genetic modification

bacterial genetic modification

Genetic manipulation of non-model bacteria represents a frontier in microbiology, going beyond the well-studied organisms like Escherichia coli and Bacillus subtilis. These non-model bacteria, which include a diverse array of species with unique metabolic capabilities, ecological roles, and potential biotechnological applications, have historically been challenging to study due to their complex genetic systems and often recalcitrant nature to traditional genetic tools.

I’m working towards this goal by using molecular biology, bioinformatics, and synthetic biology tools to enable precise genetic modifications of plant-associated bacteria. By tailoring genetic tools to the specific needs of these non-model organisms, we can explore the behaviour of microbes in their environment and harness their capabilities for innovative solutions to global challenges.

I’ve developed a wide array of tools to tag bacteria with several fluorescent protein genes and a combination of selection markers, all of them in different delivery systems (expression plasmid, Tn5-, and Tn7-plasmids). Additionally, I’m incorporating the use of the bioluminescence operon, luxCDABE, to detect bacteria and follow their growth dynamics directly on plant tissues.

Ultimately, my focus extends to the development of a CRISPR-based system tailored for generating bacterial knockouts in representative members of the Arabidopsis microbiota.

techniques

  • Molecular Cloning
    • Blunt-end and sticky-end cloning
    • Gibson assembly
    • Golden gate
  • Gene-knockout
  • Bacterial Transformation
    • Electroporation
    • Conjugation
  • Fluorescence Microscopy
  1. Stocks, C., Schlechter, R. O., & Remus-Emsermann, M. N. P. (2022). Chromatic Bacteria v.2-A Himar1 Transposon-Based Delivery Vector to Extend the Host Range of a Toolbox to Fluorescently Tag Bacteria. Bacteria, 1(1), 56–65. https://doi.org/10.3390/bacteria1010006
  2. Schlechter, R. O., Kear, E. J., Remus, D. M., & Remus-Emsermann, M. N. P. (2021). Fluorescent Protein Expression as a Proxy for Bacterial Fitness in a High-Throughput Assay. Appl. Environ. Microbiol., 87(18), e0098221. https://doi.org/10.1128/AEM.00982-21
  3. Schlechter, R. O., & Remus-Emsermann, M. N. (2019). Delivering “Chromatic Bacteria” Fluorescent Protein Tags to Proteobacteria Using Conjugation. Bio Protoc, 9(7), e3199. https://doi.org/10.21769/BioProtoc.3199
  4. Schlechter, R. O., Jun, H., Bernach, M., Oso, S., Boyd, E., Muñoz-Lintz, D. A., Dobson, R. C. J., Remus, D. M., & Remus-Emsermann, M. N. P. (2018). Chromatic Bacteria - A Broad Host-Range Plasmid and Chromosomal Insertion Toolbox for Fluorescent Protein Expression in Bacteria. Front. Microbiol., 9, 3052. https://doi.org/10.3389/fmicb.2018.03052