scholarly journals Bacillus SEVA siblings: A Golden Gate-based toolbox to create personalized integrative vectors for Bacillus subtilis

2017 ◽  
Vol 7 (1) ◽  
Author(s):  
Jara Radeck ◽  
Daniel Meyer ◽  
Nina Lautenschläger ◽  
Thorsten Mascher
Keyword(s):  
Bacillus Subtilis ◽  
Homologous Recombination ◽  
Free Choice ◽  
Fixed Combination ◽  
Golden Gate ◽  
One Pot ◽  
Natural Competence ◽  
As Species ◽  
Resistance Markers ◽  
Integrative Vectors

Abstract Bacillus subtilis combines natural competence for genetic transformation with highly efficient homologous recombination. These features allow using vectors that integrate into the genome via double homologous recombination. So far, their utilization is restricted by the fixed combination of resistance markers and integration loci, as well as species- or strain-specific regions of homology. To overcome these limitations, we developed a toolbox for the creation of personalized Bacillus vectors in a standardized manner with a focus on fast and easy adaptation of the sequences specifying the integration loci. We based our vector toolkit on the Standard European Vector Architecture (SEVA) to allow the usage of their vector parts. The Bacillus SEVA siblings are assembled via efficient one-pot Golden Gate reactions from four entry parts with the choice of four different enzymes. The toolbox contains seven Bacillus resistance markers, two Escherichia coli origins of replication, and a free choice of integration loci. Vectors can be customized with a cargo, before or after vector assembly, and could be used in different B. subtilis strains and potentially beyond. Our adaptation of the SEVA-standard provides a powerful and standardized toolkit for the convenient creation of personalized Bacillus vectors.

scholarly journals Creation of Golden Gate constructs for gene doctoring

2020 ◽  
Vol 20 (1) ◽  
Author(s):  
Nicholas M. Thomson ◽  
Chuanzhen Zhang ◽  
Eleftheria Trampari ◽  
Mark J. Pallen
Keyword(s):  
Free Choice ◽  
Gene Editing ◽  
Fluorescent Protein ◽  
Dna Fragments ◽  
Golden Gate ◽  
Donor Plasmid ◽  
Recombination System ◽  
Green Fluorescent ◽  
Λ Red

Abstract Background Gene doctoring is an efficient recombination-based genetic engineering approach to mutagenesis of the bacterial chromosome that combines the λ-Red recombination system with a suicide donor plasmid that is cleaved in vivo to generate linear DNA fragments suitable for recombination. The use of a suicide donor plasmid makes Gene Doctoring more efficient than other recombineering technologies. However, generation of donor plasmids typically requires multiple cloning and screening steps. Results We constructed a simplified acceptor plasmid, called pDOC-GG, for the assembly of multiple DNA fragments precisely and simultaneously to form a donor plasmid using Golden Gate assembly. Successful constructs can easily be identified through blue-white screening. We demonstrated proof of principle by inserting a gene for green fluorescent protein into the chromosome of Escherichia coli. We also provided related genetic parts to assist in the construction of mutagenesis cassettes with a tetracycline-selectable marker. Conclusions Our plasmid greatly simplifies the construction of Gene Doctoring donor plasmids and allows for the assembly of complex, multi-part insertion or deletion cassettes with a free choice of target sites and selection markers. The tools we developed are applicable to gene editing for a wide variety of purposes in Enterobacteriaceae and potentially in other diverse bacterial families.

scholarly journals Mutagenesis and ultraviolet inactivation of transforming DNA of Haemophilus influenzae complexed with a Bacillus subtilis protein that alters DNA conformation.

1996 ◽  
Vol 43 (1) ◽  
pp. 107-114 ◽  
Author(s):  
J K Setlow ◽  
B C Setlow ◽  
P Setlow
Keyword(s):  
Bacillus Subtilis ◽  
Haemophilus Influenzae ◽  
Dna Conformation ◽  
Erythromycin Resistance ◽  
Bacillus Subtilis Spore ◽  
Pyrimidine Dimers ◽  
Resistance Markers ◽  
Selection For

The wild-type Bacillus subtilis spore protein, SspCwt, binds to DNA in vitro and in vivo and changes the conformation of DNA from B to A. Synthesis of the cloned SspCwt gene in Escherichia coli also causes large increases in mutation frequency. Binding of SspCwt to transforming DNA from Haemophilus influenzae made the DNA resistant to ultraviolet (UV) radiation. The mutant protein, SspCala, which does not bind DNA, did not change the UV resistance. The UV sensitivity of the DNA/SspCwt complex was not increased when the recipients of the DNA were defective in excision of pyrimidine dimers. These data indicate that the H. influenzae excision mechanism does not operate on the spore photoproduct formed by UV irradiation of the complex. Selection for the streptomycin- or erythromycin-resistance markers on the transforming DNA evidenced significant mutations at loci closely linked to these, but not at other loci. SspCwt apparently entered the cell attached to the transforming DNA, and caused mutations in adjacent loci. The amount of such mutations decreased when the transforming DNA was UV irradiated, because UV unlinks linked markers.

