Name: Confirmation of Plasmid Uptake via Colony PCR
Uploaded: 2023-06-23T13:30:00
Description: Watch this Scientific Journal Video about Confirmation of Plasmid Uptake via Colony PCR at JoVE.com

confirmation of plasmid uptake via colony pcr

Limosilactobacillus reuteri DSM20016의 리포터 단백질 측정

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.

저자 스포트라이트: Methods for Electroporation and Transformation Confirmation in Limosilactobacillus reuteri DSM20016  

Limosilactobacillus reuteri DSM20016의 전기천공 및 변형 확인 방법

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 Confirmation of Plasmid Uptake via Colony PCR at JoVE.com

Confirmation of Plasmid Uptake via Colony PCR  

Colony PCR을 통한 플라스미드 흡수 확인

전기천공된 하룻밤 성장 L 회전 배양액 5마이크로리터를 96웰 플레이트에서 PCR 튜브로 옮깁니다. 펠릿을 20 밀리몰 수산화 나트륨 20 마이크로 리터에 재현 탁하기 전에 세포를 회전시키고 상청액을 버립니다. 샘플을 섭씨 95도에서 5 분 동안 끓입니다.소용돌이를 일으키고 샘플을 얼음 위에 놓기 전에 끓는 것을 한 번 반복하십시오. 세포를 회전시킨 다음 1 마이크로 리터의 수프라 나탄 트를 취하여 99 마이크로 리터의 얼음처럼 차가운 DNA와 RNA가 없는 이중 증류수로 희석합니다. 표준 PCR 반응에서 주형 DNA로서 100배 희석된 플라스미드를 사용하고, 플라스미드 특이적 프라이머를 사용하여 대장균 미니 프렙으로부터 유래된 플라스미드와 함께 양성 대조군을 포함한다.PCR 후 샘플을 얼음 위에 놓고 적절한 로딩 염료를 1 X 농도로 추가합니다. TAE 버퍼의 1%Agorase gell에서 30분 동안 110V에서 샘플을 실행합니다. 형질전환된 L로타리의 콜로니 PCR은 제조 온도를 변화시켜 PCR 성공률의 상이한 비율을 초래하였다.얼음 위에서 준비한 샘플은 실온에서 준비하거나 실온에서 준비한 샘플보다 성공률이 높았으며, PCR 전 섭씨 37도에서 30분 동안 배양했습니다.

Research

JoVE Journal

Bioengineering

Confirmation of Plasmid Uptake via Colony 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 ...

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Preparing Limosilactobacillus reuteri DSM20016 Electrocompetent Cells for Electroporation

Preparing Limosilactobacillus reuteri DSM20016 Electrocompetent Cells for Electroporation  

Limosilactobacillus reuteri DSM20016 Electroporation을 위한 Electrocompetent Cells 준비

Measurement of the Acid-Resistant Fluorescent Reporter Protein mCherry2

내산성 형광 리포터 단백질 mCherry2의 측정

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

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

리모실락토바실러스 루테리(Limosilactobacillus reuteri)에 대한 Mini-Prep 프로토콜 및 PCR에 의한 플라스미드 존재 확인