutations cannot be entirely ruled online casini. In addition, incorrect pairing of DNA fragments during assembly may also cause unanticipated mutations, especially in the PKS or NRPS genes, which contain numerous repeat sequences. In vivo DNA cloning online casini for direct capture of the target DNA fragment, which are based on the strong homologous recombination ability of E.

They represent an alternative strategy for BGC cloning. These methods mainly include phage recombinase-mediated homologous recombination cloning in E. The endogenous HR system in E. Many cloning strategies based on the endogenous HR have been created, such as in vivo cloning IVCin best preflop hands PCR products containing terminal sequences identical to the two terminals of the linearized vector were co-transfected into E. However, due to its strong exonuclease activity, the RecBCD complex can rapidly degrade exogenous linear DNA molecules.


The PCR products and linear vector can be introduced and stably maintained only in RecBCD-deficient E. In addition, RecA-dependent recombination requires a much longer homologous region approximately bp. To develop online casini more efficient and reliable HR system in E. It was later identified as an inhibitor of the Onlind subunit of RecBCD. This protein protects linear DNA from degradation by endogenous nucleases Abbasi et al. This technology was first used xasini reconstitute an entire 43 kb myxochromide BGC from two overlapping cosmids Wenzel et al. Subsequently, it has been widely applied for the cloning of a variety of NP BGCs ranging from 11 to kb from different microbes, including Streptomyces, Sorangiumand Cystobacter Binz et al.

Fu et al. Using this LLHR linear—linear homologous recombination approach, ten large NRPS and PKS BGCs with sizes from 10 to 37 acsini from the genomic DNA of Photorhabdus luminescens were directly cloned into linear expression vectors in a one-step recombination event. However, they online casini to direct clone the intact onnline salinomycin gene cluster from the genome of Streptomyces albus using LLHR. Finally, the group successfully cloned three fragments of salinomycin BGC using LLHR separately and assembled online casini into a complete one Yin et al. After exonuclease chew-back, the target DNA fragment and the vector were annealed together via online casini homology arm about 80 bp and were then transformed into E.

ExoCET is generally applicable to a broader range of direct cloning with respect to size up to kb and genome complexity Wang et al. However, it is not always easy to find appropriate restriction enzyme cutting sites. The assembly of two DNA molecules containing homologous sequences via recombination in yeast was first demonstrated by Onliine et al. A couple of years later, a convenient online casini for plasmid construction using this in vivo bimolecular recombination reaction was developed Ma et al. Motivated by this method, a transformation-associated recombination TAR strategy in yeast based on this approach was later introduced, allowing for the selective isolation of large genomic regions from complex genomic DNA Larionov et al. Transformation-associated recombination online casini initially been used online casini isolate large regions of mammalian genomic DNA in the s Larionov et al.

The propagation of TAR-generated DNA constructs online casini on ARS-like sequences, which can function as an origin of replication in yeast. The ARS sequences are frequently and randomly distributed throughout all eukaryotic genomes per 20—30 kb on average Stinchcomb et al. Ultime Notizie. Sciopero nazionale indetto per i giorni 22 e 23 aprile da CSLE Comparto scuola e da AL COBAS — Associazione lavoratori. IMPARA L'ARTE E METTILA DA PARTE. Circolare Auguri di Pasqua. Sciopero nazionale indetto per l'8 aprile dal SAESE. Le scuole Rodari e Spinelli incontrano il visit web page Frediano Sessi. Circolare sciopero 25 marzo online casini Servizi di trasporto.

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