Team:NCKU Tainan/Poster

Poster: NCKU_Tainan

Eye kNOw: Envisioning the future of glaucoma treatment
Presented by Team NCKU Tainan 2020

Yi Ling, Chou1, Sheng Yan, Sun1, Yi Lun, Huang1, Li An, Wang1, Cheng Yi, Ku1, Ren Hao, Tsai1, Yu Ling, Hsu1, Hong Yan, Huang1, Virginia Clarence Setiawan1, Yao Tien, Cheng1, Felicia Yi1, Tzu Tan, Weng1, Chin En, Yen1, Ya Chu, Yu1, Chen Yuang, Lee1, Jia Yi, Lin2, Fu Jie, Tham2, Sabrina Yeo2, Yi Jun, Lan2, Ji Yu, Heng2, Hsin Chieh, Yu2, Chia Hua, Tsai2, Dr. I-Son Ng3, Dr. Masayuki Hashimoto3, Dr. Jia-Horung Hung3, Dr. Han-Ching Wang3

1iGEM Student Team Member, 2iGEM Team Advisor, 3iGEM Team PI

Glaucoma is characterized by progressive loss of visual field without any early symptoms. A leading cause of irreversible blindness, glaucoma affects millions of people worldwide and causes heavy burden on healthcare systems. The only proven effective treatment is reducing the patients’ intraocular pressure (IOP), which is usually done by providing eye drops with IOP-lowering effect, like nitric oxide. However, due to ineffective IOP management and warning strategies, blindness is still a problem. Therefore, iGEM NCKU-Tainan developed a revolutionary solution, Eye kNOw, a pair of contact lenses that contains engineered bacteria producing nitric oxide in response to IOP fluctuation. We also developed Eye Screen, an affordable and portable detection device that utilizes ultrasound to measure IOP and identify the high-risk groups. With Eye kNOw and Eye Screen, IOP can be controlled in a real-time manner, and glaucoma can be detected and monitored with a low-cost device, saving more people from life-long darkness.
Silent Killer of Vision: Glaucoma
This year, we're targeting glaucoma, a kind of ocular disease that will lead to irreversible visual loss. Statistics show that over 80 million people are affected by glaucoma, which causes a heavy burden on the healthcare system worldwide1.

Mechanism of Glaucoma
Glaucoma2 is caused by elevated intraocular pressure (IOP), which is caused by buildup of aqueous humor inside our eyes. In normal people's eyes, the influx is the same as the efflux of aqueous humor, which maintains the IOP in the range of 10-20 mmHg. However, in glaucoma patients' eyes, the influx is larger than the efflux of aqueous humor, which leads to higher IOP and damages the optic nerve located inside the eyes. This situation is usually caused by the over-contraction of trabecular meshwork, a kind of smooth muscle that is responsible for controlling the efflux rate of aqueous humor.

Fig. 1. Eye structure.

Why Is Glaucoma So Dangerous: Lack of Detection and Ineffective Treatment
Since glaucoma is irreversible, early detection with effective treatment is an important issue. However, there's no early symptoms and signs for glaucoma, making the public unaware of its progression. What's more, current treatment utilizes eye drops, which is ineffective since IOP fluctuates in an uncertain pattern. These make glaucoma a difficult disease to cope with using current strategies.

Eye kNOw: Envision the Future Treatment of Glaucoma
To combat glaucoma, we came up with Eye kNOw and Eye Screen —— Eye kNOw, a pair of contact lenses that release drugs in accordance to IOP change, for more effective treatment; Eye Screen, a portable and affordable IOP detection device, for early detection of glaucoma. With Eye kNOw and Eye Screen, we provide a comprehensive solution to glaucoma, and glaucoma will never threaten our visions anymore.

Fig. 2. Comprehensive solution to glaucoma.

