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As B. subtilis was selected as a model bacterium, simple and efficient genetic tools have been developed in the past decades. Classical genome modification relies on the insertion of a selectable marker, usually an antibiotic resistance gene, into the chromosome of the target strain . The most commonly used scarless genetic manipulations systems for B. subtilis rely on counter-selectable markers (CSM) , while other methods include site-specific recombination systems (SSR) , and the recently developed CRISPR-Cas9 system .
CSM are often used for the markerless construction of engineered strains and have been used to construct Bacillus cell factories for various industrial applications. Selectable markers can generally be divided into positive and negative selection markers, whereby the former are most commonly antibiotic-resistance markers. In this classical approach, antibiotic-resistant strains are selected on appropriate agar plates (Fig. 2). In addition to the genomically integrated markers, Jeong et al. constructed a synthetic gene circuit consisting of a plasmid-based selection system, in which the Pxyl-lacI and neomycin resistant gene (neo) are integrated into the genome, while a Pspac-chloramphenicol (cat) resistant cassette and xylR gene are on the plasmid. In the first recombination, Pxyl-lacI and neo are integrated into the genome as a selectable marker. When xylose is added to the medium, the lacI gene is expressed then the chloramphenicol resistant gene is repressed. Consequently, the cell will survive only when the Pxyl-lacI and neo are deleted through a second round of recombination. Finally, the plasmid can be removed after several rounds of culture without chloramphenicol . This is a highly efficient method for genome engineering in B. subtilis, and it avoids the introduction of a selectable marker into the genome or the tightly controlled expression of a toxic gene. Other counter-selectable markers commonly used in B. subtilis include upp, blaI, araR, and hewI . Fabret et al. used the upp gene, which encodes uracil phosphoribosyltransferase as a counter-selection marker to achieve the transmission of unlabeled point mutations, in-frame deletions and large numbers of deletions on the chromosome . Brans et al. developed another method to knock out a single gene and introduce a new gene by combining the use of blaI, an antibiotic resistance gene, which encodes a repressor of the Bacillus licheniformis BlaP β-lactamase, with a conditional lysine-auxotrophic B. subtilis strain . However, CSM-based strategies require host pre-modification and have a low success rate due to the leaky expression of the CSM.Fig. 2
Schematic overview of genome editing methods based on counter-selectable markers. Left: genome editing (gene knockout as an example) with two integration steps. Step 1, an exogenous artificial DNA (plasmid or fragment) with up- and downstream homologous sequences is integrated into the genome, replacing the target gene. The recombinant clone can be selected under condition A. Step 2, under the selection condition B, the clone obtained in step 1 deleted the selectable marker and repressor/toxin gene through a self-recombination with the DR (direct repeats). Right: Composition of the selectable elements, the selectable marker A, toxin gene/repressor, and up- and downstream homologous sequences of the target gene can be constructed as fragments or on a plasmid. Examples of selectable markers A include: cat (chloramphenicol), phleo (phleomycin), or spe (spectinomycin). Examples of toxic genes include: upp, pyrF, or mazF. Examples of repressor genes include: xylR, blaI, araR, or lacI. The repressor can inhibit the expression of the selectable marker B, which can be integrated into the genome or a plasmid
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Site-specific recombination (SSR) systems are powerful tools for precise excision of DNA fragments. These systems, such as FLP/FRT  and Cre/loxP , have much higher recombination efficiency than the endogenous recombination systems, making them an ideal tool for many genetic manipulations. By combining a mutated Cre/lox system with the long segment fusion PCR method , Yan et al. developed a rapid and accurate B. subtilis genome engineering tool that allows operations such as targeted gene inactivation, long-fragment deletion, and in-frame deletion of target genes .
In recent years, the application of the CRISPR-related (Cas) system in B. subtilis has further enriched the gene editing toolbox. The CRISPR locus is first transcribed into a precursor CRISPR ribonucleic acid (pre-crRNA), which is then cut into small RNA units under the action of Cas protein or endonuclease. These small RNA units are mature crRNAs that contain spacer sequences and partial repeat sequences. Maturation of the crRNAs of type II CRISPR/Cas systems requires not only the participation of Cas9 and RNase, but also the guidance of a tracrRNA . Mature crRNAs and tracrRNA form double-stranded RNA structures through complementary base pairing. The resulting duplexes bind Cas9 protein to form a targeted cutting complex, specifically cutting foreign sequences to achieve the goal of identifying and eliminating invading foreign genes such as plasmids and viruses [21,22,23]. At present, there are three kinds of CRISPR/Cas9-based genome editing strategies widely used in B. subtilis. (1) The single-plasmid based system, in which Cas9, a single guide RNA (gRNA), donor DNA, and other elements are assembled into the same carrier skeleton, wherein Cas9 protein and gRNA are respectively expressed from inducible or strong constitutive promoters; (2) The two-plasmid-based system is more flexible than the single plasmid system. In this system, Cas9, gRNA, and donor DNA are assembled on two different plasmids, which are respectively used to produce Cas9 protein and deliver the gRNA transcription module and donor DNA template; (3) The chromosomally integrated system is more stable and effective than the first two systems, but it requires the use of engineered strains. The Cas9 was integrated into the genome, and then araE/R initiation subsystem was used to construct a multi-gRNA delivery vector . Furthermore, this CRISPR-Cas9 toolkit was extended to CRISPR interference (CRISPRi) for transcriptional-level regulation . The CRISPRi system is composed of a deactivated Cas9 (dCas9) protein and gRNA, enabling the targeting of dCsa9 to any target gene on the genome under the guidance of gRNA to inhibit its transcription without inducing a double-strand break, which can be applied to gene repression in metabolic engineering . So et al. developed a CRISPR-derived genome engineering technique to efficiently generate large genomic deletions in B. subtilis without the introduction of counter-selectable markers such as antibiotic-resistance genes, which had previously limited the application of B. subtilis in food engineering . This method has wide applicability for various types of site-directed mutagenesis in B. subtilis [24, 26, 27]. However, CRISPR/Cas9 has low efficiency in multi-gene editing. Liu et al. recently developed a CRISPR/Cas9n-mediated genome editing system, using Cas9n to exchange the natural Cas9 of existing constructs for B. subtilis and for iterative editing of the genome . This system is more effective than CRISPR/Cas9 in various types of gene modification and shows higher efficiency for large genomic deletions or multiplex gene editing. In terms of multi-gene editing and regulation, the newly developed CRISPR/Cpf1 system is the most powerful tool in B. subtilis. Compared with CRISPR/Cas9, CRISPR/Cpf1 has higher targeting specificity and can be used for gene editing in human cells, plant cells and many bacteria, while also offering a higher efficiency of multiplex gene editing . In fact, the system provides up to 100% efficiency of double in-frame knockouts, enables the introduction of multiple point mutations (up to six) with 100% efficiency, and can be used to simultaneously activate and/or inhibit multiple genes .
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