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JoVE Journal
Bioengineering
Methods for Electroporation and Transformation Confirmation in Limosilactobacillus reuteri
Methods for Electroporation and Transformation Confirmation in Limosilactobacillus reuteri
JoVE Journal
Bioengineering
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JoVE Journal Bioengineering
Methods for Electroporation and Transformation Confirmation in Limosilactobacillus reuteri DSM20016

Methods for Electroporation and Transformation Confirmation in Limosilactobacillus reuteri DSM20016

Full Text
4,935 Views
11:04 min
June 23, 2023

DOI: 10.3791/65463-v

Alexander Duggan1, David McMillen1

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.

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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ElectroporationTransformation ConfirmationLimosilactobacillus ReuteriDSM20016Synthetic BiologyLactobacillalesProtocolsBacterial FamilyMicrobial SensorsTherapeutic DeliveryGastrointestinal TractPhenotypic VariationGenotypic VariationCommensal Strains

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