Part:BBa_K3702206
Formaldehyde inducible ParD2 generator
Antibiotic resistance is a global problem where bacteria or fungi develop defense mechanisms against antibiotics that were designed to kill them. One of the ways this resistance develops in the environment is through the pollution of antibiotics from wastewater treatment plants (Kraemer, 2019). Since wastewater treatment plants are not specifically designed to degrade antibiotics (Kulkarni, 2017), some of the antibiotics remain even after the treatment of the water and then the water is released and often reused. This creates a problem because it puts selective pressure on bacterias in the surrounding area of the water to develop antibiotic resistance to fight against the antibiotic being released. The amount of antibiotics being released are not enough to kill every single bacteria it comes in contact with but is enough to make them stronger. Initially we had many ideas like using a filter with micropores to remove antibiotics from the effluent water, engineering bacterias to degrade antibiotics, engineering algae to absorb antibiotics and antibiotic resistant genes, or even using high sound frequency ultrasound to remove antibiotics. After doing more research about how wastewater treatment plants work, we found that hardware solutions such as UV lights or adding a chlorination chamber entails considerable funding and time, our team has decided to pursue solutions that would be easy to implement.
After discussing with the team, we narrowed down our ideas to either design algae to absorb antibiotics or design a bacteria to degrade antibiotics. After consulting with Mike Kelley, a wastewater treatment plant owner and Dr. Tang, a wastewater engineer and a professor at the FAMU-FSU college of engineering about our ideas, we found that the algae have many issues like it would need sunlight to stay alive and so would likely only remain on the water surface, this could be resolve by just providing artificial light but that could be very expensive for the treatment plant since wastewater treatment plant chambers can be massive in size, the algae also does not filter antibiotic well in fast-moving water, making it unsuitable for most wastewater treatment plants, it works better with slower-moving water or a pond. The bacteria on the other hand would be much easier to implement and cheaper to use. So we decided to design a bacteria to degrade antibiotics. Since our team did not have access to a lab, we couldn’t build and test our project, but we came up with protocols for experiments we would have done to test it.
For our chassis, we decided to go with E. coli (Escherichia coli) because it is a widely researched organism, its ability to grow fast and is cheap to grow(sciencelearn.org). E. coli can also survive in many stages of wastewater treatment plants (Anastasi, 2012) making it suitable to be the chassis of choice in our project. The pH of wastewater treatment plants is between 6 to 8 (Trygar, 2018) and the temperature range about 20 degrees celsius (Heger, 2017). The optimal pH for E. coli is between 6.5 to 7.5 (Büchs, 1970) and optimal temperature is 37 degrees celsius(sciencelearn.org).
Since the enzyme we engineered our E.coli bacteria to produce can degrade antibiotics, that means that our bacteria would be an antibiotic resistant bacteria and could be dangerous if it gets out into the environment. To counteract this, we decide to implement some kind of kill switch that would activate if the bacteria gets out of the wastewater treatment plant alive. The kill switch we decided to go with is a toxin/antitoxin module, meaning that it is made up of two genes, where one produces the toxin and the other one produces the antitoxin. We decided to model our system after team UCAS 2016 where they designed a TA module with the toxin ParE2 and antitoxin ParD2 that is dependent on the amount of tetracycline in the wastewater treatment plant, in low concentration of tetracycline, the gene will stop producing the ParD2 which leaves ParE2 by itself to kill the host bacteria. We took this kill switch design and made changes that we thought would be better for our problem. First we built a design with a stronger RBS and a different terminator. The second change was switching out the promoter. Their design used a tetracycline inducible promoter Ptet. While this promoter work for them, it might not work as well for us since our project was based on the U.S wastewater treatment plants while their project was based in China, it was also risky using this promoter since we can not confirm how much the amount of tetracycline fluctuate and that it would be the same throughout different plants in the U.S. So we decided to go with something that is more likely to work, a methane inducible system. This system was more likely to work since methane is already abundant in wastewater treatment plants since they have to treat human feces, whereas a tetracycline inducible system was dependent on something that was more out of control.
For more details on the Sewage Purification Limiting Antibiotic Spread in a Habitat (SPLASH) project click here.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 405
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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