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におけるレポータータンパク質の測定 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.

著者スポットライト:Limosilactobacillus reuteri DSM20016におけるエレクトロポレーションと形質転換確認の方法  

リ モシラクトバチルス・ロイテリ におけるエレクトロポレーションと形質転換確認の方法 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 Confirmation of Plasmid Uptake via Colony PCR at JoVE.com

Confirmation of Plasmid Uptake via Colony PCR  

コロニーPCRによるプラスミド取り込みの確認

5マイクロリットルのエレクトロポレーション一晩増殖L回転培養物を96ウェルプレートからPCRチューブに移します。細胞をスピンダウンし、上清を廃棄してから、ペレットを20マイクロリットルの20ミリモル水酸化ナトリウムに再懸濁します。サンプルを摂氏95度で5分間煮沸します。ボルテックス、そしてサンプルを氷の上に置く前に一度沸騰を繰り返します。細胞をスピンダウンし、1マイクロリットルの上清化合物を取り、99マイクロリットルの氷冷DNAおよびRNAを含まない二重蒸留水に希釈します。標準PCR反応における鋳型DNAとして100倍希釈液を使用し、プラスミド特異的プライマーを使用し、大腸菌ミニプレップ由来のプラスミドを用いたポジティブコントロールが挙げられる。PCR後、サンプルを氷上に置き、適切なローディング色素を1つのX濃度で添加します。サンプルを1%アゴラーゼゲル中のTAEバッファー中で110ボルトで30分間実行します。調製温度を変化させた形質転換LロータリーのコロニーPCRは、PCR成功率の異なる結果となった。氷上で調製したサンプルは、室温で調製したものや室温で調製したものよりも高い成功率を示し、その後、PCRの前に摂氏37度で30分間インキュベートしました。

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 ...

生物工学

Preparing Limosilactobacillus reuteri DSM20016 Electrocompetent Cells for Electroporation

Preparing Limosilactobacillus reuteri DSM20016 Electrocompetent Cells for Electroporation  

エレクトロポレーションのための リモシラクトバチルス・ロイテリ DSM20016エレクトロコンピテントセルの調製(英語)

Measurement of the Acid-Resistant Fluorescent Reporter Protein mCherry2

耐酸性蛍光レポータータンパク質mCherry2の測定

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  

リ モシラクトバチルス・ロイテリ のミニ分取プロトコルとPCRによるプラスミドの存在確認