scholarly journals A simplified method for CRISPR-Cas9 engineering of Bacillus subtilis

2021 ◽  
Author(s):  
Ankita Jagdish Sachla ◽  
Alexander Jesus Alfonso ◽  
John D Helmann
Keyword(s):  
Bacillus Subtilis ◽  
Essential Gene ◽  
Site Specific ◽  
Guide Rna ◽  
Genome Modification ◽  
Pcr Product ◽  
Simplified Method ◽  
Intergenic Regions ◽  
Short Palindromic Repeat ◽  
Repair Template

The clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 system from Streptococcus pyogenes has been widely deployed as a tool for bacterial strain construction. Conventional CRISPR-Cas9 editing strategies require design and molecular cloning of an appropriate guide RNA (gRNA) to target genome cleavage and a repair template for introduction of the desired site-specific genome modification. Here, we present a streamlined method that leverages the existing collection of nearly 4000 Bacillus subtilis strains (the BKE collection) with individual genes replaced by an integrated erythromycin (erm) resistance cassette. A single plasmid (pAJS23) with a gRNA targeted to erm allows cleavage of the genome at any non-essential gene, and at sites nearby to many essential genes. This plasmid can be engineered to include a repair template, or the repair template can be co-transformed with the plasmid as either a PCR product or genomic DNA. We demonstrate the utility of this system for generating gene replacements, site-specific mutations, modification of intergenic regions, and introduction of gene-reporter fusions. In sum, this strategy bypasses the need for gRNA design and allows the facile transfer of mutations and genetic constructions with no requirement for intermediate cloning steps.

scholarly journals A Simplified Method for CRISPR-Cas9 Engineering of Bacillus subtilis

2021 ◽  
Author(s):  
Ankita J. Sachla ◽  
Alexander J. Alfonso ◽  
John D. Helmann
Keyword(s):  
Bacillus Subtilis ◽  
Genome Editing ◽  
Model Organism ◽  
Gram Positive ◽  
Content Type ◽  
Guide Rna ◽  
Simplified Method ◽  
Repair Template

Bacillus subtilis is a well-characterized Gram-positive model organism and a popular platform for biotechnology. Although many different CRISPR-based genome editing strategies have been developed for B. subtilis , they generally involve the design and cloning of a specific guide RNA (gRNA) and repair template for each application.

scholarly journals Development of a CRISPR-Cas9 Tool Kit for Comprehensive Engineering of Bacillus subtilis

2016 ◽  
Vol 82 (16) ◽  
pp. 4876-4895 ◽  
Author(s):  
Adam W. Westbrook ◽  
Murray Moo-Young ◽  
C. Perry Chou
Keyword(s):  
Bacillus Subtilis ◽  
Gene Mutations ◽  
Biochemical Properties ◽  
Site Specific ◽  
Content Type ◽  
Host System ◽  
Gene Insertion ◽  
Guide Rnas ◽  
Transcriptional Modulation ◽  
Short Palindromic Repeat

ABSTRACTThe establishment of a clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 system for strain construction inBacillus subtilisis essential for its progression toward industrial utility. Here we outline the development of a CRISPR-Cas9 tool kit for comprehensive genetic engineering inB. subtilis. In addition to site-specific mutation and gene insertion, our approach enables continuous genome editing and multiplexing and is extended to CRISPR interference (CRISPRi) for transcriptional modulation. Our tool kit employs chromosomal expression of Cas9 and chromosomal transcription of guide RNAs (gRNAs) using a gRNA transcription cassette and counterselectable gRNA delivery vectors. Our design obviates the need for multicopy plasmids, which can be unstable and impede cell viability. Efficiencies of up to 100% and 85% were obtained for single and double gene mutations, respectively. Also, a 2.9-kb hyaluronic acid (HA) biosynthetic operon was chromosomally inserted with an efficiency of 69%. Furthermore, repression of a heterologous reporter gene was achieved, demonstrating the versatility of the tool kit. The performance of our tool kit is comparable with those of systems developed forEscherichia coliandSaccharomyces cerevisiae, which rely on replicating vectors to implement CRISPR-Cas9 machinery.IMPORTANCEIn this paper, as the first approach, we report implementation of the CRISPR-Cas9 system inBacillus subtilis, which is recognized as a valuable host system for biomanufacturing. The study enables comprehensive engineering ofB. subtilisstrains with virtually any desired genotypes/phenotypes and biochemical properties for extensive industrial application.

scholarly journals Cloning and expression of meta-cleavage enzyme (CarB) of carbazole degradation pathway from Pseudomonas stutzeri

2005 ◽  
Vol 48 (spe) ◽  
pp. 127-134 ◽  
Author(s):  
Ariane Leites Larentis ◽  
Tito Lívio Moitinho Alves ◽  
Orlando Bonifácio Martins
Keyword(s):  
Active Form ◽  
Pseudomonas Stutzeri ◽  
Degradation Pathway ◽  
Cloning And Expression ◽  
Restriction Enzyme Digestion ◽  
Site Specific ◽  
Recombination System ◽  
Pcr Product ◽  
Cleavage Enzyme ◽  
Site Specific Recombination System

In this work, the 1082bp PCR product corresponding to carBaBb genes that encode the heterotetrameric enzyme 2'-aminobiphenyl-2,3-diol 1,2-dioxygenase (CarB), involved in the Pseudomonas stutzeri ATCC 31258 carbazole degradation pathway, was cloned using the site-specific recombination system. Recombinant clones were confirmed by PCR, restriction enzyme digestion and sequencing. CarB dioxygenase was expressed in high levels and in active form in Escherichia coli BL21-SI using the His-tagged expression vector pDEST TM17 and salt induction for 4h.

