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Bioengineering

Methods for Electroporation and Transformation Confirmation in Limosilactobacillus reuteri DSM20016

Published: June 23rd, 2023

DOI:

10.3791/65463

1University of Toronto Mississauga

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 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 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.

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 Lac....

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1. Preparing L. reuteri DSM20016 electrocompetent cells

NOTE: This is based on a protocol by Berthier et al.17, with centrifugation speeds informed by Rattanachaikunsopon et al.18.

  1. In a 50 mL centrifuge tube, inoculate L. reuteri from glycerol stock into 6 mL of deMan Rogosa Sharpe (MRS) broth. Incubate aerobically overnight at 37 °C in a static incubator.
  2. The next morning, in.......

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Transformation efficiencies
L. reuteri does not require a dcm-/dam- non-methylated plasmid, as observed for other Lactobacillaceae19,20 (see Figure 1). Electroporation of L. reuteri DSM20016 with 10 µL of the 8.5 kb plasmid pTRKH3_mCherry2 (pAMβ1 theta origin of replication) should give transformation efficiencies of roughly 80 colony forming units (CFU.......

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The most critical step for the transformation of L. reuteri DSM20016 is the generation of anaerobic growth conditions after transformations are plated; colonies gained in aerobic conditions are only very occasional and generally fail to grow when inoculated in MRS broth. Plating the entire recovery volume should also be practiced to maximize the probability of colony growth. Even with these two critical steps, transformation efficiency is still a limitation on experimentation, as expected colonies can number as .......

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We greatly appreciate the valuable advice provided by Prof. J.P. van Pijkeren (University of Wisconsin-Madison), whose guidance on working with L. reuteri ATCC PTA 6475 provided a foundation for the methods described here.

....

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Name Company Catalog Number Comments
1 kb Plus DNA Ladder NEB N3200L
1mL Spectrophotometer cuvettes Thomas Scientific 1145J12
Agarose  BioShop AGR001
Allegra X-15R (refrigerated centrifuge) Beckman Allegra  N/A No longer in production
AnaeroGen 2.5 L Sachet Thermo Scientific OXAN0025A
BTX, ECM 399 electroporation system VWR 58017-984
Centrifuge tubes (50 mL) FroggaBio TB50-500
DNA gel x6 loading dye NEB B7024S
Electroporation cuvette Fisherbrand FB101
Erythromycin Millipore Sigma E5389-5G
Gel electroporation bath/dock VWR 76314-748
Glycerol  BioShop GLY001
Limosilactobacillus reuteri Leibniz Institute DSMZ DSM20016 Strain designation F275
Lysozyme BioShop LYS702.5
Microcentrifuge tubes (1.7 mL) FroggaBio LMCT1.7B
Miniprep kit (Qiagen) Qiagen 27106 slpGFP replaced with constitutive, codon optimised, mCherry2 reporter protein 
MRS Broth (Dehydrated) Thermo Scientific CM0359B
Mutanolysin Millipore Sigma M9901-5KU
NaOH  Millipore Sigma 1064691000
P100 Pipette Eppendorf 3123000047
P1000 Pipette Eppendorf 3123000063
P2.5 Pipette Eppendorf 3123000012
P20 Pipette Eppendorf 3123000039
P200 Pipette Eppendorf 3123000055
PCR tubes FroggaBio STF-A120S
Personal benchtop microcentrifuge Genlantis E200100
Petri dishes VWR 25384-088
PTC-150 Thermal Cycler MJ Research N/A No longer in production
pTRKH3_slpGFP (modified) Addgene 27168
SPECTRONIC 200 Spectrophotometer Thermo Scientific 840-281700
Storage microplate Fisher Scientific 14-222-225
Sucrose BioShop SUC507
TAE Buffer 50x Thermo Scientific B49
Vortex VWR 58816-121 No longer in production
VWR 1500E incubator VWR N/A No longer in production

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