Design of contact lens
Based on research, the increase in patient's IOP will result in the deformation of cornea, further leading to a structural change in contact lens. We utilize this feature to design a special contact lens which contains a chamber with engineered bacteria and IPTG inside. Deformation of the contact lens will lead to volume change of the chamber and push out the water, thus increasing the IPTG concentration inside the chamber. The increase of IPTG concentration will induce the bacteria to produce more Nitric Oxide Synthase (NOS), converting more L-arginine into nitric oxide, and finally lower the intraocular pressure (IOP). This design enables our contact lens to adjust the dosage of NO according to IOP.

Fig. 1. The deformation of contact lens.

The mold of our contact lens is made by 3D printing using polypropene (PP). We use Hydroxyethylmethacrylate (HEMA), a common material for manufacturing commercial contact lenses, as the main material of our contact lens. A semipermeable material is used to form the chamber, which contains engineered bacteria, IPTG, L-arginine, and DAP inside. The main body of our contact lens will then be combined and sealed with the chamber, thus becoming our final product ─ Eye kNOw.

Fig. 2. The design of contact 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.

Fig. 3. Plasmid design of NOS.

Growth switch
For our project, we need the bacteria to stay alive in our contact lenses until it can be used. However, bacteria can’t live for a long time in such a small space, especially with such limited nutrients. Hence, we designed a growth switch to put bacteria into hibernation for storage, which we can then resuscitate after exposure to external stimulation.

Fig. 1. The mechanism of growth switch.

There are three mechanisms for growth switch: Thermo sensitive-system (pCP20), Toxin-Antitoxin system (hicA-hicB), and recombinase system (FRT). The promoter on pCP20 will be activated when the temperature is higher than 36ºC. For the TA system, HicA is a toxin protein that hibernates the bacteria, and HicB is an antitoxin that can neutralize the effect of HicA. In addition, FRT can delete genes when FLP is expressed.

Fig. 2. The functions of growth switch in three steps.

During the storage, we will add arabinose in a contact lens to activate pBAD to transcribe hicA. Since pSB3K3 is a low copy number plasmid, the expression of hicA gene is stronger than hicB gene, that is why the bacteria hibernate. After patients wear the contact lens, the environment temperature rises to 36-38 ºC, the promoter on pCP20 will be activated and transcribe the FLP gene, which can activate FRT sites to delete hicA gene. Hence, our bacteria can resuscitate to function. With this design, we can prolong the survival time of our bacteria in contact lens, avoiding the possibility that bacteria may die during storage.
We chose E. coli WM3064 as our chassis, which lacks the essential gene dapA for cell growth and cell wall synthesis. Without this gene, the bacteria will have to depend on exogenous diaminopimelate (DAP) to survive. Hence, if the bacteria escape from the contact lens, they cannot survive.34

Fig. 1. dapA gene knockout.

Bacteria biofilm has been shown to exhibit extraordinary ability to help bacteria bind to biotic and abiotic surfaces5. 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 formation6, 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.

Fig. 2. Overexpress csgD and csgA.

Results and Conclusions
Nitric Oxide Synthases (NOS)
This year, our team aims to reduce intraocular pressure (IOP) through the contact lens with engineered E. coli that can produce Nitric Oxide (NO). We ordered a DNA sequence containing a lacO-T7 promoter, B0034 RBS, and bsNOS, ligating them altogether and inserted them into pSB1C3 plasmid and combined it with another sequence (pLac, RBS, csgA, RBS, csgD, LacI) from IDT, then transformed into E. coli DH5-Alpha. By doing so, NOS can convert L-arginine into nitric oxide which will be released into the eyes7.

Fig. 1. SDS-PAGE of E. coli BL21(DE3) with different concentrations of IPTG. The bacteria is cultured for two hours and induced with IPTG for 12 hours. M: Marker; Lane 1: 0.1 mM; Lane 2: 0.05 mM; Lane 3: 0.025 mM; Lane 4: 1 mM. The arrow from top to bottom indicates NOS (~40kDa), CsgD (~24kDa), and CsgA (~17kDa).

