Welcome to SIAT-SCIE

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Brief Intro

Outer membrane vesicles (OMVs) are ubiquitously produced in the world of bacteria yet have been grievously overlooked in the past; budding out as spherical containers of 20 to 500 nm in diameter from the bacterial membrane, they are potentially capable of transporting a wide array of biomolecules that awaits the academia to divulge. [1] As potent transporters, OMVs play an integral role in various biological phenomena, ranging from stress regulation to microbial interactions. [2]

Seeing that the unique properties of OMVs may revolutionize traditional delivery system, our team aims to apply the wonders of OMVs to the very frontiers of genetic engineering research - the CRISPR-Cas9 system. As natural kins to cell membranes, they can be degraded easily while preserving the shape and bioactivity of sensitive Cas9 proteins within, as well as single guide RNA (sg-RNA). We expect this technique would open up new possibilities of in vitro genetic engineering, and be of substantial aid in curing and preventing illnesses such as inflammatory bowel diseases by removing virulence genes from malignant bacteria.

The Specifics

As a preliminary step, we will perform transformation of plasmids containing genes of Cas9 and sg-RNA on pathogens, whose efficacy of gene editing may serve as a baseline for further experiments involving OMV transport of the Cas9 protein and sg-RNA.

In hopes of maximizing the yield of OMVs, insertion of hypervesiculation genes into the bacteria will be performed. [3] A SpyCatcher/SpyTag system will also be constructed with regards to Cas9, which may efficiently direct the protein into the OMV. [4]

To quantify the expression of Cas9 within the bacteria into which the OMVs have fused, a fluorophore may be attached to the protein for fluorescence analysis.

Lastly to confirm the efficacy of our OMV-CRISPR-Cas9 system, the expression of virulence gene in pathogen, which should be mostly removed, may be quantified as well.


  1. Liu, Y., Alexeeva, S., Defourny, K. A., Smid, E. J. & Abee, T. (2018). Tiny but mighty: bacterial membrane vesicles in food biotechnological applications. Current Opinion in Biotechnology, 49, 179-184
  2. Schwechheimer, C., & Kuehn, M. J. (2015). Outer-membrane vesicles from Gram-negative bacteria: biogenesis and functions. Nature Reviews. Microbiology, 13(10), 605–619.
  3. Valderrama, J. D. & Gutierrez F. R.S. (2018). Lipo Nanocarriers for Drug Targeting. 199-229.
  4. Zakeri, B., Fierer, J. O., Celik, E., Chittock, E. C., Schwarz- Linek, U., Moy, V. T. & Howarth, M. (2012) Peptide Tag Forming a Rapid Covalent Bond to a Protein, Through Engineering a Bacterial Adhesion. Proc. Natl. Acad. Sci. U. S. A. 109, e690−e697.