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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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NameCompanyCatalog NumberComments
1 kb Plus DNA LadderNEBN3200L
1mL Spectrophotometer cuvettesThomas Scientific1145J12
Agarose BioShopAGR001
Allegra X-15R (refrigerated centrifuge)Beckman Allegra N/ANo longer in production
AnaeroGen 2.5 L SachetThermo ScientificOXAN0025A
BTX, ECM 399 electroporation systemVWR58017-984
Centrifuge tubes (50 mL)FroggaBioTB50-500
DNA gel x6 loading dyeNEBB7024S
Electroporation cuvetteFisherbrandFB101
ErythromycinMillipore SigmaE5389-5G
Gel electroporation bath/dockVWR76314-748
Glycerol BioShopGLY001
Limosilactobacillus reuteriLeibniz Institute DSMZDSM20016Strain designation F275
LysozymeBioShopLYS702.5
Microcentrifuge tubes (1.7 mL)FroggaBioLMCT1.7B
Miniprep kit (Qiagen)Qiagen27106slpGFP replaced with constitutive, codon optimised, mCherry2 reporter protein 
MRS Broth (Dehydrated)Thermo ScientificCM0359B
MutanolysinMillipore SigmaM9901-5KU
NaOH Millipore Sigma1064691000
P100 PipetteEppendorf3123000047
P1000 PipetteEppendorf3123000063
P2.5 PipetteEppendorf3123000012
P20 PipetteEppendorf3123000039
P200 PipetteEppendorf3123000055
PCR tubesFroggaBioSTF-A120S
Personal benchtop microcentrifugeGenlantisE200100
Petri dishesVWR25384-088
PTC-150 Thermal CyclerMJ ResearchN/ANo longer in production
pTRKH3_slpGFP (modified)Addgene27168
SPECTRONIC 200 SpectrophotometerThermo Scientific840-281700
Storage microplateFisher Scientific14-222-225
SucroseBioShopSUC507
TAE Buffer 50xThermo ScientificB49
VortexVWR58816-121No longer in production
VWR 1500E incubatorVWRN/ANo longer in production

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