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Cell lysates from a laboratory strain of Escherichia coli can be exploited for seamless DNA cloning in vitro , which is named the seamless ligation cloning extract (SLiCE) cloning method. The SLiCE method can incorporate DNA fragments into a vector to achieve conventional DNA cloning and is more cost-effective than commercially seamless DNA cloning kits. In this study, we found that the SLiCE extracts could easily be prepared with different methods, such as 3% Triton X-100 lysis buffer, 3% SDS lysis buffer, or freeze–thaw cycles. At high E. coli transformation efficiency, the SLiCE extracts prepared using different simple and ultra-low cost methods did not affect the DNA cloning efficiency. These results further revealed that the SLiCE cloning method can be efficiently used for seamless DNA cloning in vitro. • SLiCE extracts can be prepared by different simple and ultra-low cost methods. • All SLiCE extracts prepared by different methods exhibited sufficient DNA cloning activity. • Transformation efficiency of E. coli affected the cloning efficiency of the SLiCE extracts. [ABSTRACT FROM AUTHOR]

Abstract: The genetic manipulation of cells is the most promising strategy for designing microorganisms with desired traits. The most widely used approaches for integrating specific DNA-fragments into the Escherichia coli genome are based on bacteriophage site-specific and Red/ET-mediated homologous recombination systems. Specifically, the recently developed Dual In/Out integration strategy enables the integration of DNA fragments directly into specific chromosomal loci (). To develop this strategy further, we designed a method for the precise cloning of any long DNA fragments from the E. coli chromosome and their targeted insertion into the genome that does not require PCR. In this method, the region of interest is flanked by I-SceI rare-cutting restriction sites, and the I-SceI-bracketed region is cloned into the unique I-SceI site of an integrative plasmid vector that then enables its targeted insertion into the E. coli chromosome via bacteriophage φ80 Int-mediated specialized recombination. This approach allows any long specific DNA fragment from the E. coli genome to be cloned without a PCR amplification step and reproducibly inserted into any chosen chromosomal locus. The developed method could be particularly useful for the construction of marker-less and plasmid-less recombinant strains in the biotechnology industry. [Copyright &y& Elsevier]

Abstract: The gene(s) encoding enzyme(s) involved in the initial reaction during degradation of zearalenone (ZEA) was characterized from the zearalenone utilizer Pseudomonas putida strain ZEA-1, where ZEA was transformed into product with less or no toxicity. A 5.5 kilobase-pair (kbp) Pst1–Kpn1 fragment containing gene(s) encoding for zearalenone degradation was cloned. The cloned gene(s) was actively expressed in Escherichia coli. ZEA degradation by recombinant E. coli was relatively rapid and effective, leaving no detectable ZEA after 24h. In further experiments, cell-free extract of E. coli has been used in the same way, both to confirm these observations and the enzymatic nature of the degradation activity. [Copyright &y& Elsevier]

Abstract: Type IIS restriction enzymes have been successfully used as “universal” restriction enzymes in DNA manipulations. We took a step further to develop a rapid technique for recombining DNA fragments, fully automatic single-tube recombination (FASTR), which enables multiple-fragment DNA recombination in a single step. Crude PCR products are directly mixed with both type IIS restriction endonuclease and DNA ligase to initiate a spontaneous and one-way recombination reaction. Highly efficient DNA recombination can be achieved by an inhibition of DNA polymerase with aphidicolin and a selective digestion of template DNAs by DpnI, a restriction enzyme to digest hemi-methylated DNA in the reaction solution; thereby the entire procedure takes less than 15min. Owing to its simplicity, efficiency and rapidity, one-step FASTR can be applied to a wide range of DNA manipulations including those involving high-throughput applications where significant reduction in time and cost is expected. [Copyright &y& Elsevier]

Highlights: [•] Poxvirus vectors can accommodate and express large amounts of heterologous DNA. [•] An important use of poxvirus vectors has been to characterize targets of immunity. [•] Poxvirus-based veterinary vaccines are licensed and human ones are in clinical trials. [ABSTRACT FROM AUTHOR]