Name: Author Spotlight: Methods for Electroporation and Transformation Confirmation in Limosilactobacillus reuteri DSM20016
Uploaded: 2023-06-23T13:30:00
Description: Watch this Scientific Journal Video about Methods for Electroporation and Transformation Confirmation in Limosilactobacillus reuteri DSM20016 at JoVE.com

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.

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.
<ol>
	<li>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 &#176;C in a static incubator.</li>
	<li>The next morning, in...

Measurement of Reporter Proteins in Limosilactobacillus reuteri DSM20016

<ol>
	<li>Makarova, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+genomics+of+the+lactic+acid+bacteria.">Comparative genomics of the lactic acid bacteria.</a> Proceedings of the National Academy of Sciences. 103 (42), 15611-15616 (2006).</li><li>Zheng, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+taxonomic+note+on+the+genus+Lactobacillus:+Description+of+23+novel+genera,+emended+description+of+the+genus+Lactobacillus+beijerinck+1901,+and+union+of+Lactobacillaceae+and+Leuconostocaceae.">A taxonomic note on the genus Lactobacillus: Description of 23 novel genera, emended description of the genus Lactobacillus beijerinck 1901, and union of Lactobacillaceae and Leuconostocaceae.</a> International Journal of Systematic and Evolutionary Microbiology. 70 (4), 2782-2858 (2020).</li><li>Adams, M. R., Marteau, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+safety+of+lactic+acid+bacteria+from+food.">On the safety of lactic acid bacteria from food.</a> International Journal of Food Microbiology. 27 (2-3), 263-264 (1995).</li><li>Urba&#324;ska, M., Gieruszczak-Bia&#322;ek, D., Szajewska, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systematic+review+with+meta-analysis:+Lactobacillus+reuteri+DSM+17938+for+diarrhoeal+diseases+in+children.">Systematic review with meta-analysis: Lactobacillus reuteri DSM 17938 for diarrhoeal diseases in children.</a> Alimentary Pharmacology and Therapeutics. 43 (10), 1025-1034 (2016).</li><li>Jansson, P. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probiotic+treatment+using+a+mix+of+three+Lactobacillus+strains+for+lumbar+spine+bone+loss+in+postmenopausal+women:+a+randomised,+double-blind,+placebo-controlled,+multicentre+trial.">Probiotic treatment using a mix of three Lactobacillus strains for lumbar spine bone loss in postmenopausal women: a randomised, double-blind, placebo-controlled, multicentre trial.</a> The Lancet Rheumatology. 1 (3), e154-e162 (2019).</li><li>Yang, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+non-viable+Lactobacillus+reuteri+combining+with+14-day+standard+triple+therapy+on+Helicobacterpylori+eradication:+A+randomized+double-blind+placebo-controlled+trial.">Effects of non-viable Lactobacillus reuteri combining with 14-day standard triple therapy on Helicobacterpylori eradication: A randomized double-blind placebo-controlled trial.</a> Helicobacter. 26 (6), 12856 (2021).</li><li>Nikawa, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lactobacillus+reuteri+in+bovine+milk+fermented+decreases+the+oral+carriage+of+mutans+streptococci.">Lactobacillus reuteri in bovine milk fermented decreases the oral carriage of mutans streptococci.</a> International Journal of Food Microbiology. 95 (2), 219-223 (2004).</li><li>Reuter, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Lactobacillus+and+Bifidobacterium+microflora+of+the+human+intestine:+Composition+and+succession.">The Lactobacillus and Bifidobacterium microflora of the human intestine: Composition and succession.</a> Current Issues in Intestinal Microbiology. 2 (2), 43-53 (2001).</li><li>Aukrust, T. W., Brurberg, M. B., Nes, I. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transformation+of+Lactobacillus+by+electroporation.">Transformation of Lactobacillus by electroporation.</a> Methods in Molecular Biology. 47, 201-208 (1995).</li><li>Lubkowicz, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reprogramming+probiotic+Lactobacillus+reuteri+as+a+biosensor+for+Staphylococcus+aureus+derived+AIP-I+detection.">Reprogramming probiotic Lactobacillus reuteri as a biosensor for Staphylococcus aureus derived AIP-I detection.</a> ACS Synthetic Biology. 7 (5), 1229-1237 (2018).</li><li>Walter, J., Britton, R. A., Roos, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Host-microbial+symbiosis+in+the+vertebrate+gastrointestinal+tract+and+the+Lactobacillus+reuteri+paradigm.">Host-microbial symbiosis in the vertebrate gastrointestinal tract and the Lactobacillus reuteri paradigm.</a> Proceedings of the National Academy of Sciences. 