We conducted an experiment by inducing with different concentrations of IPTG in proof of concept part. Besides, we did the experiment for NOS kinetic. Thereby, we can conclude that the IPTG inducible system can dynamically produce nitric oxide.

Fig. 2. NOS activity at different times to observe the time required for the substrate to be depleted.

  1. Kill switch
    In order to prove that our DAP-deficient strain WM3064 won’t survive without DAP8, we spread our E. coli WM3064 containing a plasmid (kmR) carrying T7 RNA polymerase to different plates. As the fig. 3. shown, the bacteria can survive on the plate with exogenous DAP. When there is no DAP supply, the bacteria cannot survive.

    Fig. 3. Conformation of DAP-deficient strain WM3064. WM3064 was streaked on two different agar plates containing (A) Kanamycin (Km) and 0.3 mM DAP; (B) Kanamycin (Km) only.

  2. Biofilm formation
    We want to measure the binding affinity of our engineered bacteria9. We simply drop the book from different heights and measure how well the bacteria can withstand different forces. According to the formula in fig. 4.(A) below, the force a free-falling object can exert is proportional to its square root of the height, by elevating the object to different heights, we were able to modify the force exerted on our engineered bacteria precisely.

    We compared the binding ability of three bacteria previously characterized by congo red staining (BW25113, csgD, NOS-csgDA). As seen in the fig. 4.(B), co-expressing csgD and csgA significantly increases the OD600 value when potential energy is applied compared to BW25113 and csgD alone, which congo red staining protocol only show minor difference between csgD and csgDA.

    Fig. 4. (A)The formula and graph of the experiment. (The weight of book is 1.6 kilogram, the collision is considered as completely inelastic collision, and the collision time is presumed as 0.1 second.); (B) The binding affinity of control, csgD, and csgA-csgD with given force from different heights.

Proof of concept
Step 1
We designed a simulation experiment to verify the deformation with respect to IOP increment, which is achieved by adjusting the drip bag filled with normal saline. The software called Vic-3D is a genre of digital image correlation software, which allows us to observe deformation of the cornea when IOP differs. (See Fig. 1.)

The result shows that there's a linear relationship between IOP and ratio of corneal deformation.

Fig. 1. The device of IOP simulation experiment.

Step 2
To prove that different concentrations of IPTG can induce our bacteria to produce different amounts of NOS, we test the NOS amount with NOS assay kit.

For preparation, we cultured the bacteria containing a plasmid with an IPTG inducible promoter and NOS gene. After culturing overnight, we induced bacteria with different concentrations of IPTG for different periods of time. We then adjusted the OD600 to one and disrupt the cell.

The substrate, mainly L-arginine and NADPH, was added to the disrupted cell. After twenty minutes of incubation, the L-arginine was transformed into NO. The buffer of NOS assay kit then transformed NO into Nitrite for Griess reagent to interact with.

We measure the OD540 and convert it into NO concentration with a standard curve.

Fig. 2. NOS induced by IPTG with different concentration at different induced time.

Step 3
We set up the eyeball of porcine as well, with a commercially available contact lens on the cornea. The solution which contained the supernatant and the L-arginine was dripped on the contact lens and covered with a plastic film, which was used to prevent the oxygen from the atmosphere getting involved in the reaction.

The result shown below (see Fig. 3.) suggested that a great amount of nitric oxide could easily penetrate to the aqueous humor within a given amount of time.

Fig. 3. The diffusion of nitric oxide from contact lens to aqueous humor.

Model is the bridge connecting experimental data and scientific theories. We use three models to simulate the working principles of Eye kNOw. The workflow of Eye kNOw contains steps shown in fig. 1.

The three main parts that need models to simulate are contact lens deformation, NO diffusion model in the ocular system, and quantitative estimation of Eye kNOw effectiveness. We’ll establish three models to simulate these processes.

