preparing limosilactobacillus reuteri dsm20016 electrocompetent cells for electroporation

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.

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Preparing Limosilactobacillus reuteri DSM20016 Electrocompetent Cells for Electroporation  

Preparing Limosilactobacillus reuteri DSM20016 Electrocompetent Cells for Electroporation

To begin, inoculate Limosilactobacillus reuteri DSM20016 from glycerol stock into six milliliters of De Man, Rogosa, Sharpe, or MRS broth, in a 50 milliliter tube. Incubate the culture aerobically overnight at 37 degrees Celsius in a static incubator. The next day, inoculate four milliliters of the overnight culture into 200 milliliters of MRS broth.Allow the culture to grow aerobically in a static 37 degrees Celsius incubator until the optical density at 600 nanometers reaches 0.5 to 0.85. Next, decant the culture into 50 milliliter centrifuge tubes and place them on ice while balancing the tubes. Centrifuge the culture in a pre chilled centrifuge and discard the supernatant.Before resuspending the pellet in 50 milliliters of prechilled double distilled water, repeat the centrifugation two times. After the last centrifugation, resuspend the pellet in 25 milliliters of double distilled water, 0.5 molar sucrose, and 10%glycerol. Centrifuge the cell suspension, discard the supernatant, and resuspend the pellet in two milliliters of double distilled water, 0.5 molar sucrose, and 10%glycerol on ice.Aliquot the suspension into 50 to 100 microliter portions in prechilled microcentrifuge tubes and store the tubes at minus 80 degrees Celsius for later use. Next, for electroporation, thaw an aliquot of electrocompetent cells on ice. Mix five to 10 microliters of the plasmid into the thawed aliquot avoiding pipetting as much as possible.Transfer the mixture to an ice chilled one milliliter gap electroporation cuvette and electroporate at 1.25 kilovolts, 400 ohms, and 25 microfarads. After electroporation, add one milliliter of MRS broth and mix by inverting the cuvette once or twice. Place the cuvettes into a static 37 degree Celsius incubator for 2.5 to three hours for recovery.Next, plate the entire suspension onto multiple MRS auger plates with appropriate selection. Place the plates inside an airtight container with a small lit candle in an anaerobic atmosphere generating sachet. Grow at 37 degrees Celsius for two to three days or until visible colonies appear.Electroporation of L.reuteri with constitutive mCherry2 producing pTRKH3 plasmid gives transformation efficiencies of roughly five to eight colonies per 200 microliters plated regardless of the plasmid methylation condition. Transformation with pNZ123 carrying no exogenous constitutive reporter protein yielded transformation efficiencies of three times 10 to the four CFU per microgram of DNA.

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236 Views.  University of Toronto Mississauga. To begin, inoculate Limosilactobacillus reuteri DSM20016 from glycerol stock into six milliliters of De Man, Rogosa, Sharpe, or MRS broth, in a 50 milliliter tube. Incubate the culture aerobically overnight at 37 degrees Celsius in a static incubator. The next day, inoculate four milliliters of the overnight culture into 200 milliliters of MRS broth.Allow the culture to grow aerobically in a static 37 degrees Celsius incubator until the optical density at 600 nanometers reaches 0.5 to ...