Name: Preparing Limosilactobacillus reuteri DSM20016 Electrocompetent Cells for Electroporation
Uploaded: 2023-06-23T13:30:00
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preparing limosilactobacillus reuteri dsm20016 electrocompetent cells for electroporation

Mesure des protéines rapporteures chez Limosilactobacillus reuteri DSM20016

The genus Lactobacillus were historically classified as gram-positive, rod-shaped, non-spore-forming, either facultative anaerobes or microaerophiles that break sugars down to primarily produce lactic acid1. These loose criteria led to Lactobacillus being, phenotypically and genotypically, an extremely diverse genus. This broad categorization resulted in the genus being reclassified, introducing 23 novel genera in 20202.
The old, broader genus included major commensal and probiotic species generally regarded as safe (GRAS) for consumption3. The Lactobacillaceae family maintains a public perception of being &#39;good bacteria&#39; due to many reported health benefits bestowed via the consumption of various strains4,5,6,7. The ease with which they can navigate the gastrointestinal tract8 and their public acceptance combine to position Lactobacillaceae strains as strong candidates as chassis organisms for ingestible medicinal, therapeutic, or diagnostic applications.
The wide range of characteristics present within the Lactobacillaceae family has led to a situation in which there is no de facto model-organism strain; research groups have tended to select species with the properties most relevant to their particular aims. (For example, dairy fermentation labs could choose L. lactis; studies of vegetable fermentation might select L. plantarum; research on probiotics might focus on L. acidophilus; and so on.)
This same wide range of characteristics across species has led to an accumulation of protocols and procedures that may work well for one subset of the Lactobacillaceae family, but require optimization to work efficiently (or perhaps to function at all) in others9. This need for optimization between family members and even within members of the same species can frustrate the efforts of unfamiliar researchers. Protocols published in the methods sections of papers can also include their own modifications10, leading to fragmented, decentralized protocol collections.
L. reuteri is considered a widely vertebrate commensal, found consistently in mammalian, avian11 and fish12 gastrointestinal (GI) tracts. L. reuteri sub-strains are often genetically specialized, via mucus adhesion protein adaptation, to more permanently colonize specific native hosts8,11,13. GI tract Limosilactobacillus species can be isolated in hosts outside their native host, but tend more toward a transient nature8.
Due to human-host specialization, L. reuteri DSM20016 positions itself very well as a chassis for diagnostic or therapeutic applications at any point in the human GI tract, and the strain DSM20016 could provide a longer-lasting window of effect for interventions when compared to more transitory strains.
In this paper, we outline a series of protocols with demonstrated effectiveness in Limosilactobacillus reuteri (strain designation: F275; other collection numbers: DSM20016, ATCC23272, CIP109823), along with centralized information on the strain from other sources to aid in molecular and systems biology applications. Procedures laid out herein should enable a researcher with no prior experience to culture L. reuteri, create electrocompetent stocks, select transformed colonies, confirm transformation via colony polymerase chain reaction (PCR), and measure designed system response via fluorescent reporter proteins.
We note that related protocols have covered CRISPR-Cas9 assisted ssDNA genome recombineering in L. reuteri (strain: ATCC-PTA-6475)14, and CRSIPR-Cas9 nickase-assisted genome editing in multiple non-L. reuteri, Lactobacillaceae family stains15,16; these do not, however, address the L. reuteri DSM20016 strain that is our focus here.

Pleins feux sur l’auteur : Méthodes d’électroporation et de confirmation de la transformation chez Limosilactobacillus reuteri DSM20016  

Méthodes d’électroporation et de confirmation de transformation chez Limosilactobacillus reuteri DSM20016

L.Reuteri, and specifically the strain, DSM20016 is a robust, natural human gut commensal, with great promise for therapeutic delivery and disease detection throughout the gastrointestinal tract. This work will help position DSM20016 as a synthetic biology chassis for those kinds of applications. Lactobacillales have no specific model organism.Preferred species do exist, but they're usually chosen with a bias towards particular research aims. The variation within the bacterial family has led to fragmented protocols, some with iterations of modifications going back through publications. It is very difficult for people starting out to filter exactly what is and is not useful.One paper for the transformation of DSM20016 does exist, but it's not modern. Modern protocols for other Lactobacillales family members exist, but they are not DSM20016. Here we strive to provide an easy to use, centralized, modernized, complete set of protocols for DSM20016 electroporation.This protocol set offers substantial modernization over previous DSM20016 exploration protocol, and elucidates some hanging threads, as well as emphasizing parts that were previously less clear. The protocol set is also complete, and specifically for DSM20016, a strain for which other non-DSM20016 protocols are not totally compatible. One of our main goals is using microbes as inexpensive sensors and delivery systems.We're working with intestinal disease experts to use DSM20016 to sense the conditions in the gastrointestinal tract and deliver therapeutics as required.

