Team:TAS Taipei/Modeling

• PROJECT

  Background

  Achievements

  Collaborations

  Parts

  

  Cataracts - the leading cause of blindness. Find out how we can both treat and prevent cataract formation.

• EXPERIMENTAL

  Construct

  Proof of Construct

  Prototype

  Notebook

  

  We conduct science - and we are proud to show it. Follow along with our journey of discovery in the lab.

• MODELING

  Crystallin

  GSR/25HC

  Nanoparticle

  Eyedrop

  

  Computational Biology provides us models that we cannot easily test. Click to see the power of math.

• HUMAN PRACTICE

  Research

  Change

  Outreach

  Entertainment

  

  We don't just grow cool bacteria. We make a difference. Find out how we tackled social aspects of this project.

• BIOSAFETY

  Researcher Safety

  Environmental Safety

  Biobrick Safety

  Local Safety

  

  Safety first. Especially in a lab. Here's how we maintained and investigated the integrity and security of our work environment.

• TEAM

  Members

  About

  Attributions

  

  Behind every tough iGem project lies a tough, hard-working yet cheerful group of students. Meet the team!

COUNTERACTS

• Overview
• Crystallin
• GSR/25HC
• Nanoparticles
• Eyedrop

Modeling

Overall Modeling Abstract

Our goal is simple: produce GSR/25HC, package it into nanoparticles, and transport into the lens. GSR/25HC is released over time, which decreases H2O2 concentration, reduces crystallin damage, and prevents cataracts. Our models approach these steps in reverse order, starting with our desired goal, and working backwards to understand the entire process.

In model 1, we quantified crystallin damage, and measured its true impact on light absorbance, to relate it to cataracts. Then in model 2 we modelled how varying amounts of GSR added impacted the amount of crystallin damage, until we found a desirable GSR level for cataract prevention. We examined the degradation of nanoparticles in model 3, and how repeated doses in the long-term contributed to sustain the desirable GSR level. Model 4 accounts for the actual transport of nanoparticles into the highly protected eye, mainly by flooding the eyedrops with GSR so sufficient amounts entered the eyes. Finally, Model 5 is a simple stochastic model for understanding gene expression in a simple circuit.

Outline

Introduction

Why Model?

In the lab, biologists are often unable to test everything experimentally. For example, in our cataracts project, cataract prevention occurs in the long-term, from 20-50 years. Obviously, although short experiments can provide us an idea of what prevention may look like, the power of computational biology allows us to model into the future. As a result, our modeling has been crucial in developing a prototype.

Focus

Most iGEM teams perform modeling on gene expression, which we accomplish in model 5. However, as our construct is not directly placed into the eyes, how our synthesized protein impacts the eyes after it is seperately transported is much more interesting. As a result, we spent the majority of our models on understanding the impacts on the eye.

Guiding Questions

1. How much GSR do we want inside the lens?
2. How do we use nanoparticles to control the amount of GSR in the lens?
3. How do we synthesize GSR, package into NP, and send it into the eye?

Model 1: Crystallin Damage

Abstract

An absorbance measurement is meaningless without knowing how clinically severe it is. Cataract severity is graded on the LOCS scale from 0-6, and our goal is to lower LOCS to 2.0, below the threshold of surgery. We relate measurements made on different scales, from LOCS to opacity, light transmittance, light transmittance, wavelength absorbance, and finally to crystallin damage, a quantity that is useful for our future models.

Purpose

What amount of crystallin damage is acceptable, to not require surgery? This will let us know how much GSR we need to maintain an acceptable amount of crystallin damage that will not require surgery (based on LOCS scale)

Background, Method, Results, Discussion

• Background
• Method
• Results Part 1
• Results Part 2
• Discussion

Background

There are three quantifiers of how severe cataract formation is, two are measurable, one is not.

1. Absorbance @ 397.5 nm, which is measured with lab equipment.
2. LOCS scale, subjectively measured by physicians on a scale from 0 - 6.
3. Crystallin Damage, which we define as the following (for any time $t$)

\[c.d.(t) = \int_{0}^{\infty} [H_2O_2]_t dt\]

In other words, 1 unit of crystallin damage, in M-h, is equal to the damage caused by 1 molar concentration of hydrogen peroxide reacting crystallin in the eyes for 1 hour.

In making this definition, we assume that crystallin damage is directly proportional to the amount of time crystallin is exposed to hydrogen peroxide. Hydrogen peroxide causes damage by forming disulfide bridges within cysteine molecules on crystallin. This changes the structure of crystallin, causing misfolding and cataract damage. Our linear assumption is valid because the rate for this reaction is first order with respect to hydrogen peroxide concentration.

Method

We will relate the three by doing the following:

1. Relate LOCS scale to opacity via literature research.
2. Relate opacity to light transmittance via literature research.
3. Relate light transmittance to absorbance via physical calculations.
4. Relate absorbance to crystallin damage via experimental data.

