Team:NCKU Tainan/Design
Overview
In our mission to address glaucoma comprehensively, we decided to provide an even more effective treatment for the disease. Inspired by existing treatments using the nitric-oxide (NO) signaling pathway to target the trabecular meshwork and reduce intraocular pressure[1], we came up with a novel treatment based on gaseous nitric oxide. However, since NO has a short half-life of 400 seconds [reference please], we are unable to use the gaseous form NO directly in our treatment.
Contact lens
Literature research stated that the increase in patients’ IOP will result in the deformation of cornea, further leading to a structural change in contact lens[1]. We utilized this phenomenon to design a pair of contact lenses that structurally change in response to fluctuations in intraocular pressure.
Our contact lens will be fitted with a tubular semipermeable chamber that is filled with our engineered bacteria, IPTG, NO precursor - L-arginine, and DAP. The volume change of the chamber will cause water to flow out thus increasing the IPTG concentration inside the chamber. The increase in IPTG will induce the bacteria to produce more Nitric Oxide Synthase, which can then convert L-arginine into nitric oxide to lower the intraocular pressure (IOP). This structural change is then able to induce dynamic drug delivery.
IOP simulation experiment
To prove the concept of our contact lens and our device, we designed an IOP simulation experiment with porcine eye.By changing the drip bag’s height, water pressure will directly increase IOP in the porcine eye, enabling precise control of IOP for experiments[2][3].
For more details, please visit proof of concept and hardware.
Gene Design
In order for our bacteria to reduce intraocular pressure, we planned to engineer our bacteria to have the ability to produce Nitric Oxide Synthase (NOS) [3], an enzyme that can convert L-arginine into NO.
For biosafety, we engineered our bacteria to overexpress csgD and csgA for securing bacteria onto the contact lens by increasing binding affinity between bacteria and lens.
Gene Design
During literature research, we found out that Bacillus subtilis carries Nitric Oxide Synthases and has the ability to produce NO. So we cloned Bacillus subtilis' NOS gene under the control of an inducible promoter, pLacI, into a plasmid and transformed it into E. coli.
In order to induce our target gene, NOS, with IPTG, we put a LacI and T7 promoter in front of NOS and LacO after it. We chose WM3064 as our bacteria strand strain because it contains T7 RNA polymerase.
Functional Test
We tested the kinetics of the enzyme using a NOS assay kit, which utilizes Griess reagents to react with NO and generate colormertic readouts by measuring O.D.540 value. For the purpose of controlling the production of NOS, we induce bacteria with different concentrations of IPTG and induce them for different times.
Biosafety
DAP-deficient strain
We chose E. coli WM3064 as our chassis, which lacks the essential gene dapA. This gene encodes for 4-hydroxy-tetrahydrodipicolinate synthase that is critical to the production of lysine through the DAP pathway.[4] Lysine is an essential amino acid in animals, including humans, but can be synthesised de novo in bacteria, lower eukaryotes and plants for utilisation in protein and peptidoglycan cell wall synthesis.[5] Without this gene, the bacteria will have to depend on exogenous diaminopimelate (DAP) to survive.
However, in our design, the expression of nos is controlled by the T7-lacI promoter which requires T7 RNA polymerase, which WM3064 doesn't have. Therefore, we transformed a plasmid carrying T7 RNA polymerase into WM3064 to meet our purpose.
Functional Test
References
- Varma R, Lee PP, Goldberg I, Kotak S. An Assessment of the Health and Economic Burdens of Glaucoma. American Journal of Ophthalmology. 2011;152(4):515-522.
- Rein DB. The Economic Burden of Major Adult Visual Disorders in the United States. Archives of Ophthalmology. 2006;124(12):1754.
- Weinreb RN, Aung T, Medeiros FA. The Pathophysiology and Treatment of Glaucoma. JAMA. 2014;311(18):1901.
- Hurt J. Your Senses Are Your Raw Information Learning Portals. 2012; Retrieved from https://velvetchainsaw.com/2012/05/23/your-senses-your-raw-information-learning-portals/
- Ha A, Kim YK, Park YJ, Jeoung JW, Park KH. Intraocular pressure change during reading or writing on smartphone. Bhattacharya S, ed. PLOS ONE. 2018;13(10):e0206061.
