2021 Virtual Undergraduate Research Symposium
2021 Virtual Undergraduate Research Symposium
Uncovering the Secrets of Biological Vesicles
Uncovering the Secrets of Biological Vesicles
PROJECT NUMBER: 20 | AUTHOR: Elizabeth Betz, Chemistry
MENTOR: Kim Williams, Chemistry
ABSTRACT
Extracellular vesicles (Evs) are nano-sized vesicles that are released by cells that have recently become an interest of study because of their important roles in cell-to-cell communication. Outer membrane vesicles (OMVs) are a type of EV that are released from gram-negative bacteria and have been shown to contain enzymes that degrade lignin. Lignin is an abundant polymer found in plants, but its complex structure makes it difficult to break down to use as a biofuel. To better understand how OMVs break down lignin, we designed a method using asymmetrical flow field-flow fractionation (AF4) and multi-angle light scattering (MALS) to count the number of Pseudomonas putida OMVs in a sample. AF4 was used to separate OMV samples based on size and MALS was used to measure the radius and to enumerate OMVs. Liposome standards were used as a model to create a calibration curve to relate concentration to MALS signal that will be applied to OMV samples. Determining the size and number of OMVs in a sample will assist in protein and lipid analyses and uncovering the mechanism P. putida OMVs use to degrade lignin.
PRESENTATION
AUTHOR BIOGRAPHY
Elizabeth (Lizzy) is a senior studying biochemistry at Mines and is graduating December 2021. She joined the Williams research group in the chemistry department in fall of 2019 through the Mines Undergraduate Research Fellowship. Her research has focused on applying asymmetrical flow field-flow fractionation and multi-angle light scattering to count biological vesicles. She is interested in medical science and hopes to pursue a career in pharmaceutical science or epidemiology after graduation.
Great presentation! At the beginning of your video, you mentioned that biofuels are an important application area for lignin degradation. Since methodologies used have proven successful, do you plan to attempt to quantify energy production from this process to determine its feasibility?
Thank you Emily! The next step in this project for us is to separate OMVs prepared by NREL and to send them off for protein and lipid analyses. Our successful particle counting method and future protein and lipid analyses will help us to better understand the role proteins and lipids play in lignin degradation by OMVs. Our lab works specifically on designing new methods using FFF to analyze a variety of nano-sized particles, so quantifying energy production from lignin degradation is more on NREL’s side of things. NREL is interested in better understanding the biochemistry of OMVs in lignin degradation and how OMVs can be utilized as a tool in the production of biofuels.
Very interesting study and nice presentation.
I wonder what are the broad implications of this research? What kind of industries will benefit from your research conclusions?
Thank you. Extracellular vesicles (EVs) are released by all types of cells and they are currently being studied in a variety of disciplines including detecting cancer and other diseases, drug delivery and lignin degradation. Overall, through this research we designed a method to count the number of EVs (OMVs are a type of EV) in a sample. This method is applicable to other EV investigations in which the number of EVs in a sample could, for example, provide insight into how a biochemical reaction occurs, how cells interact with one another, how disease spreads, or how well an EV drug delivery system works. Broadly, the pharmaceutical industry, healthcare industry, and renewable energy industry may benefit from our research conclusions.
Hello Lizzy,
Great presentation! I enjoyed how applicable this project is to the modern day problem of our dependence on fossil fuels for energy. I did have a question regarding your methodology. You mention that the experiment was tested on a standardized liposome due to their similarity to the OMVs. In your future work, how do you predict this method will translate to the OMV structures? Are the efficiency trends predicted to be the same and how scalable would this be to useful fuel? Thank you for the time.
Hi Jack,
Our AF4-MALS method was validated with standardized liposome samples, as well as silica nanoparticles. OMVs are quite similar in their shape to liposomes as both are spherical and have a lipid bilayer. However, OMVs contain proteins and other molecules inside the vesicle and in the membrane while liposomes do not. Liposomes are the most closely related biological particle that is available as a standard, and because the method worked for silica particles as well (which are spherical particles that do not contain a bilayer), we believe that this method is applicable and will translate well for OMVs despite their slight difference in structure.
We are currently working to upscale the method to larger sample volumes (from 20uL sample to 1mL sample) so our method will not be as time consuming when we separate more OMV samples (i.e. having to run one 1-hour long experiment versus having to run fifty 40-minute long experiments). This method is being used to characterize size and count the number of OMVs in samples grown in the presence of lignin. That number as well as lipid and protein analyses will help us to better understand what mechanisms OMVs use to break down lignin. A clearer picture of the mechanism will hopefully allow for utilization of those chemical pathways to produce biofuels more efficiently at a larger scale in the future. This method is not being utilized to produce fuel, but instead is providing insight into novel ways we could at some point. I hope this helps.