Construction of a vector system for molecular cloning in Bacillus subtilis and Escherichia coli

Лагодич, А. В.; Cherva, E. A.; Shtaniuk, Ya. V.; Прокулевич, В. А.; Fomichev, Yu. K.; Prozorov, A. A.; Титок, М. А.

doi:10.1007/s11008-005-0043-7

Cited by 6 publications

(3 citation statements)

References 24 publications

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“…Construction of the plasmid pJOE8999 started with the plasmid pJOE8829.1 (unpublished), consisting of the pUC18 minimal origin (20), the temperature-sensitive minimal replicon from plasmid pE194 ts (27), the spectinomycin resistance gene aad9 from pDG1730 (28), and a chloramphenicol resistance gene from pMTLBS72 (29). In the first step, the aad9 gene was replaced by a sequence consisting of two SfiI sites, separated by a SmaI site and a T7 promoter sequence.…”

Section: Methodsmentioning

confidence: 99%

Editing of the Bacillus subtilis Genome by the CRISPR-Cas9 System

Altenbuchner

2016

Appl Environ Microbiol

277

246

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The clustered regularly interspaced short palindromic repeat (CRISPR)-associated (Cas) systems are adaptive immune systems of bacteria. A type II CRISPR-Cas9 system from Streptococcus pyogenes has recently been developed into a genome engineering tool for prokaryotes and eukaryotes. Here, we present a single-plasmid system which allows efficient genome editing of Bacillus subtilis. The plasmid pJOE8999 is a shuttle vector that has a pUC minimal origin of replication for Escherichia coli, the temperature-sensitive replication origin of plasmid pE194 ts for B. subtilis, and a kanamycin resistance gene working in both organisms. For genome editing, it carries the cas9 gene under the control of the B. subtilis mannose-inducible promoter P manP and a single guide RNA (sgRNA)-encoding sequence transcribed via a strong promoter. This sgRNA guides the Cas9 nuclease to its target. The 20-nucleotide spacer sequence at the 5= end of the sgRNA sequence, responsible for target specificity, is located between BsaI sites. Thus, the target specificity is altered by changing the spacer sequences via oligonucleotides fitted between the BsaI sites. Cas9 in complex with the sgRNA induces double-strand breaks (DSBs) at its target site. Repair of the DSBs and the required modification of the genome are achieved by adding homology templates, usually two PCR fragments obtained from both sides of the target sequence. Two adjacent SfiI sites enable the ordered integration of these homology templates into the vector. The function of the CRISPR-Cas9 vector was demonstrated by introducing two large deletions in the B. subtilis chromosome and by repair of the trpC2 mutation of B. subtilis 168. IMPORTANCEIn prokaryotes, most methods used for scarless genome engineering are based on selection-counterselection systems. The disadvantages are often the lack of a suitable counterselection marker, the toxicity of the compounds needed for counterselection, and the requirement of certain mutations in the target strain. CRISPR-Cas systems were recently developed as important tools for genome editing. The single-plasmid system constructed for the genome editing of B. subtilis overcomes the problems of counterselection methods. It allows deletions and introduction of point mutations. It is easy to handle and very efficient, and it may be adapted for use in other firmicutes.

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Section: Methodsmentioning

confidence: 99%

Editing of the Bacillus subtilis Genome by the CRISPR-Cas9 System

Altenbuchner

2016

Appl Environ Microbiol

277

246

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“…Amplification of the promoter region of mtlR ( P mtlR ) was carried out using s5799/s6392 (pKAM18). All of the amplified promoter fragments were double digested by Nhe I and Afl II in order to be fused to lacZ as the reporter gene in pSUN279.2 [ 5 ], a derivative of pMTLBS72 [ 35 ]. Plasmid pMTLBS72 is a B.subtilis/E.coli shuttle vector containing a pBR322 origin of replication for E. coli and pBS72 origin of replication for B. subtilis .…”

Section: Methodsmentioning

confidence: 99%

Regulation of mtl operon promoter of Bacillus subtilis: requirements of its use in expression vectors

