Engineering is not a destination but rather a beautiful journey. An engineer isn't simply concerned with whether the light bulb is working, but also about how bright it is compared to the rest, how much power it consumes, the life of the product, the cost of manufacturing it and so much more. Our project gave us plenty of opportunities to explore this journey and understand the scalable, quantifiable and measurable aspects of our problem statement - methylmercury poisoning, whilst also trying to improve on these aspects.
Measurement and Experimentation
The backbone to engineering success is to be able to accurately establish quantifiable data. The biggest hurdle to accurate measurement is the lack of a standardised scale, followed by errors due to equipment. To mitigate these, we have included calibration protocols for all the parameters to be measured in our experiments; Fluorescence, Optical Density and Gas Chromatography measurements. Calibration provides both a scale and can help keep errors to a minimum and thus contribute to accurate measurements. If there are any errors in the measurement of gas chromatography or OD - We have included statistical analysis methods to reduce the errors caused by deviations in the measurements. To further minimize errors, we have recommended suitable controls and duplicate measurements. We have identified some of the possible unexpected outcomes and listed methods of troubleshooting them. We have included preliminary experiments to ensure that reading time points can be accurately mapped and to prevent wastage of reagents.
Nothing embodies engineering more than a piece of hardware. Systematic Analysis of Underlying Limitations or SAUL was brainstormed after understanding the limitations of the current lab scale methods of testing probiotic bacterium as well as capsules through literature.
We imagined a device which could simulate and help understand various conditions of the stomach and gut environment including the flow dynamics due to peristalsis; shear stresses; variable pH in different sections; variable flow rates due to different kinds of food consumed; variable properties of the food itself like stickiness or fibrousness and its effects on the bacteria and capsules; differences in availability of the target component (in this case - methylmercury) and how it could affect the efficiency and much more.
To achieve this, we designed a simple device to bring the mechanical conditions to life, and act as a ground to perform laboratory experiments for testing physiological conditions. Furthermore, we wrote a detailed procedure to assemble the parts, however due to lack of access to labs we were unable to build it.
However, in order to ensure the correct simulation, we used ANSYS (Static Structural Workbench) to test the compressive forces on the dialysis tube as well as stresses such as shear stress experienced by it. The results helped us understand the relationship between the two parameters and the stresses were reduced to a value comparable to our gut stresses by reducing the pipe diameter.
We then explored the properties of the stomach and added two configurations in order to simulate both the stomach and intestine. We also ensured the retention time in the device would be the same or comparable to that of the retention time of food in the stomach and gut. This journey enabled us to not only make a device but also to gain a new perspective of the gastrointestinal tract. We have also included the expected problems and solutions in detail as well as safety consideration.
Figure 1: The Engineering Flow
To ensure reproducibility of our project - completely transparent research has been conducted. Every aspect has been documented in detail in multiple handbooks. We have meticulously curated three experimentation handbooks, with each one containing both general and experiment protocols. In addition, we have curated an implementation handbook, SAUL handbook, a Model handbook containing detailed protocols, mechanical aspects, equations, as well as a safety handbook which contains the safety considerations of the project and every reagent, experiment, processes, components, etc mentioned in other handbooks. Furthermore, We have included failures, challenges, troubleshooting, unexpected outcomes and estimated price to make certain easy and effective reproducibility.
Failure is central to engineering. Successful engineering is all about understanding how things can break or fail.
As part of modelling, we considered the two aspects of our proteins: structure and function. Through extensive literature studies, we found out the bonds, bond length, residues etc to enable structural analysis as well as equations, rate constants, concentrations etc for functional analysis. We designed VMD and matlab models in order to understand these aspects. After overcoming several challenges, we improved upon the models and obtained graphs, atom placements etc. In addition, we curated a detailed handbook including all facets of the final models.
In order to assess the activity of our dual enzyme system, we have designed an experiment which includes three stages - purification, extraction and testing. To enable extraction and purification of the proteins, we have designed three parts:
|Part Number||Part Name|
|BBa_K3470022||MerA + His-tag|
|BBa_K3470023||MerB + His-tag|
|BBa_K3470024||MerA + MerB + His-tag|
This experiment would provide three graphs,
- Mercury concentration vs time is obtained for constant concentration of enzyme
- Enzyme concentration vs activity is obtained.
- Number of cells vs enzyme concentration graph is obtained.
Using the graphs we can estimate the optimal amount of enzyme required to be produced for optimal activity and thus, estimate the optimum CFU/mL to be used to get maximum activity during implementation. According to our MATLAB - SimBiology simulation, enzyme concentration of about 10-4 would give optimal activity. However, this needs to be validated experimentally.