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, we are unable to use the gaseous form NO directly in our treatment.
Contact Lens
Based on researches, the increase in patient's IOP will result in the deformation of cornea, further leading to a structural change in contact lens[2]. 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 (NOS), which can then convert L-arginine into nitric oxide to lower the intraocular pressure (IOP). This structural change is able to induce dynamic drug delivery.
IOP simulation experiment
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)[5], 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.
Nitric Oxide Synthases (NOS)
During literature research, we found out that Bacillus subtilis carries Nitric Oxide Synthases (NOS) and has the ability to produce NO, which is responsive to oxidative stress. So we cloned this gene from Bacillus subtilis' genome and designed a new biobrick which we then incorporated into our chassis, WM3064, allowing it to produce NO.
In order to dynamically express NOS as patient's IOP fluctuate, we put NOS under the control of T7 promoter and a lacO binding site, which can be controlled by IPTG-inducible T7 RNA polymerase provided by another plasmid PDT7 (Plasmid Drive T7 RNA polymerase). As previously mentioned, the IPTG concentration inside the ring-like compartment will fluctuate according to the patient’s IOP, leading to dynamic expression of NOS.
Functional Test
We tested the kinetics of the enzyme by using a NOS assay kit, which utilizes Griess reagents to react with NO and generate colorimetric readouts by measuring OD540 value. For the purpose of controlling the production of NOS, we induced bacteria with different concentrations of IPTG and cultured them for different period times.
Biosafety
DAP-deficient strain
We chose E. coli WM3064, which lacks the essential gene dapA, as our chassis. This gene encodes for 4-hydroxy-tetrahydrodipicolinate synthase that is critical to the production of lysine through the DAP pathway[6]. Lysine is an essential amino acid in animals, including humans, but can be synthesized de novo in bacteria, lower eukaryotes and plants for utilisation in protein and peptidoglycan cell wall synthesis[7]. Without this gene, the bacteria will have to depend on exogenous diaminopimelate (DAP) to survive.
Functional Test
To test whether the bacteria will survive without exogenous DAP, we made plates with and without DAP. After streaking our engineered bacteria onto these plates, we can demonstrate the result by checking its phenotype. Furthermore, we ran a SDS-PAGE to confirm the function of the T7 expression system in our engineered WM3064.
Overexpression csgD and csgA
Bacteria biofilm has been shown to exhibit extraordinary ability to help bacteria bind to biotic and abiotic surfaces[8]. We exploited this property to design one of the biosafety measures.
We engineered our bacteria to overexpress csgD, a master transcription regulator of biofilm formation, and csgA, the major subunit of curli fibers. Overexpression of these two genes have been reported to increase biofilm formation[9], which we anticipated to help the bacteria bind to the contact lenses more securely, thus preventing the leakage of bacteria if the contact lens encounters any damage.
Functional Test
We first characterized the biofilm formation by using conventional congo red staining, then we developed a simple method to assess the binding ability of our engineered bacteria. For more information, please visit our measurement page.
Growth switch
There are several things that need to be considered before selling Eye kNOw as a product. Since Eye kNOw won’t be used by the patient immediately after being manufactured, we designed a growth switch in order to control bacteria growth in different stages of our product lifetime. We were inspired by the work of iGEM NUS 2019 who used a toxin-antitoxin system, hicA-hicB, to control the growth of bacteria. By manipulating the toxin-antitoxin ratio in a bacteria, we can determine when the bacteria should hibernate or grow.
In our design, the hicB antitoxin is constitutively expressed at a basal level, while the hicA toxin is controlled by arabinose inducible promoter. The entire hicA cassette is flanked with FRT sites, which can later be deleted by the FLP recombinase. We also added a heat-activated FRT-FLP recombinase system from pCP20 as an inducible switch. This design enables us to control the bacteria growth in three stages - production, storage and medication.
The production stage is when we are culturing our bacteria, so we need the bacteria to be able to grow normally. After the production process, the bacteria needs to be stored in the contact lens until it can be used. During the storage stage, hicA will be induced to hibernate the bacteria. The last stage is the medication stage, during which the contact lens will be used. When the contact lens is placed on the patient’s eye, the body temperature will activate the recombinase system and delete the hicA cassette, which will cause the bacteria to resuscitate and start producing the therapeutic agent.
Functional Test
To verify the function of the FLP-FRT system, we will culture the bacteria with three plasmids, containing hicA, hicB, and CI857 and FLP genes respectively. As the temperature rises to 42oC, CI857 gene will be degraded, activating the FLP gene and deleting the hicA gene. If the hicA gene is present, the OD600 value will not increase since HicA protein represses the growth of bacteria. Hence, we can verify the function of the growth switch by measuring OD600 value.
Device
IOP Detector
Since there are no early symptoms of glaucoma, the public is left unaware of its presence. Therefore, early detection is needed, so we developed a brand-new IOP detector - Eye Screen. By transmitting ultrasonic waves to the patient’s cornea and analyzing the reflected signal, we can get IOP readings immediately. With Eye Screen, we can quickly find people at high risk of glaucoma, without direct contact with the eyes.
Functional Test
To validate the function of Eye Screen, we adopted a gravity model to control the IOP of porcine eyeballs using the trocar system via microincision vitrectomy surgery. By adjusting the height of the saline bag connected to the eyeball, we can measure the IOP by calculating the difference in height of the saline and the eyeball. We then tested whether the amplitude of reflected signals can be proportional to the IOP according to the change in IOP.
References
- Muenster S, Lieb WS, Fabry G, et al. The Ability of Nitric Oxide to Lower Intraocular Pressure Is Dependent on Guanylyl Cyclase. Investigative Opthalmology & Visual Science. 2017;58(11):4826.
- Lam A. The effect of an artificially elevated intraocular pressure on the central corneal curvature. Ophthalmic and Physiological Optics. 1997;17(1):18-24.
- Chen G-Z, Chan I-S, Leung LKK, Lam DCC. Soft wearable contact lens sensor for continuous intraocular pressure monitoring. Medical Engineering & Physics. 2014;36(9):1134-1139.
- Zhang J, Zhang Y, Li Y, et al. Correlation of IOP with Corneal Acoustic Impedance in Porcine Eye Model. BioMed Research International. 2017;2017:1-6.
- BRENDA - Information on EC 1.14.13.39 - nitric-oxide synthase (NADPH). Brenda-enzymes.org. https://www.brenda-enzymes.org/enzyme.php?ecno=1.14.13.39#pH%20OPTIMUM. Published 2020. Accessed September 9, 2020.
- Dante RA, Neto GC, Leite A, Yunes JA, Arruda P. Plant Molecular Biology. 1999;41(4):551-561.
- McLennan N, Masters M. GroE is vital for cell-wall synthesis. Nature. 1998;392(6672):139-139.
- DeBenedictis EP, Liu J, Keten S. Adhesion mechanisms of curli subunit CsgA to abiotic surfaces. Science Advances. 2016;2(11):e1600998.
- Brombacher E, Baratto A, Dorel C, Landini P. Gene Expression Regulation by the Curli Activator CsgD Protein: Modulation of Cellulose Biosynthesis and Control of Negative Determinants for Microbial Adhesion. Journal of Bacteriology. 2006;188(6):2027-2037.