Part 1 Results

Table 1: Data obtained from XXX relating each value of the LOCS scale, to opacity values.

LOCS	0.0	0.5	1.0	2.0	3.0	4.0	5.0	6.0
Degree	None		Trace	Mild	Surgery Suggested	Moderate	Severe	Very Severe
Opacity (%)	0.34	4.24	5.80	18.88	23.60	49.14	65.61	90+
Absorbance (a.u.)	0.001	0.019	0.026	0.091	0.117	0.294	0.464	1.3+

We wish to remain below clinically significant levels, so we will reach attempt to lower the LOCS rating of a cataract to below grade 2.5, which means we want to control GSR such that the crystallin damage results in less than 0.108 a.u. at absorbance at 397.5 nm.

Menu 3

Eaque ipsa quae ab illo inventore veritatis et quasi architecto beatae vitae dicta sunt explicabo.

Conclusion

To make sure LOCS rating remains under 2.5, which has equivalent absorbance value of 0.108 a.u, we must add enough GSR to decrease crystallin damage until it is below 1.941 M-h.

To make sure LOCS rating remains under 1.0, which has equivalent absorbance value of 0.0299 a.u., we must add enough GSR to decrease crystallin damage until it is below 1.220 M-h.

Model 2: GSR/H2O2

Abstract

Abstract

Purpose

Purpose

Background, Method, Results, Discussion

• Background
• Method
• Results Part 1
• Results Part 2
• Discussion

Background

There are three quantifiers of how severe cataract formation is, two are measurable, one is not.

1. Absorbance @ 397.5 nm, which is measured with lab equipment.
2. LOCS scale, subjectively measured by physicians on a scale from 0 - 6.
3. Crystallin Damage, which we define as the following (for any time $t$)

\[c.d.(t) = \int_{0}^{\infty} [H_2O_2]_t dt\]

In other words, 1 unit of crystallin damage, in M-h, is equal to the damage caused by 1 molar concentration of hydrogen peroxide reacting crystallin in the eyes for 1 hour.

In making this definition, we assume that crystallin damage is directly proportional to the amount of time crystallin is exposed to hydrogen peroxide. Hydrogen peroxide causes damage by forming disulfide bridges within cysteine molecules on crystallin. This changes the structure of crystallin, causing misfolding and cataract damage. Our linear assumption is valid because the rate for this reaction is first order with respect to hydrogen peroxide concentration.

Method

We will relate the three by doing the following:

1. Relate LOCS scale to opacity via literature research.
2. Relate opacity to light transmittance via literature research.
3. Relate light transmittance to absorbance via physical calculations.
4. Relate absorbance to crystallin damage via experimental data.

Part 1 Results

Table 1: Data obtained from XXX relating each value of the LOCS scale, to opacity values.

LOCS	0.0	0.5	1.0	2.0	3.0	4.0	5.0	6.0
Degree	None		Trace	Mild	Surgery Suggested	Moderate	Severe	Very Severe
Opacity (%)	0.34	4.24	5.80	18.88	23.60	49.14	65.61	90+
Absorbance (a.u.)	0.001	0.019	0.026	0.091	0.117	0.294	0.464	1.3+

We wish to remain below clinically significant levels, so we will reach attempt to lower the LOCS rating of a cataract to below grade 2.5, which means we want to control GSR such that the crystallin damage results in less than 0.108 a.u. at absorbance at 397.5 nm.

Menu 3

Eaque ipsa quae ab illo inventore veritatis et quasi architecto beatae vitae dicta sunt explicabo.

Conclusion

Conclusion

Model 3: Nanoparticles

Abstract

Abstract

Purpose

Purpose

Background, Method, Results, Discussion

• Background
• Method
• Results Part 1
• Results Part 2
• Discussion

Background

There are three quantifiers of how severe cataract formation is, two are measurable, one is not.

1. Absorbance @ 397.5 nm, which is measured with lab equipment.
2. LOCS scale, subjectively measured by physicians on a scale from 0 - 6.
3. Crystallin Damage, which we define as the following (for any time $t$)

\[c.d.(t) = \int_{0}^{\infty} [H_2O_2]_t dt\]

In other words, 1 unit of crystallin damage, in M-h, is equal to the damage caused by 1 molar concentration of hydrogen peroxide reacting crystallin in the eyes for 1 hour.

In making this definition, we assume that crystallin damage is directly proportional to the amount of time crystallin is exposed to hydrogen peroxide. Hydrogen peroxide causes damage by forming disulfide bridges within cysteine molecules on crystallin. This changes the structure of crystallin, causing misfolding and cataract damage. Our linear assumption is valid because the rate for this reaction is first order with respect to hydrogen peroxide concentration.

