Picture I: Seriousness of Carbon Monoxide Hazard Throughout Industry
Carbon monoxide is universally present in industry. Partial oxidation of compounds which contain carbon results in the production of carbon monoxide. With the lack of enough oxygen it is formed instead of carbon dioxide. Carbon monoxide is a colorless and odorless gas and therefore does not give any warning of its presence to those exposed. Physiologically, carbon monoxide acts by displacing oxygen from the haemoglobin molecule. When encountered it is toxic for humans in concentrations as low as 35 parts per million (ppm). This is because haemoglobin has an affinity for the poisonous gas that is 210 times higher compared to oxygen. High blood level concentrations of the compound prevents sufficient amounts of oxygen from reaching vital organs. In America alone several thousand workers die each year from carbon monoxide exposure, making it one of the most dangerous industrial hazards.
Picture II: Carbon Monoxide is Bound by Our CooA Protein
The carbon monoxide detection system that we have been developing for the iGEM competition contains modified bacteria in a specific medium. It is this medium in which the carbon monoxide from the polluted area will dissolve. The bacteria in the system synthesize the receptor protein CooA, a member of the cAMP receptor protein family, which functions in a similar way as haemoglobin. Besides its capacity to bind carbon monoxide, the CooA protein also functions as a transcription factor. Therefore, CooA will not only detect the carbon monoxide from the polluted area, but also regulate the rate of an output signal.
Picture III: Bound Carbon Monoxide Initiates the Production of Carbon Monoxide
Binding of carbon monoxide to the CooA protein initiates the transcription of the protein pyruvate decarboxylase. As a result, the synthesized pyruvate decarboxylase will convert the pyruvate that is present in the medium into CO2 gas. The medium is flanked by two electrodes that produce a constant electrical current through the medium. When the CO2 gas production reaches a certain threshold, gas will accumulate and replace the medium at the location between the two electrodes. This results in a change in the resistance between the electrodes. Detection of the changing resistance by the hardware will set off an alarm.
Picture IV: Too Much Production of CO2 Leads to an Alarm: No more false alarms
Present-day carbon monoxide detectors experience complications in differentiating between carbon monoxide and hydrogen gas. Because hydrogen gas is a by-product in many industries, this interference problem can result in many false alarms. The developed device will selectively detect carbon monoxide with the use of the CooA protein and therefore has a great advantage over currently used detectors in industry.
Selective Carbon Monoxide Detection
This project is focussed on manufacturing a carbon monoxide detection system. Despite the fact that there are already detection systems for carbon monoxide on the market. There is nothing wrong with the sensitivity of the present-day detection systems. However, carbon monoxide detectors that are used in various industries have difficulty in differentiating between carbon monoxide and hydrogen gas. Because hydrogen gas is a by-product in many industries, this interference problem can result in many false alarms.
The CooA Protein
In order to create a device that can selectively detect carbon monoxide in a biological pathway, it is crucial to have a protein that can sense carbon monoxide molecules. The receptor protein CooA, a member of the cAMP receptor protein (CRP) family, is such a protein. In nature this heme-containing CooA protein is synthesised by the bacteria Rhodospirillum rubrum and functions in a similar way as haemoglobin. Besides its capacity to bind to CO, the CooA protein also functions as a transcription factor. When a CooA protein binds to a carbon monoxide molecule it alters its own structure. Due to this change in structure the CooA receptor can bind to a specific DNA sequence: a CooA-dependent promotor. In the R. rubrum bacteria the location of the CooA-binding site on the DNA forms the foundation for the transcription of the cooFSCTJ and cooMKLXUH operons, which code for the synthesis of proteins that oxidize CO to CO2. Thus, in R. rubrum bacteria CooA regulates the rate of the transcription of the genetic information that encodes for proteins that allow growth of this organism on CO as a sole energy source.
Gas Detection and Gas Production
For a CO detection system the binding of CO to the CooA protein has to initiate the transcription of proteins that can deliver an output signal, instead of the original mechanism where proteins are synthesised that can oxidize CO to CO2. As a replacement for the original operons, a gene was ligated into the DNA sequence that enables the synthesis of the enzyme pyruvate decarboxylase (pdc). Accordingly, pdc can convert the pyruvate that is present in the medium into CO2 and acetaldehyde. The released CO2 gas in the medium will be collected in a compartment where a constant electrical current goes through. As a result, the medium in the compartment will be replaced by the produced CO2 gas. When a certain threshold is reached in the amount of produced gas a change in the resistance will occur, which can be detected by the hardware. Minimal concentrations of carbon monoxide in unpolluted air already result in the production of CO2 gas that can add up and eventually reach the threshold. Therefore it was also necessary to design a constant draining system.
Other output signal molecules were taken into consideration as well. For instance, producing an electrical signal by bacteria that release electrons which subsequently can be detected by the hardware. However, oxygen molecules in the medium can capture these released electrons. Designing an anaerobic environment to counter this problem resulted in additional complications.