Name: Mini-Prep Protocol for Limosilactobacillus reuteri and Confirmation of Plasmid Presence by PCR
Uploaded: 2023-06-23T13:30:00
Description: Watch this Scientific Journal Video about Mini-Prep Protocol for Limosilactobacillus reuteri and Confirmation of Plasmid Presence by PCR at JoVE.com

mini-prep protocol for limosilactobacillus reuteri and confirmation of plasmid presence by pcr

Medição de proteínas repórter em 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.

Autor em destaque: Métodos para eletroporação e confirmação de transformação em Limosilactobacillus reuteri DSM20016  

Métodos para Confirmação de Eletroporação e Transformação em 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 Mini-Prep Protocol for Limosilactobacillus reuteri and Confirmation of Plasmid Presence by PCR at JoVE.com

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

Protocolo Mini-Prep para Limosilactobacillus reuteri e Confirmação da Presença de Plasmídios por PCR

Inocular L.reuteri em 10 mililitros de caldo MRS contendo um antibiótico apropriado e incubar durante a noite aerobicamente a 37 graus Celsius. No dia seguinte, centrifugar a cultura em centrífuga pré-resfriada Descarte o sobrenadante e lave o pellet em dois mililitros de buffer P1 padrão Centrifuge novamente e descarte o sobrenadante antes de ressuspender o pellet em 250 microlitros de tampão P1 modificado. Incubar por uma a duas horas a 37 graus Celsius.Em seguida, adicione 250 microlitros de tampão P2 e misture invertendo de quatro a seis vezes. Após cinco minutos, adicione 350 microlitros de tampão N3 e misture invertendo o tubo. Centrifugar a 10.000 G por 10 minutos e transferir o máximo de sobrenadante possível para uma coluna de spin.Centrifugar novamente por 60 segundos e descartar o fluxo. Lave a coluna de spin com 500 microlitros de PB tampão e centrífuga, descartando o flow-through. Lave a coluna de spin com 750 microlitros de PE tampão e centrífuga a 10.000 G por 60 segundos.Descarte o fluxo e centrifuga novamente por 60 segundos para remover qualquer buffer residual. Coloque a coluna de spin em um tubo de microcentrífuga de 1,5 mililitro. Adicione 20 a 30 microlitros de água bidestilada sem Dnase e RNase ao filtro da coluna de spin e deixe por um a dois minutos antes de centrifugar a 10.000 G por 60 segundos.Realizar uma reação de PCR padrão usando primers plasmidiais específicos com eluato fornecendo o DNA do molde, incluindo um controle positivo com um plasmídeo derivado da mini-preparação de E.coli. Executar o eluato de mini-preparação através de um gel de agarose resulta em uma mancha, em vez de bandas observáveis. No entanto, a PCR subsequente usando o eluato como um molde de DNA produz tamanhos de banda esperados.

Research

JoVE Journal

Bioengineering

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

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 ...