Excess nutrients, especially those that result from fertilizer runoff, can cause rapid growth of algae and cyanobacteria - a condition called an algal bloom. Thriving in both saltwater and freshwater, algal blooms have been reported on the shores of every coastal state and in countless lakes in the US alone. They are considered a major environmental threat in all 50 states. Recently, algal blooms have received notable media attention due to the severe devastation they have caused. They damage the economy by decreasing tourism and fishing in coastal communities and by hurting the business of industries that depend on clean water [3]. Additionally, when these blooms decompose, they deplete oxygen in the water, killing marine life, which may cause severe damage to the local ecosystem.
Harmful algal blooms, or HABs, are a subset of blooms which also produce toxins that are detrimental to human and animal health. One class of these toxins -- microcystins -- cannot be sufficiently broken down in the liver of many organisms, including humans, domesticated animals, and fish. Humans and animals have gotten sick and even died from swimming in or consuming water containing microcystins. Additionally, although algal blooms have short lifespans, microcystins remain in water systems for much longer periods of time, in which they may continue to contaminate local aquatic ecosystems as well as drinking and irrigation water.
Microcystin-LR, or MC-LR, is considered to be the most common and most toxic of microcystins [1]. Upon entering the liver, it inhibits two protein phosphatases, PP1 and PP2A. These proteins dephosphorylate other proteins, a common step in many biochemical reactions [2]. By inhibiting PP1 and PP2A, the toxin causes a build up of phosphorylated proteins, which prevents these essential biological reactions from occurring. This results in extensive damage to the liver, which, in extreme cases, may lead to kidney and lung damage and even heart failure.
Furthermore, it is impossible to distinguish between harmful and non-harmful blooms by sight alone. Extensive lab testing is required to detect the presence of toxins, and there are few protocols in place to remove these toxins from water.
Clearly, there is a pressing need for more rapid detection and remediation of MC-LR in both recreational and drinking water. To this end, we have developed a rapid colorimetric aptamer based detection system and - improving upon the work of several past iGEM teams - engineered a series of E. coli strains which progressively degrade MC-LR. We further encapsulated our strains in alginate beads in order to maximize reaction efficiency within our bioreactor, HABLab.
Our project this year aims to design and implement a bacterial solution for harmful algal blooms. Our proposed system consists of two components: a biological sensor to detect the presence of microcystins in water, and a filter containing engineered bacteria to degrade the microcystins (specifically microcystin-LR, the most common of these toxins).
The biological sensor consists of a DNA aptamer conjugated to gold nanoparticles, which specifically binds microcystin-LR. Binding of the aptamer to microcystin results in a colorimetric change. The color change can be quantified and related to the microcystin concentration via a standard curve.
Second, to engineer the bacteria for our filter, we have introduced genetic constructs coding for a specific cassette of enzymes. These enzymes, which are endogenous to Sphingopyxis sp., have been shown to sequentially break down microcystins. The enzymes are named mlrA through mlrF. MlrA, B, and C are proteases that degrade MC-LR. MlrD is a transporter protein that carries MC-LR into the cell. The functions of mlrE and F are yet to be elucidated, but both are believed to assist with MC-LR degradation. The filter consists of a packed-bed bioreactor, in which water is passed through a chamber containing our engineered strain immobilized on alginate beads. Additionally, we have attached a tag from the bacterial twin arginine translocase pathway to target the enzymes to the bacterial periplasm. Our hope is that moving the enzymes close to the periphery of the bacterial cell, and therefore closer to the flow of contaminated water, will increase the efficiency of the reaction. We hope these improvements will maximize our system’s potential for environmental remediation.
[1] Wikipedia contributors. (2019, July 21). Microcystin-LR. In Wikipedia, The Free Encyclopedia. Retrieved 21:38, October 18, 2019, from https://en.wikipedia.org/w/index.php?title=Microcystin-LR&oldid=907208202.
[2] Guidelines for Drinking-Water Quality, 2nd ed. Addendum to Vol. 2. Health criteria and other supporting information. World Health Organization, Geneva, 1998.
[3] Harmful Algal Blooms. (2019, July 17). Retrieved from https://www.epa.gov/nutrientpollution/harmful-algal-blooms.