Abstract
Genetic modification of lactic acid bacteria is an evolving and highly relevant field of research that allows the engineered bacteria to be equipped with the desired functions through the controlled expression of the recombinant protein. Novel genetic engineering techniques offer the advantage of being faster, easier and more efficient in incorporating modifications to the original bacterial strain. Here, we have developed a modified BglBrick system, originally introduced in Escherichia coli and optimized it for the lactic acid bacterium Lactococcus lactis. Six different expression cassettes, encoding model proteins, were assembled in different order as parts of a modified BglBrick system in a novel plasmid pNBBX. All cassettes included nisin promoter, protein encoding gene and transcription terminator. We demonstrated successful intracellular expression of the two fluorescent proteins and display of the four protein binders on the bacterial surface. These were expressed either alone or concomitantly, in combinations of three model proteins. Thus, a modified BglBrick system developed herein enables simple and modular construction of multigene plasmids and controlled simultaneous expression of three proteins in L. lactis.
Highlights
Introduction of Modified BglBrick System inLactococcus lactis for Straightforward Assembly of Multiple Gene CassettesTina Vida Plavec 1, Tim Ključevšek 1,2 and Aleš Berlec 1,2*Edited by: Siddhesh B
Lactic acid bacteria have been used as hosts for heterologous protein expression in various biotechnological and therapeutic applications
Plasmid-based genetic engineering systems in L. lactis are mostly based on the use of restriction endonucleases
Summary
Introduction of Modified BglBrick System inLactococcus lactis for Straightforward Assembly of Multiple Gene CassettesTina Vida Plavec 1, Tim Ključevšek 1,2 and Aleš Berlec 1,2*Edited by: Siddhesh B. A modified BglBrick system developed enables simple and modular construction of multigene plasmids and controlled simultaneous expression of three proteins in L. lactis. Lactic acid bacteria (LAB) are considered safe and valuable host organisms in biotechnology with various potential applications Their use as hosts for the expression of heterologous proteins that act as biosensors, biocatalysts and even therapeutics has been demonstrated (Stahl and Uhlen, 1997; Lee et al, 2003; Nguyen et al, 2016; Plavec and Berlec, 2019). We achieved the straightforward assembly of multiple gene cassettes in a single pNBBX plasmid by using the modified BglBrick system and confirmed successful expression of multiple proteins. Constructed pNBBX plasmid represents a valuable tool for faster genetic engineering and effective protein expression in L. lactis
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