Methods for Electroporation and Transformation Confirmation in Limosilactobacillus reuteri DSM20016
June 23rd, 2023
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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Overview
This article presents protocols for working with Limosilactobacillus reuteri DSM20016, a robust human gut commensal. The protocols include growth, plasmid transformation, colony PCR, and fluorescent reporter protein measurement.
Key Study Components
Area of Science
- Microbiology
- Synthetic Biology
- Gastrointestinal Health
Background
- Limosilactobacillus reuteri DSM20016 is a natural human gut commensal.
- This strain shows promise for therapeutic delivery and disease detection.
- Protocols in this area are often fragmented and difficult to navigate.
- Research aims can bias the choice of bacterial species.
Purpose of Study
- To provide clear protocols for working with DSM20016.
- To facilitate the measurement of reporter proteins and colony PCR confirmation.
- To address common issues and troubleshooting in the protocols.
Methods Used
- Growth of Limosilactobacillus reuteri DSM20016.
- Plasmid transformation techniques.
- Colony PCR for confirmation of genetic modifications.
- Measurement of fluorescent reporter proteins.
Main Results
- Successful growth and transformation protocols were established.
- Colony PCR confirmed the presence of desired genetic constructs.
- Fluorescent reporter proteins were effectively measured.
- Troubleshooting guidelines were provided for common issues.
Conclusions
- The protocols enhance the usability of DSM20016 in research.
- They provide a foundation for future synthetic biology applications.
- Clear guidance helps researchers navigate fragmented literature.
Frequently Asked Questions
Chapters in this video
0:00
Introduction
2:04
Preparing Limosilactobacillus reuteri DSM20016 Electrocompetent Cells for Electroporation
5:27
Measurement of the Acid‐Resistant Fluorescent Reporter Protein mCherry2
7:03
Confirmation of Plasmid Uptake via Colony PCR
8:45
Mini‐Prep Protocol for Limosilactobacillus reuteri and Confirmation of Plasmid Presence by PCR