Fig. 1. Workflow of Eye kNOw.

Model 1: Contact Lens Deformation
  • Design of Contact Lens
    The main body of Eye kNOw is a contact lens with a designed chamber. Fig. 2. shows the design of Eye kNOw. The contact lens will deform when IOP increases, and we use integration to calculate the deformation ratio of the chamber inside.

    Fig. 2. Design of contact lens.

  • Principle
    • 1 mmHg increment of IOP will lead to 3 micron increment of corneal radius of curvature.
    • The contact lens will perfectly fit the shape of the cornea, which means that our contact lens will also deform when IOP increases.

  • Results
    The chamber will have 1% volume decrease when IOP increases 1 mmHg.
Model 2: NO Diffusion Model
  • Overview
    • The four major factors that will affect the concentration of NO: Degradation, Convection, Diffusion, and Production. We use partial differential equations to establish the dynamic concentration profile of NO.

  • Results
    • Assumptions
      1. The four major factors that will affect the concentration of NO: Degradation, Convection, Diffusion, and Production.

      2. Factor r in the formula represents the characteristic of interfaces in the ocular system, namely Eye kNOw-cornea and cornea-aqueous humor interfaces.

    • Parameters
      Parameters are found in current research and our experimental data.

      Table. 1. Parameters used in model 2.

    • Dynamic NO Concentration Profile
      By Laplace transform method, we found out the solutions of our PDEs, which describe the dynamic NO concentration profile. Non-steady state means that the production rate of NO by Eye kNOw is still changing by time.

      Fig. 3. Dynamic NO concentration profile with Eye kNOw worn.

  • Model 3: Effectiveness of Eye kNOw
    • Principle
      • Model 1 provides quantitative IPTG concentration change while IOP is raised.
      • Model 2 provides dynamic NO concentration profile while IPTG concentration is changed.
      • Experimental data provides statistics for calculating effectiveness of Eye kNOw.
    • Results
      • Effectiveness of Eye kNOw

        Fig. 4. NO concentration profile at trabecular meshwork from 0s to 1000s.

        Fig. 5. NO concentration profile at trabecular meshwork from 0s to 50s.

        Results show that it takes less than a minute for Eye kNOw to reach minimum effective concentration, which indicates that Eye kNOw can provide treatment in real-time manner.
Eye Screen

Fig. 1. Device overview.

A major risk factor of glaucoma is the elevation of intraocular pressure (IOP). Therefore, measuring IOP is the most important method to predict glaucoma development. However, current IOP detectors are only available in clinics or hospitals and are inefficient due to untimely IOP monitoring, and also cause significant inconvenience and inaccessibility to many patients. To make IOP detection more accessible, iGEM NCKU Tainan 2020 came up with Eye Screen.

Eye Screen is an affordable, portable, and user-friendly device providing IOP measurement via ultrasound10. It transmits ultrasonic waves onto the eye and analyzes the reflected signal. The result is displayed on the LCD and uploaded onto Eye Cloud, an application that can record the user’s IOP value in real-time.

According to research, there is a proportional correlation between IOP and amplitude of reflected ultrasound signals from the cornea. Based on this theorem, we transmit ultrasonic waves onto the cornea and analyze the reflected signal. After establishing the relationship between IOP and amplitude of the reflected signal, we are able to get IOP readings immediately.

Fig. 2. Principle of Eye Screen.

Experiment and Result
To validate the function of Eye Screen, we adopted a gravity model to control the IOP of porcine eyeballs using the trocar system. By adjusting the height of the saline bottle connected to the eyeball, we can measure the IOP by calculating the difference in height of the saline and the eyeball. And we successfully verified that there is a positive correlation between IOP and average amplitude.