Development of nuclease-mediated site-specific genome modification

2012 ◽  
Vol 24 (5) ◽  
pp. 609-616 ◽  
Author(s):  
Stacey E Wirt ◽  
Matthew H Porteus
Keyword(s):  
Site Specific ◽  
Genome Modification

scholarly journals Targeted Isolation of a Designated Region of the Bacillus subtilis Genome by Recombinational Transfer

2004 ◽  
Vol 70 (4) ◽  
pp. 2508-2513 ◽  
Author(s):  
Satoshi Tomita ◽  
Kenji Tsuge ◽  
Yo Kikuchi ◽  
Mitsuhiro Itaya
Keyword(s):  
Bacillus Subtilis ◽  
Copy Number ◽  
Positional Cloning ◽  
Deletion Mutant ◽  
Essential Gene ◽  
Transfer System ◽  
Copy Number Plasmid ◽  
Low Copy Number ◽  
Subtilis Genome

ABSTRACT A method for positional cloning of the Bacillus subtilis genome was developed. The method requires a set of two small DNA fragments that flank the region to be copied. A 38-kb segment that carries genes ppsABCDE encoding five enzymes for antibiotic plipastatin synthesis and another genome locus as large as 100 kb including one essential gene were examined for positional cloning. The positional cloning vector for ppsABCDE was constructed using a B. subtilis low-copy-number plasmid that faithfully copied the precise length of the 38-kb DNA in vivo via the recombinational transfer system of this bacterium. Structure of the copied DNA was confirmed by restriction enzyme analyses. Furthermore, the unaltered structure of the 38-kb DNA was demonstrated by complementation of a ppsABCDE deletion mutant.

scholarly journals Bacillus subtilis and Escherichia coli essential genes and minimal cell factories after one decade of genome engineering

Microbiology ◽  
2014 ◽  
Vol 160 (11) ◽  
pp. 2341-2351 ◽  
Author(s):  
Mario Juhas ◽  
Daniel R. Reuß ◽  
Bingyao Zhu ◽  
Fabian M. Commichau
Keyword(s):  
Escherichia Coli ◽  
Bacillus Subtilis ◽  
Essential Gene ◽  
Building Blocks ◽  
Essential Genes ◽  
Minimal Cell ◽  
E Coli ◽  
Cell Factories ◽  
Gene Sets ◽  
K 12

Investigation of essential genes, besides contributing to understanding the fundamental principles of life, has numerous practical applications. Essential genes can be exploited as building blocks of a tightly controlled cell ‘chassis’. Bacillus subtilis and Escherichia coli K-12 are both well-characterized model bacteria used as hosts for a plethora of biotechnological applications. Determination of the essential genes that constitute the B. subtilis and E. coli minimal genomes is therefore of the highest importance. Recent advances have led to the modification of the original B. subtilis and E. coli essential gene sets identified 10 years ago. Furthermore, significant progress has been made in the area of genome minimization of both model bacteria. This review provides an update, with particular emphasis on the current essential gene sets and their comparison with the original gene sets identified 10 years ago. Special attention is focused on the genome reduction analyses in B. subtilis and E. coli and the construction of minimal cell factories for industrial applications.

scholarly journals Whole-Genome Sequencing and Phenotypic Analysis of Bacillus subtilis Mutants following Evolution under Conditions of Relaxed Selection for Sporulation

2011 ◽  
Vol 77 (19) ◽  
pp. 6867-6877 ◽  
Author(s):  
Christopher T. Brown ◽  
Laura K. Fishwick ◽  
Binna M. Chokshi ◽  
Marissa A. Cuff ◽  
Jay M. Jackson ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Chromosomal Rearrangements ◽  
Whole Genome ◽  
Nucleotide Polymorphisms ◽  
Relaxed Selection ◽  
Phenotypic Analysis ◽  
Competitive Fitness ◽  
Content Type ◽  
Intergenic Regions ◽  
Selection For

ABSTRACTLittle is known about how genetic variation at the nucleotide level contributes to competitive fitness within species. During a 6,000-generation study ofBacillus subtilisevolved under relaxed selection for sporulation, a new strain, designated WN716, emerged with significantly different colony and cell morphologies; loss of sporulation, competence, acetoin production, and motility; multiple auxotrophies; and increased competitive fitness (H. Maughan and W. L. Nicholson, Appl. Environ. Microbiol.77:4105–4118, 2011). The genome of WN716 was analyzed by OpGen optical mapping, whole-genome 454 pyrosequencing, and the CLC Genomics Workbench. No large chromosomal rearrangements were found; however, 34 single-nucleotide polymorphisms (SNPs) and +1 frameshifts were identified in WN716 that resulted in amino acid changes in coding sequences of annotated genes, and 11 SNPs were located in intergenic regions. Several classes of genes were affected, including biosynthetic pathways, sporulation, competence, and DNA repair. In several cases, attempts were made to link observed phenotypes of WN716 with the discovered mutations, with various degrees of success. For example, a +1 frameshift was identified at codon 13 ofsigW, the product of which (SigW) controls a regulon of genes involved in resistance to bacteriocins and membrane-damaging antibiotics. Consistent with this finding, WN716 exhibited sensitivity to fosfomycin and to a bacteriocin produced byB. subtilissubsp.spizizeniiand exhibited downregulation of SigW-dependent genes on a transcriptional microarray, consistent with WN716 carrying a knockout ofsigW. The results suggest that propagation ofB. subtilisfor less than 2,000 generations in a nutrient-rich environment where sporulation is suppressed led to rapid initiation of genomic erosion.

Sign in / Sign up

Export Citation Format

Share Document