Intramolecular homologous recombination in Bacillus subtilis 168

1992 ◽  
Vol 236 (1) ◽  
pp. 60-64 ◽  
Author(s):  
Juan C. Alonso ◽  
Gerhild Lüder ◽  
Thomas A. Trautner
Keyword(s):  
Bacillus Subtilis ◽  
Homologous Recombination ◽  
Bacillus Subtilis 168

scholarly journals Genetic Recombination in Bacillus subtilis 168: Effect of ΔhelD on DNA Repair and Homologous Recombination

2001 ◽  
Vol 183 (19) ◽  
pp. 5772-5777 ◽  
Author(s):  
Begoña Carrasco ◽  
Silvia Fernández ◽  
Marie-Agnes Petit ◽  
Juan C. Alonso
Keyword(s):  
Dna Repair ◽  
Bacillus Subtilis ◽  
Homologous Recombination ◽  
Genetic Recombination ◽  
Methyl Methanesulfonate ◽  
Plasmid Transformation ◽  
Dna Damaging Agents ◽  
Bacillus Subtilis 168 ◽  
Recombination Repair ◽  
Helicase Iv

ABSTRACT The B. subtilis ΔhelD allele rendered cells proficient in transformational recombination and moderately sensitive to methyl methanesulfonate when present in an otherwise Rec+ strain. The ΔhelD allele was introduced into rec-deficient strains representative of the α (recF strain), β (addA addB), γ (recH), ɛ (ΔrecU), and ζ (ΔrecS) epistatic groups. The ΔhelDmutation increased the sensitivity to DNA-damaging agents ofaddAB, ΔrecU, and ΔrecS cells, did not affect the survival ofrecH cells, and decreased the sensitivity ofrecF cells. ΔhelD also partially suppressed the DNA repair phenotype of other mutations classified within the α epistatic group, namely the recL, ΔrecO, and recR mutations. The ΔhelD allele marginally reduced plasmid transformation (three- to sevenfold) of mutations classified within the α, β, and γ epistatic groups. Altogether, these data indicate that the loss of helicase IV might stabilize recombination repair intermediates formed in the absence of recFLOR and renderrecFLOR, addAB, andrecH cells impaired in plasmid transformation.

scholarly journals Optimization of Golden Gate assembly through application of ligation sequence-dependent fidelity and bias profiling

2018 ◽  
Author(s):  
Potapov Vladimir ◽  
Jennifer L. Ong ◽  
Rebecca B. Kucera ◽  
Bradley W. Langhorst ◽  
Katharina Bilotti ◽  
...  
Keyword(s):  
Regulatory Elements ◽  
Dna Assembly ◽  
Golden Gate ◽  
End Joining ◽  
One Pot ◽  
Data Set ◽  
Flexible Assembly ◽  
Comprehensive Method ◽  
Sequence Dependent ◽  
Single Reaction

ABSTRACTModern synthetic biology depends on the manufacture of large DNA constructs from libraries of genes, regulatory elements or other genetic parts. Type IIS restriction enzyme-dependent DNA assembly methods (e.g., Golden Gate) enable rapid one-pot, ordered, multi-fragment DNA assembly, facilitating the generation of high-complexity constructs. The order of assembly of genetic parts is determined by the ligation of flanking Watson-Crick base-paired overhangs. The ligation of mismatched overhangs leads to erroneous assembly, and the need to avoid such pairings has typically been accomplished by using small sets of empirically vetted junction pairs, limiting the number of parts that can be joined in a single reaction. Here, we report the use of a comprehensive method for profiling end-joining ligation fidelity and bias to predict highly accurate sets of connections for ligation-based DNA assembly methods. This data set allows quantification of sequence-dependent ligation efficiency and identification of mismatch-prone pairings. The ligation profile accurately predicted junction fidelity in ten-fragment Golden Gate assembly reactions, and enabled efficient assembly of a lac cassette from up to 24-fragments in a single reaction. Application of the ligation fidelity profile to inform choice of junctions thus enables highly flexible assembly design, with >20 fragments in a single reaction.

RecA is required for the assembly of RecN into DNA repair complexes on the nucleoid

2021 ◽  
Author(s):  
Emma K. McLean ◽  
Justin S. Lenhart ◽  
Lyle A. Simmons
Keyword(s):  
Dna Damage ◽  
Bacillus Subtilis ◽  
Homologous Recombination ◽  
Normal Growth ◽  
Strand Break ◽  
Double Strand Break ◽  
Focus Formation ◽  
Processing Enzyme ◽  
Break Repair

