Part:BBa_K3702205
Modified EreA+EreB 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).
We wanted to target specifically the antibiotic erythromycin. EreA and EreB are erythromycin esterase type 1 and type 2. These enzymes grant resistance to erythromycin through inactivation by hydrolyzing the lactone ring of the antibiotic. They are both derived from E. coli. The EreB has been noted to work optimally in an acidic environment. Since EreA is a less well documented enzyme, it was difficult to find the optimal condition. We decided to use both EreA and EreB to degrade antibiotics since we don’t know which one may work better.
For more details on the Sewage Purification Limiting Antibiotic Spread in a Habitat (SPLASH) project click here.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30
Illegal NotI site found at 397 - 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 1475
Illegal XhoI site found at 1885
Illegal XhoI site found at 2475 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 1602
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