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

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 Confirmation of Plasmid Uptake via Colony PCR at JoVE.com

Confirmation of Plasmid Uptake via Colony PCR  

Transfer five microliters of electroporated overnight growth L rotary culture from a 96 well plate to a PCR tube. Spin down the cells and discard the supernatant before resuspending the pellet in 20 microliters of 20 millimolar sodium hydroxide. Boil the samples at 95 degrees Celsius for five minutes.Vortex, and repeat the boiling once before placing the samples on ice. Spin down the cells then take one microliter of the supra natant and dilute into 99 microliters of ice cold DNAs and RNAs free double distilled water. Use the 100 times dilution as the template DNA in a standard PCR reaction, using plasmid specific primers and include a positive control with a plasmid derived from the E coli mini prep.After PCR, place the samples on ice and add an appropriate loading dye at one X concentration. Run the samples in 1%Agorase gell in TAE buffer at 110 volts for 30 minutes. Colony PCR of transformed L Rotary varying the preparation temperature resulted in differing rates of PCR success.Samples prepared on ice showed a higher percentage of success rate than that prepared at room temperature or prepared at room temperature, and then incubated at 37 degrees Celsius for 30 minutes before PCR.

confirmation-of-plasmid-uptake-via-colony-pcr

Research

JoVE Journal

Bioengineering

Lactobacillus were an incredibly large, diverse genus of bacteria comprising 261 species, several of which were commensal strains with the potential for use as a chassis for synthetic biological endeavors within the gastrointestinal tract. The wide phenotypic and genotypic variation observed within the genus led to a recent reclassification and the introduction of 23 novel genera.
Due to the breadth of variations within the old genera, protocols demonstrated in one member may not work as advertised with other members. A lack of centralized information on how exactly to manipulate specific strains has led to a range of ad hoc approaches, often adapted from other bacterial families. This can complicate matters for researchers starting in the field, who may not know which information does or does not apply to their chosen strain.
In this paper, we aim to centralize a set of protocols with demonstrated success, specifically in&#160;the Limosilactobacillus reuteri strain designation F275 (other collection numbers: DSM20016, ATCC23272, CIP109823), along with troubleshooting advice and common issues one may encounter. These protocols should enable a researcher with little to no experience working with L. reuteri DSM20016 to transform a plasmid, confirm transformation, and measure system feedback in a plate reader via a reporter protein.

Here, we present protocols for working with Limosilactobacillus reuteri DSM20016, detailing growth, plasmid transformation, colony PCR, fluorescent reporter protein measurement, and limited plasmid mini-prep, as well as common issues and troubleshooting. These protocols allow the measurement of reporter proteins in DSM20016, or confirmation via colony PCR if no reporter is involved.

Lactobacillus were an incredibly large, diverse genus of bacteria comprising 261 species, several of which were commensal strains with the potential for ...

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.

Measurement of the Acid-Resistant Fluorescent Reporter Protein mCherry2

Pick any colony from the MRS agar plate containing electroporated L.reuteri cells and inoculate into a 96 well microplate containing filter sterilized MRS broth per well and an appropriate selection antibiotic. Incubate aerobically for 24 hours at 37 degrees Celsius without shaking. The next day, L.reuteri will precipitate out of the media in the stationary phase.Resuspend the culture by pipetting. Add 200 microliters of culture into a flat, clear, bottom 96 well plate. Transfer the plate to a plate reader and measure the optical density and fluorescence of M cherry two and other relevant reporters.Colony morphology noticeably changes in the presence of oxygen. Under low oxygen conditions colonies appear white, opaque, smooth and round. Under partial or leaky conditions, colonies appear undulate, and umbanet.Whereas under atmospheric oxygen levels colonies always appear low bait, round and dry. In wild Type L.rueteri, growth and reporter protein expression vary between colonies making accurate deductions difficult. Plasmid curing, and re-transformation can result in more stable expression but it is an uncommon and unique occurrence reliant on more stable mutant phenotypes spontaneously occurring.

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.