Development of<i>Lactococcus lactis</i>encoding fluorescent proteins, GFP, mCherry and iRFP regulated by the nisin-controlled gene expression system

Martinez-Jaramillo, Elvis; Garza-Morales, Rodolfo; Loera‐Arias, María de Jesús; Saucedo‐Cárdenas, Odila; Montes‐de‐Oca‐Luna, Roberto; McNally, Lacey R.; Gómez-Gutiérrez, Jorge G.

doi:10.1080/10520295.2017.1289554

Cited by 10 publications

(7 citation statements)

References 24 publications

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“…Notably, the Lpb. plantarum strain labeled with mCherry was not pink under natural light, unlike the color of other mCherry-labeled bacteria in previous research (Castro-Bravo et al, 2017;Martinez-Jaramillo et al, 2017;Mohedano et al, 2019); we speculate that the unique cell wall structure of WCFS1 may be the main reason.…”

Section: Discussioncontrasting

confidence: 58%

Construction of an Integrated mCherry Red Fluorescent Protein Expression System for Labeling and Tracing in Lactiplantibacillus plantarum WCFS1

et al. 2021

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Thorough intestinal adhesion and colonization greatly promote the probiotic properties of lactic acid bacteria (LAB). Labeling and tracing with fluorescent proteins are effective and reliable for studying the in vivo physiological activities of LAB including localization, adhesion, and colonization. Lactiplantibacillus plantarum WCFS1 was successfully traced with a red fluorescent protein (RFP), which was expressed by the bacteria-carrying recombinant plasmids. In this study, we aimed to construct a stable RFP mCherry expression system, whose encoding gene was integrated into the bacterial chromosome via double-crossed homologous recombination, and use it for labeling WCFS1 with the goal of avoiding the potential loss of non-chromosomal plasmids along with intestinal growth. First, the constitutive expression of the mCherry protein was improved after adjusting the length of the spacer between the promoter and the gene start codon. Then, the optimized mCherry gene expression cassette was integrated into the chromosome of WCFS1. The resulting strain had normal unimpaired growth and strong fluorescent signals, even after 100 generations, indicating its stability. Furthermore, quantitative polymerase chain reaction (PCR) results revealed a strong positive correlation between the fluorescence intensity of the strain and the number of viable cells, demonstrating its potential usage for the quantification of in vivo WCFS1 cells. Finally, the increased adhesion ability of WCFS1 due to the recombinant expression of the bsh gene was visualized and evaluated using fluorescence intensity, the results of which were consistent with those obtained using the previously established quantification methods. These results suggest that the chromosomal-integrated mCherry labeling system can be extensively used to examine the distribution, colonization, and survival of LAB in vivo in order to determine the mechanism of its probiotic function.

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Section: Discussioncontrasting

confidence: 58%

Construction of an Integrated mCherry Red Fluorescent Protein Expression System for Labeling and Tracing in Lactiplantibacillus plantarum WCFS1

et al. 2021

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“…Codon-optimized GFP has been effectively expressed in the cytoplasm and nuclei of Paramecium caudatum to visualize protein trafficking and localization [ 44 ]. The optimized sequences of genes for reporter molecules GFP, near-infrared fluorescent protein (iRFP), and mCherry , were expressed in Lactoccocus lactis [ 45 ]. In our previous experiment, we constructed RFP-labeled V. dahliae , but the red fluorescence was not strong and did not meet experimental requirements because of the lack of codon optimization.…”

Section: Discussionmentioning

confidence: 99%

mCherry-Labeled Verticillium dahliae Could Be Utilized to Investigate Its Pathogenicity Process in Nicotiana benthamiana

Rehman

et al. 2018

Genes

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Verticillium dahliae is a soil-borne phytopathogenic fungus that causes a destructive vascular wilt, but details of the molecular mechanism behind its pathogenicity are not very clear. Here, we generated a red fluorescent isolate of V. dahliae by protoplast transformation to explore its pathogenicity mechanism, including colonization, invasion, and extension in Nicotiana benthamiana, using confocal microscopy. The nucleotide sequences of mCherry were optimized for fungal expression and cloned into pCT-HM plasmid, which was inserted into V. dahliae protoplasts. The transformant (Vd-m) shows strong red fluorescence and its phenotype, growth rate, and pathogenicity did not differ significantly from the wild type V. dahliae (Vd-wt). Between one and three days post inoculation (dpi), the Vd-m successfully colonized and invaded epidermal cells of the roots. From four to six dpi, hyphae grew on root wounds and lateral root primordium and entered xylem vessels. From seven to nine dpi, hyphae extended along the surface of the cell wall and massively grew in the xylem vessel of roots. At ten dpi, the Vd-m was found in petioles and veins of leaves. Our results distinctly showed the pathway of V. dahliae infection and colonization in N. benthamiana, and the optimized expression can be used to deepen our understanding of the molecular mechanism of pathogenicity.

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“…Both upstream and downstream assembly was applied, and the evolutionary tree depicting the construction of plasmids containing multiple gene cassettes is shown in Figure 2. Model proteins included four protein binders (affibody against HER2 (Orlova et al, 2006;Feldwisch et al, 2010), affitin against EpCAM (Kalichuk et al, 2018), evasin-3 against IL-8 ( Škrlec et al, 2017) and affibody against IL-6 (Zahirović et al, manuscript in preparation) and two fluorescent proteins (IRFP (Berlec et al, 2015) and mCherry (Martinez-Jaramillo et al, 2017)).…”

Section: Bglbrick Plasmid Constructionmentioning

confidence: 99%

Introduction of Modified BglBrick System in Lactococcus lactis for Straightforward Assembly of Multiple Gene Cassettes

Plavec

Ključevšek

Berlec

2021

Front. Bioeng. Biotechnol.

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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.

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Development ofLactococcus lactisencoding fluorescent proteins, GFP, mCherry and iRFP regulated by the nisin-controlled gene expression system

Cited by 10 publications

References 24 publications

Construction of an Integrated mCherry Red Fluorescent Protein Expression System for Labeling and Tracing in Lactiplantibacillus plantarum WCFS1

Construction of an Integrated mCherry Red Fluorescent Protein Expression System for Labeling and Tracing in Lactiplantibacillus plantarum WCFS1

mCherry-Labeled Verticillium dahliae Could Be Utilized to Investigate Its Pathogenicity Process in Nicotiana benthamiana

Introduction of Modified BglBrick System in Lactococcus lactis for Straightforward Assembly of Multiple Gene Cassettes

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