Team:Rotterdam HR/Design
Design
Design
The Carbon Monoxide Problem
Carbon monoxide is an odourless, colorless and toxic gas which is known as
the silent killer.
There are already a lot of carbon monoxide detectors for the industry but they have a lot of
interference with hydrogen gas. The detector can't see the difference between carbon monoxide and hydrogen gas,
this results in false alarms and unsafe work environments. We decided to build a selective carbon monoxide
detector without the interference of hydrogen gas.
How our constructs detect carbon monoxide
In our search 
to find a means to detect carbon monoxide we learned about the
transcription factor CooA, a heme-group containing protein normally made by Rhodospirillum rubrum, which becomes
active after binding to carbon monoxide (Shelver et al. 1997). Thus it becomes able to interact with the carbon
monoxide regulated transcriptional units cooMKLXUH (also known as pCooM) and cooFSCTJ (also known as pCooF).
Cloning these promoters so that they regulate reporter genes, ensures these reporters are only
expressed when carbon monoxide is present.
How our biological constructs work together with our hardware
Gas production
When CO is bound to CooA, the structure of CooA will change and this will bind with a CooA dependent promoter CooF or CooM. By placing a signal generating gene after CooF or CooM, this signal will only be expressed if carbon monoxide is present.
We looked into bacteria which give an electrical signal but this is very difficult measuring since bacteria always give some electrical signal.
After 
this option we thought of other ways to produce a signal. We found
some biobricks (K173003, K173013, K133071, K133116, K173013 etc.) which most likely produce CO2 if the required
substrate is present. We used the genes coding for the enzymes: pyruvate decarboxylase (pdc) and alcohol
dehydrogenase II (adhB). Pyruvate decarboxylase catalyses pyruvic acid to acetaldehyde and carbon dioxide.
Alcohol dehydrogenase II (adhB) catalyses the acetaldehyde reduction to ethanol.
Gas measurement
Our initial idea was to have the bacteria produce electrons, which could be easily picked up by our hardware. Another upside was the relative short duration of the signal. Sadly we never were able to go through with this idea due to the fact that E.coli produces generates electrons after a (long) acclimation time. We did look to see if we could use the electron production of other Exoelectrogens to our advantage. But all of the bacteria had had these genes in their genome and not in the plasmid. Which made it impossible for us to modify. Due to this and and the uncertainty as to when the electrons are created in E.coli We've decided to drop working with electrons and work with the gas production.
To measure the gas production, we collect the gas in a tube filled with medium, and after it hits a specific level of medium displacement, we release it in a controlled manner using a peristaltic pump. By measuring the interval between the tube filling two times, we can determine the amount of gas produced. Because we’re using a peristaltic pump, we can increase the accuracy of the measurement by releasing a smaller amount of gas. This makes the interval shorter, and allows us to make more measurements. For more information, visit our hardware page.
How our hardware control the environment
To make sure the only variable controlling the gas production is the concentration of carbon monoxide, we need to keep the other variables constant. To achieve this, we have made a device to keep the temperature stable and a device that will occasionally shake the tube containing the bacteria. This will make sure that the bacteria won't pile up on the bottom of the tube, which would cause a decrease in gas production.
Things we wanted to build
Killswitch for the safety
To ensure the safety of our end product we wanted to take a look at biological containment. Our eye quickly fell on so called "kill-switches", genetic circuits capable of killing synthetic bacteria when they are not in the presence of certain chemicals.One of the most interesting kill switches we found is the Deadman Kill Switch (Chan et al. 2015), which seemed to be one of the most easily adaptable kill switches at the moment. Though kill switches generally have problem with staying active in successive generations. Considering these kill switches are generally lost upon successive generations (around ~140 generations, which translates to around 3 days using E.coli), we decided not to include a killswitch in our own constructs, though it would be a practical addition if this product would be used in a real environment.
ATP sensor for the bacteria
We wanted a way to make sure our bacteria live inside the hardware. We wanted this because a dead cell wouldn't give off a signal, which could lead to a false positive feeling of safety. A way to make sure a cell lives is by using an ATP sensor. A living cell produces ATP which can be used for processes inside the cell. When the bacteria carries an ATP sensor, ATP will bind to the sensor and will produces a fluorescent protein. A living cell will be fluorescent and can be detected. When there is a low concentration of living bacteria, a low output will be generated. The measured CO concentration can be corrected for the number of living bacteria
References
Shelver, D., Kerby, R. L., He, Y., & Roberts, G. P. (1997). CooA, a CO-sensing transcription factor from Rhodospirillum rubrum, is a CO-binding heme protein. Proceedings of the National Academy of Sciences, 94(21), 11216–11220. doi:10.1073/pnas.94.21.11216.
Coyle, C. M., Puranik, M., Youn, H., Nielsen, S. B., Williams, R. D., Kerby, R. L., … Spiro, T. G. (2003). Activation Mechanism of the CO Sensor CooA. Journal of Biological Chemistry, 278(37), 35384–35393. doi:10.1074/jbc.m301000200.
Chan, C. T. Y., Lee, J. W., Cameron, D. E., Bashor, C. J., & Collins, J. J. (2015). " Deadman" and "Passcode" microbial kill switches for bacterial containment. Nature Chemical Biology, 12(2), 82–86. doi:10.1038/nchembio.1979.