2020 Virtual undergraduate Research symposium
Layer-by-Layer Growth of Bacterial Biofilms
* THIRD PLACE BEST IN SHOW *
PROJECT NUMBER: 20
AUTHOR: Tony Tien, Chemical and Biological Engineering | MENTOR: Kevin Cash, Chemical and Biological Engineering
ABSTRACT
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Bacterial biofilms pose barriers to the current antibiotic selection strategies for treating affected clinical patients quickly. Pseudomonas aeruginosa, in particular, is a bacteria commonly found in the environment that poses risks of lung, blood, and other bodily infections with high contraction risk for people with compromised immune systems, open or recovering wounds, or exposure to contaminated medical devices. P. aeruginosa also colonizes the lungs of people with cystic fibrosis, providing a targeted interest for a type of biofilm that has proven difficult to treat. The exploration of a layer-by-layer growth technique for an artificial biofilm aims to mimic in vivo biofilm characteristics to improve the diagnosis and treatment of patients infected with biofilms. A layer-by-layer dipping technique involving a sequence of polymers, GFP-expressing P. aeruginosa PA01GFP, and water rinses provided a basis for the growth of biofilms on circular, glass microscope coverslips. Biofilm deposition and bacterial incorporation within the biofilm were then analyzed with fluorescence and confocal spectroscopy. A future implementation of these biofilms aims to take advantage of metabolite-sensitive nanosensors developed by the Cash Lab for the development of an effective antibiotic screening test for clinical biofilm treatment.
VISUAL PRESENTATION
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AUTHOR BIOGRAPHY
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Tony is a Chemical Engineering sophomore performing research within the department in the lab of Dr. Kevin Cash. His work in the Cash Lab has involved the development of analyte nanosensors to be applied to biological systems in vivo. He has also worked on a layer-by-layer biofilm incorporating Pseudomonas aeruginosa, designed to replicate a natural P. aeruginosa biofilm for improved diagnosis of biofilms in clinical settings. In the future, he intends to go to grad school to continue research in fields related to biotechnology and is considering working towards a Ph.D.
Great Job Tony!
Thanks, Megan! It was great to work with you on this project!
Hi Tony, very nice poster! I am curious, why do you think the order of deposition (PSS -> PEI -> GFP vs PEI -> PSS -> GFP) influenced the bacterial growth?
Hi David,
I am not very sure about why the PSS -> PEI -> GFP variation worked better than PEI -> PSS -> GFP, though my thinking is that starting with PSS was more conducive to the overall construction of the biofilm — allow me to explain further.
Both the PSS and PEI purchased were dissolved in water and are very viscous in nature. The PEI had to be diluted with a relatively large amount of water to balance charges with PSS properly and create the overall neutral matrix that was desired. The glass microscope coverslips are neutrally charged, so while they may not have a particular affinity to positive ions over negative ions, or vice versa, the fact that the PSS did not have to be nearly as dilute meant that the viscous polymer could stay on the surface of the glass better than the PEI could. As a result, the PEI would be able to attach better as more PSS ions would stay on the surface. In contrast, the PEI solution had a lot more water, so starting with PEI and then going into the water bath may have washed away more of the PEI from the surface since it was already more dilute and pose a barrier to proper layering and charge balancing through the dipping process. The solutions were stirred as necessary to ensure the polymers stayed in a consistent concentration for proper charge balancing.
Very early tests in this project used positively charged glass microscope slides that were about 4x the size of the coverslips ultimately used, though different polymers were being tested with the microscope slides. Microscope coverslips were chosen over glass slides since we already had trays that could fit the microscope coverslips for analysis in our microplate reader, whereas continuing with the glass slides required a 3D printed tray that we designed but had trouble printing. PSS and PEI were chosen for further testing due to more information about them being used in other layer-by-layer applications, great availability, and easier preparation for the bath solutions, though other polymer combinations may be considered in the future.
Thanks for checking out my poster!
Tony
Nice paper, well presented, well organized. The motivation was clear, the procedures, the conclusion. Easy for the nonspecialist to understand. I did not understand why the cation and the anion baths, and the analysis was not crystal clear, in part because of a few undefined initialisms. All in all, however, splendid paper!
Hi Professor Young,
I apologize for any confusion on unexplained acronyms — PSS, PEI, and M9 are acronyms for chemical components that were used for this project. The absorbance units (a.u. = absorbance units) are related to optical depth, while the fluorescence units (RFU = relative fluorescence units) are a relative measure of intensity. Both units are considered dimensionless.
The cation and anion baths were chosen to create charged layers of biofilm medium that can balance out with each other through the dipping process. Since the oppositely charged ions would have an affinity for each other in the biofilm structure, they contribute to the overall structure of the biofilm as well as entrapping other material (in this case, bacteria) by layering on top of non-ion components in the biofilm. The intent was to choose this method to be able to create biofilm layers that could stack on top of each other with more dipping cycles, creating a biofilm of a relatively desired thickness (more dipping for more bacterial incorporation).
The quantitative and qualitative analysis of the results, particularly with regards to the confocal analysis, showed that the bacteria appeared to successfully incorporate within the biofilm material. With reference to Figure 5 on the poster, the green areas show GFP-fluorescing material (the bacteria), whereas the gray areas show material outside of the GFP fluorescence range (the PSS and PEI matrix components). The cross part of the picture that protrudes from the plane shows the depth of the entire biofilm, with the large squares hollowed out to show what the fluorescence looks like depth-wise towards the center of the biofilm. The fluorescence and absorbance analysis were used as quick measures for the relative deposit of bacteria and biofilm material, respectively, essentially providing a glimpse of what the biofilm looks like from an overhead, 2D view.
I had intended to provide a portion of the analysis orally, so I could have done a better job balancing the information to include greater analysis on the poster. Thank you for your feedback on the analysis and acronyms — it is much appreciated!
Tony
Very good, thanks!
Hello Tony, very nice and professional looking poster!
I was curious why in the motivation section you mentioned planktonic bacteria and bolded it, but did not include in further in the poster. Is this an important distinction?
Hi Ben,
I just bolded that part to highlight that one of the main motivations for this project is that these biofilms are typically harder to treat than planktonic bacteria. I also bolded it for readability purposes.
Biofilms tend to be harder to treat than planktonic bacteria because the medium in which the bacteria grow in provide protection for the bacteria from the surroundings. Some consequences of this characteristic include poor bacteria quantification in a biofilm and poor antibiotic effectivity due to inhibited antibiotic diffusion and/or antibiotic neutralization from reacting with biofilm matrix components. I had intended to expand upon this in an oral component to this presentation, so I apologize for any confusion.
Thank you for checking out my poster, and I appreciate your feedback!
Tony
Amazing work Tony! Very cool
Thanks, Pilar!
Nice job Tony! Cash lab represent.
Thanks, Lexie!
Nice work Tony! Can’t wait to see where this research takes you.
Thanks, Ashley!
Great job, Tony! Very impressed.
Thanks, Tyler!