2011

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BackgroundSeveral vector systems have been developed to express any gene desired to be studied in Bacillus subtilis. Among them, the transcriptionally regulated promoters involved in carbohydrate utilization are a research priority. Expression systems based on Bacillus promoters for xylose, maltose, and mannose utilization, as well as on the heterologous E. coli lactose promoter, have been successfully constructed. The promoter of the mtlAFD operon for utilization of mannitol is another promising candidate for its use in expression vectors. In this study, we investigated the regulation of the mtl genes in order to identify the elements needed to construct a strong mannitol inducible expression system in B. subtilis.ResultsRegulation of the promoters of mtlAFD operon (PmtlA) and mtlR (PmtlR) encoding the activator were investigated by fusion to lacZ. Identification of the PmtlA and PmtlR transcription start sites revealed the σA like promoter structures. Also, the operator of PmtlA was determined by shortening, nucleotide exchange, and alignment of PmtlA and PmtlR operator regions. Deletion of the mannitol-specific PTS genes (mtlAF) resulted in PmtlA constitutive expression demonstrating the inhibitory effect of EIICBMtl and EIIAMtl on MtlR in the absence of mannitol. Disruption of mtlD made the cells sensitive to mannitol and glucitol. Both PmtlA and PmtlR were influenced by carbon catabolite repression (CCR). However, a CcpA deficient mutant showed only a slight reduction in PmtlR catabolite repression. Similarly, using PgroE as a constitutive promoter, putative cre sites of PmtlA and PmtlR slightly reduced the promoter activity in the presence of glucose. In contrast, glucose repression of PmtlA and PmtlR was completely abolished in a ΔptsG mutant and significantly reduced in a MtlR (H342D) mutant.ConclusionsThe mtl operon promoter (PmtlA) is a strong promoter that reached a maximum of 13,000 Miller units with lacZ as a reporter on low copy plasmids. It is tightly regulated by just one copy of the mtlR gene on the chromosome and subject to CCR. CCR can be switched off by mutations in MtlR and the glucose transporter. These properties and the low costs of the inducers, i.e. mannitol and glucitol, make the promoter ideal for designing regulated expression systems.

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“…Vector pMTL7-1 [15] is able to be replicated in B. subtilis, so when confirming the effectiveness of transformation, pMTL7-1 was used as a positive control of transformation in this study. The vector pMTL7-1 with chloramphenicol resistance could be able to use for screening transformants.…”

Section: Methodsmentioning

confidence: 93%

Analysis of the efficiency factors of electrotransformation of Bacillus subtilis to inactivate the aroK gene by the method of homologous recombination

Chao¹,

Lahodzich

2021

Journal of the Belarusian State University. Biology

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A hyper-osmotic electrotransformation method was developed for strain Bacillus subtilis. Sorbitol and mannitol are included in the hyper-osmotic electroporation medium and recovery medium. In this study, the hyper-osmotic electroporation method was optimised to increase the transformation efficiency of B. subtilis strain 5434 (non-transformable by chemical methods) by 430 fold, with a maximum value of 8.6 ⋅ 105 CFU/µg of integrative plasmid DNA. With the electroporation setted 25 µF, 23 kV/cm, 200 Ω, the method was optimised as follows: a) the OD600 value of the bacterial culture solution was increased to about 1.2, which significantly enhanced survival of bacteria and quantity of viable B.subtilis strain 5434 cells after electroporation; b) the elution frequency of washing solution (hyper-osmotic electroporation medium) for complement cells was increased from 3 to 5 times, resulted in significantly reducing the conductivity of the hyper-osmotic electoporation medium with competent cells (electrocompetent cultue), and effectively extending the pulse time under the same electric field strength; c) quantity of integrative plasmid DNA added to hyper-osmotic electrocompetent culture was optimised. These results indicate that increasing the number of viable B. subtilis strain 5434 cells and reducing the number of metal ions in the electroporation solution mix (integrative plasmid DNA, competent cells of B. subtilis strain 5434, electroporation medium) are useful approach to improve transfomation efficiency of B. subtilis strain 5434. Concentration of shikimic acid in the fermentation medium was quantified by high performance liquid chromatography. Quantification of shikimic acid revealed that B. subtilis strain 5434p4SA produced 403.98 ± 9.1 µg/mL of shikimic acid.

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Construction of a vector system for molecular cloning in Bacillus subtilis and Escherichia coli

Cited by 6 publications

References 24 publications

Editing of the Bacillus subtilis Genome by the CRISPR-Cas9 System

Editing of the Bacillus subtilis Genome by the CRISPR-Cas9 System

Regulation of mtl operon promoter of Bacillus subtilis: requirements of its use in expression vectors

Analysis of the efficiency factors of electrotransformation of Bacillus subtilis to inactivate the aroK gene by the method of homologous recombination

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