A versatile mini-mazF-cassette for marker-free targeted genetic modification in Bacillus subtilis

Lin, Zhiwei; Deng, Bin; Jiao, Zhihua; Wu, Bingbing; Xu, Xin; Yu, Dongmin; Li, Weifen

doi:10.1016/j.mimet.2013.07.020

Cited by 13 publications

(14 citation statements)

References 25 publications

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“…Genome engineering using the mazF system resulted in many false-positive clones in our experimental condition, and was not a proper method. Recently, an improved method using a mini- mazF -cassette was reported ( 29 ). They used direct repeat (DR) sequences for the in vivo recombination in the amyE deletion and gfp insertion experiments, which resulted in the DR remaining in the chromosome during deletion of the selectable marker system.…”

Section: Discussionmentioning

confidence: 99%

Genome engineering using a synthetic gene circuit in Bacillus subtilis

Jeong

Park

Pan

et al. 2014

Nucleic Acids Research

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Genome engineering without leaving foreign DNA behind requires an efficient counter-selectable marker system. Here, we developed a genome engineering method in Bacillus subtilis using a synthetic gene circuit as a counter-selectable marker system. The system contained two repressible promoters (B. subtilis xylA (Pxyl) and spac (Pspac)) and two repressor genes (lacI and xylR). Pxyl-lacI was integrated into the B. subtilis genome with a target gene containing a desired mutation. The xylR and Pspac–chloramphenicol resistant genes (cat) were located on a helper plasmid. In the presence of xylose, repression of XylR by xylose induced LacI expression, the LacIs repressed the Pspac promoter and the cells become chloramphenicol sensitive. Thus, to survive in the presence of chloramphenicol, the cell must delete Pxyl-lacI by recombination between the wild-type and mutated target genes. The recombination leads to mutation of the target gene. The remaining helper plasmid was removed easily under the chloramphenicol absent condition. In this study, we showed base insertion, deletion and point mutation of the B. subtilis genome without leaving any foreign DNA behind. Additionally, we successfully deleted a 2-kb gene (amyE) and a 38-kb operon (ppsABCDE). This method will be useful to construct designer Bacillus strains for various industrial applications.

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Section: Discussionmentioning

confidence: 99%

Genome engineering using a synthetic gene circuit in Bacillus subtilis

Jeong

Park

Pan

et al. 2014

Nucleic Acids Research

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“…For B. subtilis 168, a mazF toxin-based ( 58 ), a Cre-Lox based ( 59 ) and a CRISPR based ( 60, 61 ) engineering methods were selected. The toxin-antibiotic cassette assembly was achieved by HiFi assembly (NEB) of all the parts.…”

Section: Supplementary Materials Formentioning

confidence: 99%

Versioning Biological Cells for Trustworthy Cell Engineering

Tellechea‐Luzardo

Velázquez

et al. 2021

Preprint

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Abstract“Full-stack”biotechnology platforms for cell line (re)programming are on the horizon, due mostly to (a) advances in gene synthesis and editing techniques as well as (b) the growing integration with informatics, the internet of things and automation. These emerging platforms will accelerate the production and consumption of biological products. Hence, transparency, traceability and -ultimately-trustworthiness is required -from cradle to grave- for engineered cell lines and their engineering processes. We report here the first version control system for cell engineering that integrates a new cloud-based version control software for cell lines’ digital footprint with molecular barcoding of living samples. We argue that version control for cell engineering marks a significant step towards more open, reproducible, easier to trace and share, and more trustworthy engineering biology.One Sentence SummaryWe demonstrate a transparent and open way of engineering and sharing cell lines.

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“…BW25113 strain was used as a barcode receiver and as genomic DNA template source for homologous regions cloning for E. coli experiments. For B. subtilis experiments, 168 strain was used as a barcode receiver and as genomic DNA template source for homologous regions, strain ZPM6 was used for toxin/antitoxin experiment (Lin et al, 2013).…”

Section: Strains and Plasmidsmentioning

confidence: 99%

“…Using SOE-PCR a cassette containing both homologous arms, a mazF-ZeoR cassette and the barcode sequence was created and amplified following an adaptation of the protocol described in (Lin et al, 2013). After transformation colonies were restreaked on LB/Zeocin (20 µg/mL) plates and tested for the integration of the recombinant DNA by PCR.…”

Section: Crisprmentioning

confidence: 99%

Linking Engineered Cells to Their Digital Twins: a Version Control System for Strain Engineering

Tellechea‐Luzardo

Widera

Krasnogor

2019

Preprint

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As DNA sequencing and synthesis become cheaper and more easily accessible, the scale and complexity of biological engineering projects is set to grow. Yet, although there is an accelerating convergence between biotechnology and computing science, a deficit in software and laboratory techniques diminishes the ability to make biotechnology more agile, reproducible and transparent while, at the same time, limiting the security and safety of synthetic biology constructs. To partially address some of these problems, this paper presents an approach for physically linking engineered cells to their digital footprint -we called it digital twinning. This enables the tracking of the entire engineering history of a cell line in a specialised version control system for collaborative strain engineering via simple barcoding protocols.

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A versatile mini-mazF-cassette for marker-free targeted genetic modification in Bacillus subtilis

Cited by 13 publications

References 25 publications

Genome engineering using a synthetic gene circuit in Bacillus subtilis

Genome engineering using a synthetic gene circuit in Bacillus subtilis

Versioning Biological Cells for Trustworthy Cell Engineering

Linking Engineered Cells to Their Digital Twins: a Version Control System for Strain Engineering

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