The purpose of our project was to test the effects of antifreeze proteins and circumin and their ability to disrupt biofilm formation. There were many different aspects of this project that needed to be broken down into smaller experiments. One part of the team focused on finding out how biofilms grew on leafy crops and how to quantify the biofilms, while another part of the team looked at the antimicrobial properties of antifreeze proteins and circumin. Lastly, part of the team built a gene gun to deliver the proteins into a plant to then have that plant express the proteins.
Biofilm Formation on Crops
Biofilms are formed by a community of bacteria, protists, or fungal cells that create a cellular matrix and cling to a surface. Biofilms are found everywhere; on teeth, your gut, metals, leafy vegetables, the surface of ponds, etc. however, the more polar and textured the surface, the higher the probability of bacteria sticking to the material (Jamal, 2015). When a biofilm is formed, the bacteria produce a sticky substance that attaches and holds the cells together and adheres them to a surface (Jamal, 2015). Development of biofilms takes place through a four-stage process: attachment of bacteria, micro-colony growth, ESP production and networking, and finally release of encased bacteria (Jamal, 2015). Typically biofilms are mostly made of water, and equal parts of cells and a mixture of complex carbohydrates, proteins, and nucleic acids (Jamal, 2015). Within the cellular portion of the biofilm (which is usually the core of the film), substances critical for bacterial survival go around by water-filled tunnels among cells, with an appearance like a spider web (Jamal. 2015). Biofilms can grow and divide in order to grow in other areas. Biofilms form to increase their survival under stress; such as a lack of water, the presence of toxic substances, low or high pH, and more (Jamal, 2015). Quantifying biofilms can be difficult because their contents are small and there are not many available procedures to follow. The thickness of a biofilm and the rate at which it grows is important to know. We choose to quantify biofilms by growing cultures from a number of bacterial strains, normalizing the absorbance levels to 0.1 and plating them in round bottom well plates to grow for 48 hours. We used crystal violet to dye the biofilms and compared the results from the plate reader to a standard curve made with the crystal violet dye and acidic acid.
Antifreeze Proteins' Antimicrobial Properties
Antifreeze proteins are a class of proteins produced by certain vertebrates, such as fish and insects, plants, bacteria, and fungi. The proteins bind to ice crystals to prevent the growth and recrystallization of ice that would harm the organism’s ability to grow and survive. Antifreeze proteins are known to have anti-virulent properties and be able to disrupt the formation of biofilms. The proteins may bind to the biofilm in a similar way in which they bind to ice disrupting the development of the film. This project is based on multiple journal articles that look at the antimicrobial action of antifreeze proteins. However, there is not a lot of published research on the mechanism of action of these proteins against microbes. We wanted to connect this new research on antifreeze proteins’ antimicrobial action to a real-world application; on crops (Sinha, 2016). Below as an example of one of the constructs we worked with, AraC regulated by IAGFP. In this plasmid, antifreeze protein expression is regulated by the inducible promoter AraC, which in the presence of arabinose will produce protein. In the absence of arabinose, no protein is created.
Impact of Curcumin on Biofilms
Curcumin apparently achieves its antibacterial nature from lysing the cell membranes of both Gram-positive and Gram-negative bacteria (Tyagi, 2015). Though the composition of bacterial types differs, this compound perforates bacterial membranes to allow the contents of each cell type to eject into the extracellular space, thereby killing the cells in the process (Tyagi, 2015). Specifically, by virtue of possessing an amphipathic chemistry, curcumin can selectively insert into lipid bilayers and disrupt the native structure (Tyagi, 2015). As biofilms develop through quorum sensing mechanisms (the communication pathways between bacteria that selectively activates gene transcription via signaling molecules), the breakdown of cellular membranes does away the entryways of such molecules (Solano, 2014). Therefore, if bacteria survive initial membrane perforation, they lose the ability to accurately secrete and absorb signaling molecules to further biofilm growth ("QS").
Creating Transgenic Plants
There are two methods we researched to make plants express antifreeze proteins. The first approach is known as a biolistic method and it involves using a gene gun. We chose to build our own gene gun instead of purchasing one. The plasmid of interest is bound to very small particles made of gold or tungsten and shot into the plant's cells. The other method we chose to investigate involves applying a solution of bacteria containing the plasmid that can infect the plant and transmit the plasmid. This method will hopefully be used in future experiments if time permits.
Jamal, M., Tasneem, U., Hussain, T., & Andleeb, S. (2015). Bacterial Biofilm: Its Composition, Formation, and Role in Human Infections. Research & Reviews: Journal of Microbiology and Biotechnology, 4(3). Retrieved from http://www.rroij.com/open-access/bacterial-biofilm-its-composition-formation-and-role-in-human-infections.php?aid=61426
Sinha, P., Muralidharan, S., Sengupta, S., & Veerappapillai, S. (2016). A Brief Review on Antifreeze Proteins: Structure, Function and Applications. [Abstract]. Research Journal of Pharmaceutical, Biological and Chemical Sciences, 7(3), 914-919. Retrieved October 16, 2018, from https://www.researchgate.net/publication/304935678_A_brief_review_on_antifreeze_proteins_Structure_function_and_applications.
Solano, C., Echeverz, M., & Lasa, I. (2014). Biofilm dispersion and quorum sensing. Current Opinion in Microbiology, 18, 96–104. https://doi.org/10.1016/J.MIB.2014.02.008
Tyagi, P., Singh, M., Kumari, H., Kumari, A., & Mukhopadhyay, K. (2015). Bactericidal Activity of Curcumin I Is Associated with Damaging of Bacterial Membrane. Plos One,10(3). doi:10.1371/journal.pone.0121313
What is QS? (n.d.). Retrieved October 3, 2018, from https://www.nottingham.ac.uk/quorum/what.html