Homologous recombination requires the coordinated effort of several proteins to complete break resection, homologous pairing and resolution of DNA crossover structures. RecN is a conserved bacterial protein important of double strand break repair and a member of the Structural Maintenance of Chromosomes (SMC) protein family. Current models in Bacillus subtilis propose that RecN responds to double stranded breaks prior to RecA and end processing suggesting that RecN is among the very first proteins responsible for break detection. Here, we investigate the contribution of RecA and end processing by AddAB to RecN recruitment into repair foci in vivo . Using this approach, we found that recA is required for RecN-GFP focus formation on the nucleoid during normal growth and in response to DNA damage. In the absence of recA function, RecN foci form in a low percentage of cells, RecN localizes away from the nucleoid, and RecN fails to assemble in response to DNA damage. In contrast, we show that the response of RecA-GFP foci to DNA damage is unchanged in the presence or absence of recN . In further support of RecA activity preceding RecN we show that ablation of the double-strand break end processing enzyme addAB results in a failure of RecN to form foci in response to DNA damage. With these results, we conclude that RecA and end processing function prior to RecN establishing a critical step for the recruitment and participation of RecN during DNA break repair in Bacillus subtilis . IMPORTANCE Homologous recombination is important for the repair of DNA double-strand breaks. RecN is a highly conserved protein that has been shown to be important for sister chromatid cohesion and for survival to break-inducing clastogens. Here, we show that the assembly of RecN into repair foci on the bacterial nucleoid requires the end processing enzyme AddAB and the recombinase RecA. In the absence of either recA or end processing RecN-GFP foci are no longer DNA damage inducible and foci form in a subset of cells as large complexes in regions away from the nucleoid. Our results establish the stepwise order of action, where double-strand break end processing and RecA association precede the participation of RecN during break repair in Bacillus subtilis .

scholarly journals Natural Genetic Competence in Bacillus subtilis Natto OK2

2000 ◽  
Vol 182 (9) ◽  
pp. 2411-2415 ◽  
Author(s):  
Sayaka Ashikaga ◽  
Hideaki Nanamiya ◽  
Yoshiaki Ohashi ◽  
Fujio Kawamura
Keyword(s):  
Bacillus Subtilis ◽  
Base Change ◽  
Competence Development ◽  
Transformation Frequency ◽  
Genetic Competence ◽  
Natural Competence ◽  
Bacillus Subtilis Natto ◽  
High Transformation ◽  
Single Base Change ◽  
Natural Genetic Competence

ABSTRACT We isolated a Bacillus subtilis natto strain, designated OK2, from a lot of commercial fermented soybean natto and studied its ability to undergo natural competence development using acomG-lacZ fusion at the amyE locus. Although transcription of the late competence genes was not detected in theB. subtilis natto strain OK2 during competence development, these genes were constitutively transcribed in the OK2 strain carrying either the mecA or the clpC mutation derived from B. subtilis 168. In addition, both OK2 mutants exhibited high transformation frequencies, comparable with that observed for B. subtilis 168. Moreover, as expected from these results, overproduction of ComK derived from strain 168 in strain OK2 resulted in a high transformation frequency as well as in induction of the late competence genes. These results clearly indicated that ComK produced in both the mecA and clpC mutants of strain OK2 (ComKOK2) could activate the transcription of the whole set of late competence genes and suggested that ComKOK2 was not activated in strain OK2 during competence development. We therefore sequenced the comS gene of OK2 and compared it with that of 168. The comS OK2had a single-base change, resulting in the replacement of Ser (strain 168) by Cys (strain OK2) at position 11.

scholarly journals Natural Competence Rates Are Variable Among Xylella fastidiosa Strains and Homologous Recombination Occurs In Vitro Between Subspecies fastidiosa and multiplex

2017 ◽  
Vol 30 (7) ◽  
pp. 589-600 ◽  
Author(s):  
Prem P. Kandel ◽  
Rodrigo P. P. Almeida ◽  
Paul A. Cobine ◽  
Leonardo De La Fuente
Keyword(s):  
Homologous Recombination ◽  
Fluorescent Protein ◽  
Xylella Fastidiosa ◽  
Dna Recombination ◽  
Bacterial Cells ◽  
Disease Emergence ◽  
Natural Competence ◽  
In Silico Studies ◽  
Green Fluorescent

Xylella fastidiosa, an etiological agent of emerging crop diseases around the world, is naturally competent for the uptake of DNA from the environment that is incorporated into its genome by homologous recombination. Homologous recombination between subspecies of X. fastidiosa was inferred by in silico studies and was hypothesized to cause disease emergence. However, no experimental data are available on the degree to which X. fastidiosa strains are capable of competence and whether recombination can be experimentally demonstrated between subspecies. Here, using X. fastidiosa strains from different subspecies, natural competence in 11 of 13 strains was confirmed with plasmids containing antibiotic markers flanked by homologous regions and, in three of five strains, with dead bacterial cells used as source of donor DNA. Recombination frequency differed among strains and was correlated to growth rate and twitching motility. Moreover, intersubspecific recombination occurred readily between strains of subsp. fastidiosa and multiplex, as demonstrated by movement of antibiotic resistance and green fluorescent protein from donor to recipient cells and confirmed by DNA sequencing of the flanking arms of recombinant strains. Results demonstrate that natural competence is widespread among X. fastidiosa strains and could have an impact in pathogen adaptation and disease development.

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