108, 4645-4652 (2011).</li><li>Ahmad, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+bimodal+molecular+weight+levan+by+a+Lactobacillus+reuteri+isolate+from+fish+gut.">Production of bimodal molecular weight levan by a Lactobacillus reuteri isolate from fish gut.</a> Folia Microbiologica. 67 (1), 21-31 (2022).</li><li>MacKenzie, D. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strain-specific+diversity+of+mucus-binding+proteins+in+the+adhesion+and+aggregation+properties+of+Lactobacillus+reuteri.">Strain-specific diversity of mucus-binding proteins in the adhesion and aggregation properties of Lactobacillus reuteri.</a> Microbiology. 156 (11), 3368-3378 (2010).</li><li>Oh, J. H., van Pijkeren, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CRISPR-Cas9-assisted+recombineering+in+Lactobacillus+reuteri.">CRISPR-Cas9-assisted recombineering in Lactobacillus reuteri.</a> Nucleic Acids Research. 42 (17), 131 (2014).</li><li>Song, X., Huang, H., Xiong, Z., Ai, L., Yang, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CRISPR-Cas9D10A+nickase-assisted+genome+editing+in+Lactobacillus+casei.">CRISPR-Cas9D10A nickase-assisted genome editing in Lactobacillus casei.</a> Applied and Environmental Microbiology. 83 (22), e01259 (2017).</li><li>Goh, Y. J., Barrangoua, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Portable+CRISPR-Cas9N+system+for+flexible+genome+engineering+in+Lactobacillus+acidophilus,+Lactobacillus+gasseri,+and+Lactobacillus+paracasei.">Portable CRISPR-Cas9N system for flexible genome engineering in Lactobacillus acidophilus, Lactobacillus gasseri, and Lactobacillus paracasei.</a> Applied and Environmental Microbiology. 87 (6), e02669 (2021).</li><li>Berthier, F., Zagorec, M., Champomier-Verg, M., Ehrlich, S. D., Morel-Devillel, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+transformation+of+Lactobacillus+sake+by+electroporation.">Efficient transformation of Lactobacillus sake by electroporation.</a> Microbiology. 142 (5), 1273-1279 (1996).</li><li>Rattanachaikunsopon, P., Phumkhachorn, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glass+bead+transformation+method+for+gram-positive+bacteria.">Glass bead transformation method for gram-positive bacteria.</a> Brazilian Journal of Microbiology. 40 (4), 923 (2009).</li><li>Pfl&#252;gl, S., Marx, H., Mattanovich, D., Sauer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+engineering+of+Lactobacillus+diolivorans.">Genetic engineering of Lactobacillus diolivorans.</a> FEMS Microbiology Letters. 344 (2), 152-158 (2013).</li><li>Spath, K., Heinl, S., Grabherr, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+cloning+in+Lactobacillus+plantarum:+Electroporation+with+non-methylated+plasmid+DNA+enhances+transformation+efficiency+and+makes+shuttle+vectors+obsolete.">Direct cloning in Lactobacillus plantarum: Electroporation with non-methylated plasmid DNA enhances transformation efficiency and makes shuttle vectors obsolete.</a> Microbial Cell Factories. 11, 141 (2012).</li><li>Ortiz-Velez, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome+alterations+associated+with+improved+transformation+efficiency+in+Lactobacillus+reuteri.">Genome alterations associated with improved transformation efficiency in Lactobacillus reuteri.</a> Microbial Cell Factories. 17 (1), 138 (2018).</li><li>O'Sullivan, D. J., Klaenhammer, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+mini-prep+isolation+of+high-quality+plasmid+DNA+from+Lactococcus+and+Lactobacillus+spp.">Rapid mini-prep isolation of high-quality plasmid DNA from Lactococcus and Lactobacillus spp.</a> Applied and Environmental Microbiology. 59 (8), 2730-2733 (1993).</li><li>Lizier, M., Sarra, P. G., Cauda, R., Lucchini, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+expression+vectors+in+Lactobacillus+reuteri+strains.">Comparison of expression vectors in Lactobacillus reuteri strains.</a> FEMS Microbiology Letters. 308 (1), 8-15 (2010).</li><li>Roberts, R. J., Vincze, T., Posfai, J., Macelis, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=REBASE-a+database+for+DNA+restriction+and+modification:+Enzymes,+genes+and+genomes.">REBASE-a database for DNA restriction and modification: Enzymes, genes and genomes.</a> Nucleic Acids Research. 43, D298-D299 (2015).</li><li>Ahrne, S., Molin, G., Axelsson, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transformation+of+Lactobacillus+reuteri+with+electroporation:+Studies+on+the+erythromycin+resistance+plasmid+pLUL631.">Transformation of Lactobacillus reuteri with electroporation: Studies on the erythromycin resistance plasmid pLUL631.</a> Current Microbiology. 24, 199-205 (1992).</li></ol>

