Team:CU Boulder/Design

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The BioStake

CU iGEM BioStake

The general design of the stake was inspired this paper published in Biotechnology Progress which describes a miniature bioreactor to detect toxicity by using bioluminescent E. coli. Our design, shown in the image, will holds cells in a gel at the top of the long ground-insertion portion and will include homeostatic controls on the top of the stake. The outer filtration and pump aspects are not shown in the design above, but will be included to protect the cells from harmful environmental influences.

Biological Design Considerations

Cells which contain logic gates and take part in cell-to-cell signaling demand a very specific environment in order for the system to work properly. Since the logic gates are intended to be used as information storage as to whether a contaminant has passed and are designed so monitoring does not need to be continuous, the cells must be able to survive and remain active for several days inside the device. Furthermore, the cell-to-cell singaling requires that the cells are suspended in such a manner that AHL diffusion is efficient and that they can induce one-another in an efficient manner. In order to support both of these, we designed a special hydrogel scaffold made out of crosslinked polyvinyl alcohol. PVA hydrogels are commonly used in biomaterials based on its hyrdophilic nature which can attract moisture and nutrients to keep the cells alive, its pore size which allows for the diffusion of proteins and sugars while holding the cells in a relatively fixed position, and its biodegradability, which is attractive in the context of making an environmentally-conscious product. Additionally, by developing a hydrogel with relatively small pore size such as with PVA, it can act as a filter to keep out larger competetive bacteria that may infiltrate the device as well as prevent solid sedimentation from blocking cell-to-cell interaction and nutrient diffusion.

In early September, PVA scaffolds were made which contained cells with the pBad inducible promoter in front of the RFP gene, and experimental results support the hypothesis that the engineered E. coli were able to live inside and respond to arabinose when soaked in the solution. Further experimentation described below is still necessary to confirm that this 

In addition to designing the spatial arrangment of the cells, it's also important to consider elements that will allow for their survival over the time period which the stake is in place to collect data. If E. coli cells are not kept in highly sterile conditions within a narrow temperature, humidity, and nutrient concentration range, they will not sustain a functional cell count for the system to work properly. Therefore, a control system that can sense and automatically adjust these conditions must be incorporated. The proposed plan is to use a solar-powered stake so batteries do not have to be replaced, creating a more sustainable device that can be used repeadetly. However, the incorporation of such technology could potentially drive up the cost of the BioStake which would not be favorable considering its intended use is for quick, easy, and low-cost results available to anybody. Nonetheless, it seems to be a feasible solution to work with the cells that we have already developed.

Designing for Environmental Functionality

The types of locations in which the BioStake will be placed, and how it tracks contamination at the sites is crucial to the design of the stake itself. To create the best device, it should be able to intake an air or water sample that is consistent with the bioavaiable concentrations of fracking contaminants, while excluding harmful components from affecting the bacteria. The BioStake would contain a small pump that would attract water or air through a filter wraped around the base of the stake, and then small tubes would draw up the fluid to the cell-containing gel via capillary action.   

Creating a User-Friendly Device

As described on the motivation page, the market has high demand for a biosensor that is easy to use and available to the general public to detect fracking contaminants. Apart from the design of the pump and homeostasis mechanism, many components of the stake are designed specifically to make the BioStake inexpensive and easy-to-use.

The physical structure of the stake is intended to be simple enough that the main components could be manufactured in a plastic injection mold. It's also designed for the hydrogel to be held in the compartment between the top and the long ground-insertion portion so that the user can know whether or not napthalene is present based on visual inspection of the device to see whether the gel has changed colors. The stake itself would be less than 6 inches in length and would weigh less than a pound. Research still has to be done to balance being small enough to be portable, and long enough for it to be able to collect continuous fluid samples from air, water, or soil in the environments around fracking sites.

Future Directions for Development

Further experimentation is required to determine the feasibility of using PVA to allow AHL diffusion in cell-to-cell signaling, extensive cell growth studies to see if the system can continue. If we had more time, measurements would be taken to determine cell growth rates over time, and then the hydrogel chemistry could be adjusted to control nutrient diffusion appropriately.

Additionally, the proposed method to maintain homeostasis within the device that would allow the cells to survive over long periods of time may be tedious to maintain and the aspect of the design that is most vulnerable to failure. It may be beneficial to move the design from E. coli into a bacterium that can survive in much harsher conditions, perhaps a bacteria that already thrives in natural water sources.

Ideally, this product could be prototyped and put into mass production. However, without enough information to specify exact design criteria, its difficult to develop an entrepreneurial plan, despite environmental agencies and oil companies expressing interest in the product.


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