Team:Cornell/Demonstrate
The bioreactor is composed of immobilized E. coli which are enabled to express the mlr cassette into their periplasm. Immobilization was achieved by adapting a technique to encapsulate cells within millimeter-sized alginate beads [7]. The immobilized cells were then packed into a cylindrical bioreactor and lake water was passed through. As water passes through the bioreactor, microcystins diffuse into the beads and then into the E.coli periplasm where it is then degraded by the mlr enzymes.
To ensure that encapsulation was achieved, and that E. coli was capable of surviving within the alginate beads over an extended period of time, the beads were placed in a water bath with comparable water temperature for cyanobacterial growth (27.5°C) [8]. Every day a select number of beads were dissolved in sodium citrate, creating a solution of bacteria. This solution was then plated. Presence of colonies on the plates indicated survival of the E. Coli and successful encapsulation. To further test viability, wet cultures of the beads were successfully made by piercing the bead and placing the sample into LB.
To test the efficacy of the bioreactor, water treated with a known concentration of MC-LR was then passed through a bioreactor model packed with beads containing strains expressing either either mlrA (BBa_k2960012) or mlrA with translocase tag (BBa_k2960001), and the outflow then underwent aptamer testing. The results of the treated sample were compared to the initial results. This test was then replicated multiple times (n=3) to validate the bioreactor’s efficacy. To estimate the microcystinase activity, we compared the MC-LR concentration of the outflow to that of a control reaction, in which the same solution was passed through the empty chamber.
As compared to the control, the outflow from the bioreactor packed with beads containing mlrA has lower relative MC-LR concentration. Relative concentration was determined by normalizing the test (mlrA) absorbance values in the outflow by those of the control and then using the equation given by the standard curve to calculate corresponding relative concentration.
[1] Detection Methods for Cyanotoxins. (2019, August 12). Retrieved from https://www.epa.gov/ground-water-and-drinking-water/detection-methods-cyanotoxins.
[2] Gold Nanoparticle Properties. (n.d.). Retrieved October 19, 2019, from http://www.cytodiagnostics.com/store/pc/viewcontent.asp?idpage=2.
[3] Pamies, R., Cifre, J. G. H., Espín, V. F., Collado-González, M., Baños, F. G. D., & Torre, J. G. D. L. (2014). Aggregation behaviour of gold nanoparticles in saline aqueous media. Journal of Nanoparticle Research, 16(4). doi: 10.1007/s11051-014-2376-4
[4] Turner, A., Dhanji-Rapkova, M., O’Neill, A., Coates, L., Lewis, A., & Lewis, K. (2018). Analysis of Microcystins in Cyanobacterial Blooms from Freshwater Bodies in England. Toxins, 10(1), 39. doi: 10.3390/toxins10010039
[5] US Department of Commerce, Noaa, Great Lakes Environmental Research Laboratory, & Institute for Limnology and Ecosystems Research. (n.d.). Microcystin Guidelines. Retrieved October 19, 2019, from https://www.glerl.noaa.gov/res/HABs_and_Hypoxia/microcystinGuidelines.html.
[6] Li, X., Cheng, R., Shi, H., Tang, B., Xiao, H., & Zhao, G. (2016). A simple highly sensitive and selective aptamer-based colorimetric sensor for environmental toxins microcystin-LR in water samples. Journal of Hazardous Materials, 304, 474–480. doi: 10.1016/j.jhazmat.2015.11.016
[7] Dziga, D., Sworzen, M., Wladyka, B., & Wasylewski, M. (2013). Genetically Engineered Bacteria Immobilized in Alginate as an Option of Cyanotoxins Removal. International Journal of Environmental Science and Development, 360–364. https://doi.org/10.7763/IJESD.2013.V4.371
[8] Jiaqi You, Kevin Mallery, Jiarong Hong, Miki Hondzo, Temperature effects on growth and buoyancy of Microcystis aeruginosa, Journal of Plankton Research, Volume 40, Issue 1, January-February 2018, Pages 16–28, https://doi.org/10.1093/plankt/fbx059