Fig. 3. Experimental setup for the gravity model. (A) Real experimental setup; (B) Schematic diagram for experimental setup

Fig. 4. Mean amplitude of reflected signals from different porcine eyes. (N=4)

Human Practice
  • Education
    • Synthetic Biology in 5 Levels
      To raise synthetic biology awareness, we made Synthetic Biology in 5 Levels, a video where we explained synthetic biology to people with 5 different levels of understanding, students from middle school, high school, college, grad school and an expert. This way, everyone can understand synthetic biology despite their background knowledge.

      Fig. 1. Synthetic biology in 5 levels.

    • I’ve Gotta PhD
      In collaboration with CSMU Taiwan, we launched I’ve Gotta PhD or I’ve Gotta public health eDucation, a social media platform sharing topics related to public health, to achieve SDGs 3 and 4, which are Good health and well-being and Quality Education. We collected submissions in the form of infographics, articles, video and so on. Through this platform, we were able to work with more teams to ensure that we were providing correct information about public health for the public. We collaborated with more than 10 teams in total. Moreover, we also took a chance to share about glaucoma on this platform.

      Fig. 2. Partnership with CSMU Taiwan for I’ve Gotta PhD.

    • Into the Darkness
      To raise the awareness of blindness, we created a LARP (Live action role-playing) event called Into the Darkness, with an intriguing story line and sensory engaging experience, players could experience the life of the visually impaired. More than that, they could also learn about synthetic biology in a new and fresh approach.

      Fig. 3. Into the darkness LARP.

Future directions
  • Application 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, we can also add a biosensing system into our contact lenses, allowing for the detection of pathogens or inflammatory biomarkers in the eye. This way, our dynamic drug release system can be used for more applications. Thus, more people can experience the benefit of Eye kNOw.
  • Eyesaac Biotechnologies
    Eye kNOw project will be developed into a start-up company, named Eyesaac Biotechnologies. Eyesaac Biotechnologies will aim to expand the portfolio of medical products which employs our core biosensing technology to other forms of disease or medical conditions. Eyesaac Biotechnologies aims to expand the area served to cover most of the countries around the world, starting with China and Japan, which represent the largest glaucoma prevalence rates in Asia. Further expansion will be focused on the EMEA and the Americas.

    The short-term goal for Project Eye kNOw is to successfully complete clinical trials and have the patents granted as soon as possible.

    The long-term goals for Eyesaac Biotechnologies are to expand Eye kNOw into larger markets as well as adapting the technologies and frameworks established into treating other types of diseases such as diabetes and sepsis.
Funding Attributions and Acknowledgements
Dr. Huey Jen Su,
Dr. Fong Chin Su,
Dr. Chung I Lin,
Dr. Cheng Wen Wu,
Dr. Ming Derg Lai,
The ministry of education,
Diamond BioFund Incorporated,
GSL Biotech LLC,
National Cheng Kung University,

  1. 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.
  2. Weinreb RN, Aung T, Medeiros FA. The Pathophysiology and Treatment of Glaucoma. JAMA. 2014;311(18):1901.
  3. Dante RA, Neto GC, Leite A, Yunes JA, Arruda P. Plant Molecular Biology. 1999;41(4):551-561.
  4. McLennan N, Masters M. GroE is vital for cell-wall synthesis. Nature. 1998;392(6672):139-139.
  5. DeBenedictis EP, Liu J, Keten S. Adhesion mechanisms of curli subunit CsgA to abiotic surfaces. Science Advances. 2016;2(11):e1600998.
  6. 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.
  7. BRENDA - Information on EC - nitric-oxide synthase (NADPH). Published 2020. Accessed September 9, 2020.
  8. McLennan N, Masters M. GroE is vital for cell-wall synthesis. Nature. 1998;392(6672):139-139.
  9. DeBenedictis EP, Liu J, Keten S. Adhesion mechanisms of curli subunit CsgA to abiotic surfaces. Science Advances. 2016;2(11):e1600998.
  10. He, X., & Liu, J. (2011). Correlation of corneal acoustic and elastic properties in a canine eye model. Investigative ophthalmology & visual science. 52(2), 731-736.