In vitro assembly of plasmid DNA for direct cloning in Lactiplantibacillus plantarum WCSF1

Lactobacilli are gram-positive bacteria that are growing in importance for the healthcare industry and genetically engineering them as living therapeutics is highly sought after. However, progress in this field is hindered since most strains are difficult to genetically manipulate, partly due to the...

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Bibliographic Details
Published in:PloS one 2023-02, Vol.18 (2), p.e0281625-e0281625
Main Authors: Blanch-Asensio, Marc, Dey, Sourik, Sankaran, Shrikrishnan
Format: Article
Language:English
Subjects:
Amplification
Antibiotics
Assembly
Bacteria
Biology and Life Sciences
Cell walls
Cloning
Cloning, Molecular
Cold
Deoxyribonucleic acid
DNA
DNA methylation
DNA, Recombinant
E coli
Engineering and Technology
Escherichia coli - genetics
Genes
Genetic engineering
Genetic transformation
Gram-positive bacteria
In vitro methods and tests
Lab Protocol
Laboratories
Lactobacilli
Lactobacillus plantarum - genetics
Medicine and Health Sciences
Plasmids
Plasmids - genetics
Recombinant DNA
Research and Analysis Methods
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Summary:Lactobacilli are gram-positive bacteria that are growing in importance for the healthcare industry and genetically engineering them as living therapeutics is highly sought after. However, progress in this field is hindered since most strains are difficult to genetically manipulate, partly due to their complex and thick cell walls limiting our capability to transform them with exogenous DNA. To overcome this, large amounts of DNA (>1 μg) are normally required to successfully transform these bacteria. An intermediate host, like E. coli, is often used to amplify recombinant DNA to such amounts although this approach poses unwanted drawbacks such as an increase in plasmid size, different methylation patterns and the limitation of introducing only genes compatible with the intermediate host. In this work, we have developed a direct cloning method based on in-vitro assembly and PCR amplification to yield recombinant DNA in significant quantities for successful transformation in L. plantarum WCFS1. The advantage of this method is demonstrated in terms of shorter experimental duration and the possibility to introduce a gene incompatible with E. coli into L. plantarum WCFS1.
ISSN:1932-6203
1932-6203
DOI:10.1371/journal.pone.0281625