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 ...

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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1 kb Plus DNA Ladder</td>
			<td>NEB</td>
			<td>N3200L</td>
			<td></td>
		</tr>
		<tr>
			<td>1mL Spectrophotometer cuvettes</td>
			<td>Thomas Scientific</td>
			<td>1145J12</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose&#160;</td>
			<td>BioShop</td>
			<td>AGR001</td>
			<td></td>
		</tr>
		<tr>
			<td>Allegra X-15R (refrigerated centrifuge)</td>
			<td>Beckman Allegra&#160;</td>
			<td>N/A</td>
			<td>No longer in production</td>
		</tr>
		<tr>
			<td>AnaeroGen 2.5 L Sachet</td>
			<td>Thermo Scientific</td>
			<td>OXAN0025A</td>
			<td></td>
		</tr>
		<tr>
			<td>BTX, ECM 399 electroporation system</td>
			<td>VWR</td>
			<td>58017-984</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge tubes (50 mL)</td>
			<td>FroggaBio</td>
			<td>TB50-500</td>
			<td></td>
		</tr>
		<tr>
			<td>DNA gel x6 loading dye</td>
			<td>NEB</td>
			<td>B7024S</td>
			<td></td>
		</tr>
		<tr>
			<td>Electroporation cuvette</td>
			<td>Fisherbrand</td>
			<td>FB101</td>
			<td></td>
		</tr>
		<tr>
			<td>Erythromycin</td>
			<td>Millipore Sigma</td>
			<td>E5389-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>Gel electroporation bath/dock</td>
			<td>VWR</td>
			<td>76314-748</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol&#160;</td>
			<td>BioShop</td>
			<td>GLY001</td>
			<td></td>
		</tr>
		<tr>
			<td>Limosilactobacillus reuteri</td>
			<td>Leibniz Institute DSMZ</td>
			<td>DSM20016</td>
			<td>Strain designation F275</td>
		</tr>
		<tr>
			<td>Lysozyme</td>
			<td>BioShop</td>
			<td>LYS702.5</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge tubes (1.7 mL)</td>
			<td>FroggaBio</td>
			<td>LMCT1.7B</td>
			<td></td>
		</tr>
		<tr>
			<td>Miniprep kit (Qiagen)</td>
			<td>Qiagen</td>
			<td>27106</td>
			<td>slpGFP replaced with constitutive, codon optimised, mCherry2 reporter protein&#160;</td>
		</tr>
		<tr>
			<td>MRS Broth (Dehydrated)</td>
			<td>Thermo Scientific</td>
			<td>CM0359B</td>
			<td></td>
		</tr>
		<tr>
			<td>Mutanolysin</td>
			<td>Millipore Sigma</td>
			<td>M9901-5KU</td>
			<td></td>
		</tr>
		<tr>
			<td>NaOH&#160;</td>
			<td>Millipore Sigma</td>
			<td>1064691000</td>
			<td></td>
		</tr>
		<tr>
			<td>P100 Pipette</td>
			<td>Eppendorf</td>
			<td>3123000047</td>
			<td></td>
		</tr>
		<tr>
			<td>P1000 Pipette</td>
			<td>Eppendorf</td>
			<td>3123000063</td>
			<td></td>
		</tr>
		<tr>
			<td>P2.5 Pipette</td>
			<td>Eppendorf</td>
			<td>3123000012</td>
			<td></td>
		</tr>
		<tr>
			<td>P20 Pipette</td>
			<td>Eppendorf</td>
			<td>3123000039</td>
			<td></td>
		</tr>
		<tr>
			<td>P200 Pipette</td>
			<td>Eppendorf</td>
			<td>3123000055</td>
			<td></td>
		</tr>
		<tr>
			<td>PCR tubes</td>
			<td>FroggaBio</td>
			<td>STF-A120S</td>
			<td></td>
		</tr>
		<tr>
			<td>Personal benchtop microcentrifuge</td>
			<td>Genlantis</td>
			<td>E200100</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dishes</td>
			<td>VWR</td>
			<td>25384-088</td>
			<td></td>
		</tr>
		<tr>
			<td>PTC-150 Thermal Cycler</td>
			<td>MJ Research</td>
			<td>N/A</td>
			<td>No longer in production</td>
		</tr>
		<tr>
			<td>pTRKH3_slpGFP (modified)</td>
			<td>Addgene</td>
			<td>27168</td>
			<td></td>
		</tr>
		<tr>
			<td>SPECTRONIC 200 Spectrophotometer</td>
			<td>Thermo Scientific</td>
			<td>840-281700</td>
			<td></td>
		</tr>
		<tr>
			<td>Storage microplate</td>
			<td>Fisher Scientific</td>
			<td>14-222-225</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>BioShop</td>
			<td>SUC507</td>
			<td></td>
		</tr>
		<tr>
			<td>TAE Buffer 50x</td>
			<td>Thermo Scientific</td>
			<td>B49</td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex</td>
			<td>VWR</td>
			<td>58816-121</td>
			<td>No longer in production</td>
		</tr>
		<tr>
			<td>VWR 1500E incubator</td>
			<td>VWR</td>
			<td>N/A</td>
			<td>No longer in production</td>
		</tr>
	</tbody>
</table>

methods for electroporation and transformation confirmation in limosilactobacillus reuteri dsm20016

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.

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.

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 &#181;L of the 8.5 kb plasmid pTRKH3_mCherry2 (pAM&#946;1 theta origin of replication) should give transformation efficiencies of roughly 80 colony forming units (CFU...

Watch this Scientific Journal Video about Methods for Electroporation and Transformation Confirmation in Limosilactobacillus reuteri DSM20016 at JoVE.com

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 ...