Plasmid Tools for Gram-Positive Bacteria to Expand Microbiome Editing
Carlotta Ronda, Principal Investigator
Innovative Genomics Institute
Closed. This professor is continuing with Spring 2025 apprentices on this project; no new apprentices needed for Fall 2025.
Gram-positive microbes are important members of host-associated and environmental microbiomes, yet many remain genetically intractable. This project will implement novel microbiome editing tools by developing modular plasmids that can either replicate or integrate into the genomes of these difficult-to-engineer bacteria, and by establishing workflows for introducing them into strains that currently lack genetic access. The plasmids will include visible reporters (e.g., fluorescent proteins) to provide clear readouts of success and stability. Beyond single-strain tests, the system will also be evaluated in in vitro bacterial communities to assess performance in more complex environments. By creating reliable toolkits for non-model Gram-positive microbes, this work expands the microbiome engineering toolbox and opens new opportunities for microbial community modulation.
Qualifications: The undergraduate apprentice will:
* Plasmid Construction: Design plasmid maps with mentor guidance, assemble modular plasmids using cloning methods (e.g., Gibson Assembly, restriction–ligation), and verify by sequencing.
* Outcome: At least two sequence-verified plasmids carrying distinct reporter modules.
* Delivery Workflow Development: Test different plasmid introduction approaches and track outcomes using colony growth and reporter expression.
* Outcome: A clear, stepwise workflow comparing delivery strategies.
* Validation in Single Strains and Communities: Monitor plasmid stability and reporter activity over short passages and extend testing to in vitro bacterial communities to evaluate performance in mixed populations.
* Outcome: Proof-of-principle demonstration of plasmid functionality in Gram-positive microbes and preliminary data on community-level behavior.
* Documentation: Compile results in figures, concise protocol notes, and workflow templates to support reproducibility.
Learning Outcomes: Students will gain hands-on experience in plasmid engineering, microbial genetics, and workflow design. They will also learn how to evaluate genetic tools in the context of bacterial communities, strengthening skills in experimental planning, record-keeping, and protocol writing while contributing to the development of novel microbiome editing platforms.
Day-to-day supervisor for this project: Zhixiang Yao , Ph.D. candidate
Hours: 9-11 hrs
Related website: https://