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

Scientific Reports
Jara RadeckThorsten Mascher

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 t...Continue Reading

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Citations

Sep 28, 2018·Microbial Biotechnology·Luísa Czamanski NoraRafael Silva-Rocha
Aug 23, 2019·Biotechnology Advances·Luísa Czamanski NoraRafael Silva-Rocha
Feb 25, 2021·Synthetic Biology·Marcos Valenzuela-Ortega, Christopher French
Apr 3, 2021·ACS Synthetic Biology·Cristina Hernández-RollánMorten H H Nørholm
Nov 19, 2021·ACS Synthetic Biology·Kristoffer Bach FalkenbergSheila Ingemann Jensen

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Datasets Mentioned

BETA
ECE701-26
KY995178
KY995203

Methods Mentioned

BETA
PCR
Destination
PCRs
Fluorescence

Software Mentioned

Gen5 TM
SEVA
Bacillus SEVA siblings

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