inPOSE: A Flexible Toolbox for Chromosomal Cloning and Amplification of Bacterial Transgenes.
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microorganisms-10-00236-v3.pdf
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3.53 MB
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Resource type
Journal article
Date published
February 2022
Abstract
Cloning the genes and operons encoding heterologous functions in bacterial hosts is now almost exclusively carried out using plasmid vectors. This has multiple drawbacks, including the need for constant selection and variation in copy numbers. The chromosomal integration of transgenes has always offered a viable alternative; however, to date, it has been of limited use due to its tedious nature and often being limited to a single copy. We introduce here a strategy that uses bacterial insertion sequences, which are the simplest autonomous transposable elements to insert and amplify genetic cargo into a bacterial chromosome. Transgene insertion can take place either as transposition or homologous recombination, and copy number amplification is achieved using controlled copy-paste transposition. We display the successful use of IS1 and IS3 for this purpose in Escherichia coli cells using various selection markers. We demonstrate the insertion of selectable genes, an unselectable gene and a five-gene operon in up to two copies in a single step. We continue with the amplification of the inserted cassette to double-digit copy numbers within two rounds of transposase induction and selection. Finally, we analyze the stability of the cloned genetic constructs in the lack of selection and find it to be superior to all investigated plasmid-based systems. Due to the ubiquitous nature of transposable elements, we believe that with proper design, this strategy can be adapted to numerous other bacterial species.
Funder
| Funder name | Awards |
National Research, Development and Innovation Office, Hungary | 2020-1.1.6-JÖVŐ-2021-00003 - Grants No. K119298 |
European Union | GINOP-2.3.2-15-2016-00001 |
Emberi Eroforrások Minisztériuma, Hungary | Biotechnological National Laboratory Grant |
Journal title
Microorganisms
Volume
10
Issue
202
Article number
236
Publisher
MDPI AG
Place of publication
Basel, Switzerland
eISSN
2076-2607
Date accepted
January 18, 2022
Official URL
Related URL
Rights statement
In Copyright
Additional information
This article belongs to the Special Issue Current Prokaryotic Genome Engineering.