Project Description:

Organisms such as Aphanizomenon flos-aquae and Bacillus megaterium naturally produce gas vesicles employed in floatation based functions, under different environmental conditions. Gas vesicle production is associated with a range of primary (GvpA, C) and secondary Gvp genes (GvpN, R, G, etc.) in its wildtype strains. These proteins are conserved across different species and have been targeted for a variety of applications, including waste effluent separation from water, bio-processing and metabolic accumulation or degradation of potentially toxic by-products. This flotation has many potential pragmatic applications in industries such as pharmaceutical production, wastewater processing, and the mining industry where removal of organic macromolecules or heavy metals is required. These industrial processes are often costly and/or inefficient. We aim to utilize this cellular behaviour as a separation technique for wastewater processing in various industries where the efficiency of current processes is cause for large financial and environmental burden, as reduction of pollution becomes increasingly important.

The Shapiro Lab has demonstrated that gas vesicles produced by their lab’s synthetically produced Arg1 operon can induce flotation in Escherichia coli. The Arg1 operon consists of repeats of the primary GvpA and GvpC genes derived from A. flos-aquae, as well as secondary Gvp genes from B. megaterium. We propose that combining cell-surface engineering of Escherichia coli with Arg1 transformation could yield a cellular bioremediation platform superior in both monetary and energetic efficiency compared to current approaches. It is known that E. coli can be engineered to express metal-binding peptides on its surface with certain specificity to particular metals. Given this, introducing a biomass of E. coli transformed with Arg1 and expressing metal-binding peptides on its surface into a bioreactor with effluent containing a particular metal of interest, the binding of metal particles to surface proteins could induce a downstream signal cascade that upregulates Arg1 expression. Expression of Arg1 would then induce flotation due to overexpression of gas vesicle proteins and subsequent assembly of gas vesicles. Future investigations could develop analogous mechanisms which would allow for the potential application of the removal of organic molecules which would produce a more modular and widespread applicability of this system. Investigation of the specific signaling mechanisms that could be leveraged for this process could be explored as well.

Optimization of the Arg1 construct for flotation in E. coli should yield the function of efficient, inducible flotation in transformed E. coli cells. The Arg1 operon is greater than 7kb and the secondary Gvp genes have no known fundamental role in the production of the gas vesicles. We aim to reduce the size of the Arg1 operon to exclude the secondary proteins, thereby relying on the primary proteins, and test flotation in E. coli cells. In addition to laboratory experimentation for characterization of the Arg1 construct, gas vesicles expressed by it, and the function/necessity of the gas vesicle proteins that go into forming the vesicles, our team will be doing extensive mathematical modelling and computational analysis of data to model growth dynamics of the engineered cell strain and the kinetics of metal binding surface proteins, as well as to determine the buoyant force imparted by gas vesicle expression. These secondary models will be fed into a larger model that aims to predict the potential performance and operating characteristics for our proposed cellular bioremediation platform on an industrial scale.