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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 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.

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Document Type:Preprint
Author:Marc Blanch-Asensio, Sourik DeyORCiD, Shrikrishnan SankaranORCiD
Parent Title (English):protocols.io
Issue:PROTOCOL integer ID: 70493
First Page:1
Last Page:29
Date of first Publication:2023/01/31
Release Date:2023/02/03
Funding Information:Deutsche Forschungsgemeinschaft (DFG) Research grant [Project # 455063657], Collaborative Research Centre, SFB 1027 [Project # 200049484] and the Leibniz Science Campus on Living Therapeutic Materials [LifeMat].
Scientific Units:Bioprogrammable Materials
DDC classes:500 Naturwissenschaften und Mathematik / 570 Biowissenschaften, Biologie
Open Access:Open Access
Signature:INM 2023/023_protocol
Licence (German):License LogoCreative Commons - CC BY - Namensnennung 4.0 International