Method

We will relate the three by doing the following:

1. Relate LOCS scale to opacity via literature research.
2. Relate opacity to light transmittance via literature research.
3. Relate light transmittance to absorbance via physical calculations.
4. Relate absorbance to crystallin damage via experimental data.

Part 1 Results

Table 1: Data obtained from XXX relating each value of the LOCS scale, to opacity values.

LOCS	0.0	0.5	1.0	2.0	3.0	4.0	5.0	6.0
Degree	None		Trace	Mild	Surgery Suggested	Moderate	Severe	Very Severe
Opacity (%)	0.34	4.24	5.80	18.88	23.60	49.14	65.61	90+
Absorbance (a.u.)	0.001	0.019	0.026	0.091	0.117	0.294	0.464	1.3+

We wish to remain below clinically significant levels, so we will reach attempt to lower the LOCS rating of a cataract to below grade 2.5, which means we want to control GSR such that the crystallin damage results in less than 0.108 a.u. at absorbance at 397.5 nm.

Menu 3

Eaque ipsa quae ab illo inventore veritatis et quasi architecto beatae vitae dicta sunt explicabo.

Conclusion

Conclusion

Model 4: Eyedrops

Abstract

Abstract

Purpose

Purpose

Background, Method, Results, Discussion

• Background
• Method
• Results Part 1
• Results Part 2
• Discussion

Background

There are three quantifiers of how severe cataract formation is, two are measurable, one is not.

1. Absorbance @ 397.5 nm, which is measured with lab equipment.
2. LOCS scale, subjectively measured by physicians on a scale from 0 - 6.
3. Crystallin Damage, which we define as the following (for any time $t$)

\[c.d.(t) = \int_{0}^{\infty} [H_2O_2]_t dt\]

In other words, 1 unit of crystallin damage, in M-h, is equal to the damage caused by 1 molar concentration of hydrogen peroxide reacting crystallin in the eyes for 1 hour.

In making this definition, we assume that crystallin damage is directly proportional to the amount of time crystallin is exposed to hydrogen peroxide. Hydrogen peroxide causes damage by forming disulfide bridges within cysteine molecules on crystallin. This changes the structure of crystallin, causing misfolding and cataract damage. Our linear assumption is valid because the rate for this reaction is first order with respect to hydrogen peroxide concentration.

Method

We will relate the three by doing the following:

1. Relate LOCS scale to opacity via literature research.
2. Relate opacity to light transmittance via literature research.
3. Relate light transmittance to absorbance via physical calculations.
4. Relate absorbance to crystallin damage via experimental data.

Part 1 Results

Table 1: Data obtained from XXX relating each value of the LOCS scale, to opacity values.

LOCS	0.0	0.5	1.0	2.0	3.0	4.0	5.0	6.0
Degree	None		Trace	Mild	Surgery Suggested	Moderate	Severe	Very Severe
Opacity (%)	0.34	4.24	5.80	18.88	23.60	49.14	65.61	90+
Absorbance (a.u.)	0.001	0.019	0.026	0.091	0.117	0.294	0.464	1.3+

We wish to remain below clinically significant levels, so we will reach attempt to lower the LOCS rating of a cataract to below grade 2.5, which means we want to control GSR such that the crystallin damage results in less than 0.108 a.u. at absorbance at 397.5 nm.

Menu 3

Eaque ipsa quae ab illo inventore veritatis et quasi architecto beatae vitae dicta sunt explicabo.

Conclusion

Conclusion

Human GzmB Inhibitor ACT3m

Section 1

• Home
• Menu 1
• Menu 2
• Menu 3

HOME

Given our model of Serpina3n, we also wanted to create a model for the protein used in our device, ACT3m. The dataset obtained from the ACT3m paper (Marcet-Palacios et al., 2014) is the result of a colorimetric assay. Data was presented as absorbance values (A₄₀₅), which correspond to the concentration of free GzmB, at different inhibitor concentrations. The paper used this to prove that their novel ACT3m inhibitor was the strongest out of their entire pool of possible candidates: treatment with ACT3m resulted in the lowest A₄₀₅ values, which suggests the strongest inhibition of GzmB.

Menu 1

To model this set of data, similar adjustments had to be made. First, the dataset was scaled so the numbers ranged between 0 and 1 (in this case, increased by a factor of 2). The same modified Hill equation was used from the Serpina3n analysis. Our model returned a Kd of ≈ 13.336 and n ≈ 0.605, so the equation looks like (Figure 2).

Menu 2

Figure 2. Model: ACT3m Inhibition of GzmB. Using data from the Marcet-Palacios et al. paper, we developed an equation that models relative GzmB activity as a function of ACT3m concentration.concentration.

Menu 3

Eaque ipsa quae ab illo inventore veritatis et quasi architecto beatae vitae dicta sunt explicabo.

Conclusion

Yay

Citations

Prevention

GSR Eyedrop

Treatment

25HC Eyedrop

LOCS: 0

Eyedrops