Watch this Scientific Journal Video about Preparing Limosilactobacillus reuteri DSM20016 Electrocompetent Cells for Electroporation at JoVE.com

Preparing Limosilactobacillus reuteri DSM20016 Electrocompetent Cells for Electroporation  

Préparation de cellules électrocompétentes de Limosilactobacillus reuteri DSM20016 pour l’électroporation

Pour commencer, inoculez Limosilactobacillus reuteri DSM20016 du stock de glycérol dans six millilitres de bouillon De Man, Rogosa, Sharpe ou MRS, dans un tube de 50 millilitres. Incuber la culture en aérobiose pendant la nuit à 37 degrés Celsius dans un incubateur statique. Le lendemain, inoculez quatre millilitres de la culture de nuit dans 200 millilitres de bouillon MRS.Permettre à la culture de croître en aérobiose dans un incubateur statique de 37 degrés Celsius jusqu’à ce que la densité optique à 600 nanomètres atteigne 0,5 à 0,85. Ensuite, décantez la culture dans des tubes à centrifuger de 50 millilitres et placez-les sur de la glace tout en équilibrant les tubes. Centrifuger la culture dans une centrifugeuse pré-réfrigérée et jeter le surnageant.Avant de remettre le granulé en suspension dans 50 millilitres d’eau distillée double prérefroidie, répétez la centrifugation deux fois. Après la dernière centrifugation, remettre la pastille en suspension dans 25 millilitres d’eau distillée double, 0,5 molaire de saccharose et 10% de glycérol. Centrifuger la suspension cellulaire, jeter le surnageant et remettre en suspension la pastille dans deux millilitres d’eau distillée double, 0,5 molaire de saccharose et 10% de glycérol sur glace.Aliquote la suspension en portions de 50 à 100 microlitres dans des tubes microcentrifuges prérefroidis et stocker les tubes à moins 80 degrés Celsius pour une utilisation ultérieure. Ensuite, pour l’électroporation, décongeler une partie aliquote de cellules électrocompétentes sur de la glace. Mélanger cinq à 10 microlitres du plasmide dans l’aliquote décongelée en évitant autant que possible de pipeter.Transférer le mélange dans une cuvette d’électroporation d’un millilitre refroidie par glace et électroporate à 1,25 kilovolts, 400 ohms et 25 microfarads. Après électroporation, ajouter un millilitre de bouillon MRS et mélanger en retournant la cuvette une ou deux fois. Placez les cuvettes dans un incubateur statique à 37 degrés Celsius pendant 2,5 à trois heures pour la récupération.Ensuite, plaquez la suspension entière sur plusieurs plaques de tarière MRS avec la sélection appropriée. Placez les assiettes à l’intérieur d’un récipient hermétique avec une petite bougie allumée dans une atmosphère anaérobie générant un sachet. Croître à 37 degrés Celsius pendant deux à trois jours ou jusqu’à ce que des colonies visibles apparaissent.L’électroporation de L.reuteri avec le plasmide constitutif mCherry2 produisant pTRKH3 donne des efficacités de transformation d’environ cinq à huit colonies par 200 microlitres plaqués, quelle que soit la condition de méthylation plasmidique. La transformation avec pNZ123 ne portant aucune protéine rapporteure constitutive exogène a donné des efficacités de transformation de trois fois 10 à quatre UFC par microgramme d’ADN.

Research

JoVE Journal

Bioengineering

Preparing Limosilactobacillus reuteri DSM20016 Electrocompetent Cells for Electroporation

Methods for Electroporation and Transformation Confirmation in Limosilactobacillus reuteri DSM20016

Lactobacillus were an incredibly large, diverse genus of bacteria comprising 261 species, several of which were commensal strains with the potential for ...

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Mini-Prep Protocol for Limosilactobacillus reuteri and Confirmation of Plasmid Presence by PCR  

Protocole de mini-préparation pour Limosilactobacillus reuteri et confirmation de la présence de plasmides par PCR