serial cloner construct digest only one end

subtilis can be cultivated under the same conditions as Escherichia coli in LB broth at 37☌. Special handling of the BGM vector system is not required. subtilis genome is engineered to have homologous cloning site sequences, a target DNA fragment flanked by homologous sequences is easily cloned into the BGM. subtilis genome by homologous recombination. The recombinogenic DNA is incorporated into the B. The transformation machinery non-specifically binds and imports extracellular DNA fragments into the cytoplasm in single-stranded form. subtilis expresses competence-related genes at the late stage of cell growth, and their products, transformation machinery molecules, are assembled in the cell membrane. The cloning strategy for this vector system is based on unique B. The Bacillus subtilis genome (BGM) vector system has been developed as a novel cloning system using a unique concept in which the entire 4.2 Mb genome of B. Thus, these two systems have complementary advantages over each other in terms of cloning capacity and insert stability.

Generally, the isolation of intact YACs is difficult because of their linear form and contamination with endogenous yeast chromosomes. Although the inserts can be easily modified by homologous recombination, YACs often suffer from insert chimerism and unwanted rearrangements due to potent and constitutive yeast recombination activity. In contrast to the BAC system, genomic DNA inserts of up to 2 Mb can be used with the YAC system. The BAC system can generally accommodate up to 300 kb genomic inserts. The methods for modifying BAC inserts require additional recombination components, e.g., RecA or phage-derived recombination proteins. BAC clones are easy to manipulate and retrieve due to their plasmid form and the stability of the cloned DNA. However, there are several technological limitations in their cloning size, genetic modification and insert stability. BAC and YAC transgenesis techniques have contributed greatly to genome research. Artificial chromosomes, such as bacterial artificial chromosomes (BACs) and yeast artificial chromosomes (YACs), have been used for these purposes in combination with transgenesis. Technological developments in chromosome engineering are essential for the manipulation and functional analysis of genomic DNA fragments. Using this system, we provide the first experimental evidence of a cis-acting element for a class I OR gene. Thus, the BGM vector system can be an alternative platform for engineering large DNA fragments in addition to conventional systems such as bacterial and yeast artificial chromosomes. In addition, we demonstrate that this system can be used for mouse transgenesis. We offer a complete genetic modification method for the BGM vector system, including insertion, deletion, inversion and fusion, to engineer genomic DNA fragments without any trace of modifications. Transgenic mice carrying the enlarged transgene recapitulated the expression and axonal projection patterns of the target class I OR gene in the main olfactory system. We further demonstrated that the modified, reconstructed genomic DNA fragments could be used to generate transgenic mice. DNA sequencing revealed that no mutations occurred during gene manipulations with the BGM vector. Because they were oriented in opposite directions, we performed an inversion modification to align their orientation and then fused them to enlarge the genomic structure. Using two contiguous bacterial artificial chromosome clones containing several class I OR genes, we constructed two transgenes in the BGM vector by inserting a reporter gene cassette into one class I OR gene. Although a cis-acting locus control region is expected to regulate transcription, this has not yet been determined experimentally. To explore the potential of the BGM vector, we focused on the fish-like odorant receptor (class I OR) gene family, which consists of 158 genes and forms a single gene cluster. In this study, we developed important additions to the genetic modification methods of the BGM vector system. However, genetic modifications using the BGM vector system have not been fully established, and this system has not been applied to transgenesis.

The BGM vector system has several attractive properties, such as a large cloning capacity of over 3 Mb, stable propagation of cloned DNA and various modification strategies using RecA-mediated homologous recombination. The Bacillus subtilis genome (BGM) vector is a novel cloning system for large DNA fragments, in which the entire 4.2 Mb genome of B.

Serial cloner construct digest only one end