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

Measurement of Reporter Proteins in &lt;em&gt;Limosilactobacillus reuteri&lt;/em&gt; 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.

Author Spotlight: Methods for Electroporation and Transformation Confirmation in Limosilactobacillus reuteri DSM20016  

Methods for Electroporation and Transformation Confirmation in 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  

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

Inoculate L.reuteri into 10 milliliters of MRS broth containing an appropriate antibiotic and incubate overnight aerobically at 37 degrees Celsius. The next day, centrifuge the culture in a pre-chilled centrifuge Discard the supernatant and wash the pellet in two milliliters of standard P1 buffer Centrifuge again and discard the supernatant before resuspending the pellet in 250 microliters of modified P1 buffer. Incubate for one to two hours at 37 degrees Celsius.Next, add 250 microliters of buffer P2 and mix by inverting four to six times. After five minutes, add 350 microliters of buffer N3 and mix by inverting the tube. Centrifuge at 10, 000 G for 10 minutes and transfer as much supernatant as possible to a spin column.Centrifuge again for 60 seconds and discard the flow-through. Wash the spin column with 500 microliters of buffer PB and centrifuge, discarding the flow-through. Wash the spin column with 750 microliters of buffer PE and centrifuge at 10, 000 G for 60 seconds.Discard the flow-through and centrifuge again for 60 seconds to remove any residual buffer. Place the spin column in a 1.5-milliliter microcentrifuge tube. Add 20 to 30 microliters of Dnase-and RNase-free double-distilled water to the spin column filter and leave for one to two minutes before centrifuging at 10, 000 G for 60 seconds.Perform a standard PCR reaction using plasmid-specific primers with eluate providing the template DNA, including a positive control with a plasmid-derived from the E.coli mini-prep. Running the mini-prep eluate through an agarose gel results in a smear, rather than observable bands. However, subsequent PCR using the eluate as a DNA template produces expected band sizes.

mini-prep-protocol-for-limosilactobacillus-reuteri-confirmation

Research

JoVE Journal

Bioengineering

166 조회수.  University of Toronto Mississauga. 적절한 항생제가 들어있는 MRS 브로스 10 밀리리터에 L.reuteri를 접종하고 섭씨 37도에서 하룻밤 동안 호기성으로 배양합니다. 다음날, 배양물을 예비-냉장 원심분리기에서 원심분리하고, 상청액을 버리고, 펠릿을 2밀리리터의 표준 P1 완충액으로 다시 세척하고, 펠릿을 250 마이크로리터의 변형된 P1 완충액에 재현탁시키기 전에 상청액을 폐기한다. 섭씨 37도에서 1-2 시간 동안 배...