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...
| Main Authors: | , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Public Library of Science (PLoS)
2023-01-01
|
| Series: | PLoS ONE |
| Online Access: | https://doi.org/10.1371/journal.pone.0281625 |
| _version_ | 1797895517778214912 |
|---|---|
| author | Marc Blanch-Asensio Sourik Dey Shrikrishnan Sankaran |
| author_facet | Marc Blanch-Asensio Sourik Dey Shrikrishnan Sankaran |
| author_sort | Marc Blanch-Asensio |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-04-10T07:27:04Z |
| format | Article |
| id | doaj.art-f804b54c291e48feb990600722d07185 |
| institution | Directory Open Access Journal |
| issn | 1932-6203 |
| language | English |
| last_indexed | 2024-04-10T07:27:04Z |
| publishDate | 2023-01-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS ONE |
| spelling | doaj.art-f804b54c291e48feb990600722d071852023-02-24T05:31:40ZengPublic Library of Science (PLoS)PLoS ONE1932-62032023-01-01182e028162510.1371/journal.pone.0281625In vitro assembly of plasmid DNA for direct cloning in Lactiplantibacillus plantarum WCSF1.Marc Blanch-AsensioSourik DeyShrikrishnan SankaranLactobacilli 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.https://doi.org/10.1371/journal.pone.0281625 |
| spellingShingle | Marc Blanch-Asensio Sourik Dey Shrikrishnan Sankaran In vitro assembly of plasmid DNA for direct cloning in Lactiplantibacillus plantarum WCSF1. PLoS ONE |
| title | In vitro assembly of plasmid DNA for direct cloning in Lactiplantibacillus plantarum WCSF1. |
| title_full | In vitro assembly of plasmid DNA for direct cloning in Lactiplantibacillus plantarum WCSF1. |
| title_fullStr | In vitro assembly of plasmid DNA for direct cloning in Lactiplantibacillus plantarum WCSF1. |
| title_full_unstemmed | In vitro assembly of plasmid DNA for direct cloning in Lactiplantibacillus plantarum WCSF1. |
| title_short | In vitro assembly of plasmid DNA for direct cloning in Lactiplantibacillus plantarum WCSF1. |
| title_sort | in vitro assembly of plasmid dna for direct cloning in lactiplantibacillus plantarum wcsf1 |
| url | https://doi.org/10.1371/journal.pone.0281625 |
| work_keys_str_mv | AT marcblanchasensio invitroassemblyofplasmiddnafordirectcloninginlactiplantibacillusplantarumwcsf1 AT sourikdey invitroassemblyofplasmiddnafordirectcloninginlactiplantibacillusplantarumwcsf1 AT shrikrishnansankaran invitroassemblyofplasmiddnafordirectcloninginlactiplantibacillusplantarumwcsf1 |