Team:NCKU Tainan/Future
Overview
This year, we have tried our best to turn our project from an idealistic concept to implementation. However, we believe that there is still a long way to go to improve our project. We have listed several strategies for further project improvement. We expect our project can make a significant impact on the world in the future.
Eye kNOW
Nitric Oxide Production
We cloned bsNOS into E. coli in order to makeitproduce nitric oxide. We chose bsNOS as a target gene because Bacillus subtilis is the most closely-related species that can produce a substantial amount of Nitric Oxide. Eventually, we managed to engineer E. coli toproduce Nitric Oxide. In the future, we would like to try cloning nitric oxide synthase from GH3 pituitary cells[1]. The NOS from GH3 has a lower Km value with higher efficiency. It may increase the nitric oxide production efficiency of our engineered E. coli.
Growth Switch
In the current design, we used CI857 as a heat-sensing protein as a switch that regulates the promoter pR. Therefore, we have to culture our engineered bacteria under 30°C to keep the switch off. This will lead to low culture efficiency. According to research, using mutant pR can increase the culture temperature up to 36°C[2].
A mutation in the OR2 operator region of the rightward pR promoter increases the temperature stability of the pR/cI857 gene expression system. Thus gene expression is stringently repressed at temperatures up to 36°C. By doing so, we can get better production efficiency.
Applications for Ocular Diseases
Eye kNOw boasts a dynamic drug release system, one that can be applied in other situations. If we replace NOS with other enzymes, we can also develop additional dynamic treatments for intraocular pressure diseases.
Not only that, but we can also add a biosensing system into our contact lenses, allowing for the detection of pathogens or inflammation biomarkers in the eye. This way, our dynamic drug release system can be used for more applications so that more people can benefit from Eye kNOw.
Reduce the Number of Plasmidsh
We have introduced several plasmids into our engineered bacteria so that it can produce not only NO but also increase its binding affinity to the contact lens. We have also established a growth switch and kill switch so our bacteria can safely function in the contact lenses. The whole system looks perfect; however, we have designed too many plasmids to introduce into the bacteria, which may increase the burden of bacteria and cause harmful effects on them. Therefore, we hope that some genes can be integrated into the chromosome, while some plasmids can be merged to reduce the number of plasmids in the future.
We plan to integrate csgD, csgA, and NOS into the chromosome and merge the three plasmids of the growth switch into one, that is, clone both hicA and hicB into pCP20. This way, our total number of plasmids will be significantly reduced to one, making our engineered bacteria expression more efficient.
Eye Screen
We hope to promote the device to the general public in the future. With Eye Screen, users can monitor their IOP level by themselves, quickly and easily, wherever they are. These IOP values around-the-day will automatically be synchronized to the smartphone app, Eye Cloud, where they are stored and analyzed, enabling users to be informed of their risks of glaucoma in a real-time manner. It can also be placed in medical centers to increase the public's willingness to track their IOP level. Healthcare practitioners can also bring Eye Screen to long-term care institutions for IOP monitoring. We designed a compact function generator to drive the transducers, omitting the need for a conventional setup. We will also improve Arduino signal processing settings to optimize the precision of IOP readings.
Besides, collaboration with various institutions related to glaucoma will increase the willingness to measure IOP for anyone and effectively raise awareness of glaucoma. Furthermore, artificial intelligence (AI) in healthcare and medicine is booming. Keeping up with this trend is our next step. Therefore, Eye Screen will also be an additional tool to collect data as the samples for machine learning. In the future, Eye Screen 2.0 will be launched with the ability to carry out big data analytics and offer better precision. This portable signal processing system could be applied in various fields, bringing benefits to society.
References
- BRENDA - Reference to 1.14.13.39; Id = 440214. Brenda-enzymes.org. Published 2017. https://www.brenda-enzymes.org/literature.php?e=1.14.13.39&r=440214
- Jechlinger W, Szostak MP, Witte A, Lubitz W. Altered temperature induction sensitivity of the lambdapR/cI857 system for controlled geneEexpression inEscherichia coli. FEMS Microbiology Letters. 1999;173(2):347-352.
