Difference between revisions of "Team:IISER-Tirupati India/Parts"

Introduction

Natural systems are highly complex to understand as well as to experiment with. To make predictions of the outcomes, we use the available information and the knowledge of physics, mathematics, chemistry, and computer science to build a theoretical model. This page deals with the models built on the different aspects of our project namely :

1. GMO delivery
2. GMO growth and colonisation
3. Protease production
4. Diffusion and ovum Hardening
5. Kill switch for reversibility
6. Kill switch for Biosafety

Delivery & Colonisation

INTRODUCTION: THE JOURNEY OF GMO BEGINS

PART A: Delivery

OVERVIEW

Delivery is an essential part of our project as it determines the audience we reach. We tried to develop multiple ways to deliver GMO in a user-friendly way with minimum invasion. Minimum invasion means it should not colonize the whole reproductive tract or reach the ovaries. The device should ensure bacterial colonization in the ampulla of the fallopian tube. One of the constraints that we faced is the high viscosity of the oviductal fluid.

After calculating the time taken for delivery in each method, talking to a couple of IVF experts, and getting their inputs into it,we thought hysteroscopic techniques would be the best for our purpose. This method gives us an advantage by delivering the bacteria directly into the ampulla region.

DEVICE DESIGNING (How to deliver?)

GMO will be delivered directly to the ampulla by a process called Hysteroscopy. It’s done with medical assistance and requires constant X-Ray monitoring to ensure the catheter is inserted without any issues. The reason is that the Ostia and the Fallopian tube are dynamic structures and that there is no chemical or significant physical difference between the ampulla and the other components of the Fallopian tube.

A solution of GMO of a specific concentration is prepared. Then the catheter is inserted under medical guidance up to the ampulla. Then an injector is projected with narrow long incisions on both sides of it, which ensures that the GMO forms a ring along the circumference of the tube. Ovum is now 360° surrounded by the GMO.

The GMO Lactobacillus Acidophilus has pili which expectantly forms hydrogen bonds at the tube circumference with mucin found in mucus. The GMO is now adhered to the walls and multiplies once it adjusts to the introduced environment (lag phase)

FUTURE ASPECTS:

Since we aim to reach a larger population, the hysteroscopic technique, we believe, is too expensive. We also considered using hydrogels for the delivery of bacteria at a pH-specific region.

Why is this system compatible with the delivery of bacteria in the ampulla of the Fallopian tube region?

We found out that the pH in the oviductal Ampulla region is close to 8.0. That’s why we considered using hydrogels for bacterial delivery, which swells up at a pH range of 7.8 to 8.0.

Amongst the stimuli-responsive hydrogels, pH-sensitive hydrogels are the most studied hydrogels. The rate at which hydrogels respond depends upon their size, shape, cross-linking density, number of ionic groups, and composition, which can be tailored by varying these factors. The response rate increases with increasing pore size and number of ionic groups and decreasing their size and cross-linking density.

For a delivery in a basic medium, we planned to use anionic hydrogels, such as carboxymethyl chitosan, which swell at higher pH (basic medium) due to ionization of the acidic groups. As a result, the ionized negatively charged pendant groups on the polymer chains cause repulsion leading to swelling. This property of hydrogels can be exploited for GMO delivery at pH 7.4 in the ampulla of the fallopian tube.

CELL ENCAPSULATION (What to deliver?)

Our goal is to design a live-bacteria entrapment system. More than just encapsulating bacteria, we want to entirely prevent their escape from the bead body into the surroundings.

The Oviductal Fluid is slightly alkaline (ph 7.4 to 7.7), so we need our encapsulating membrane to dissolve away at this pH. Potential candidates for hydrogel materials include chitosan, guar gum, and xanthan. Chitosan forms Hydrogen bonds with the mucin protein in the mucus allowing for the anchoring at the walls and preventing the hydrogels from getting washed away.

PART B: Colonization

OVERVIEW (why is it necessary to study growth and colonization)

In biosynthesis, growth kinetics is a crucial study to be conducted. The growth of a cell comprises both the size and the number. These growths are affected by external factors such as temperature, the chemical composition of the growth nutrient, etc., and by different physiological factors such as growth factor proteins[1][2]. The cells in a particular environment extract the nutrients from the growth media and produce biomolecules, which humans utilize for different purposes. This phenomenon has applications, from producing delicious Italian wine to getting antibiotics which saves millions of lives every year. We will be using this simple formula to reach the goals of our project. To have a qualitative understanding of the protein production by the Genetically Modified anism(GMO), we need to have a theoretical approach. We need to understand how we can calculate the growth of GMOs [3]. This, in turn, will help us figure out protein production. This detailed study of growth kinetics will help us calculate the initial inoculation required for the production of a protein that we need.

For a better understanding, we will be considering two cases. The first comprises the study of the growth of Bacillus Subtilis in normal petri dish conditions. This is the known environment and we can have this condition easily in computer simulation and/or lab and reconfirm our model. The second case of study is the continuous culture model where we study the growth of Lactobacillus Acidophilus in the fallopian tube, which is our target region.

GROWTH MODELS

PETRI DISH MODEL (Bacillus Subtilis, curve fitting)

CONTINUOUS CULTURE MODEL (lactobacillus acidophilus + fallopian tube environment)

INOCULUM CALCULATIONS (connection with 2nd module)

Protease production

OVERVIEW ( overall picture)

Well known for extracellular protease production[1], Bacillus subtilis is our model organism for the proof of concept experiments. Moreover, it is a gram-positive bacteria which even if not too close but is closer than e coli (gram-negative) to our proposed bacteria lactobacillus Acidophilus. This module deals with the whole mechanism of action ideally the GMO must follow for contraception to be attained. To know how this module developed to what it is, please refer to the engineering success (hyperlink).

Let's create the flow, you must know by now that we plan to produce the protease known as ovastacin, an indigenous protease[2] to the human ovum. It is known to cause Zona pellucida hardening naturally used by the ovum to prevent polyspermy[3]. To see the mechanism of zona pellucida hardening click here (goes to project overview where it is explained).

1] Total amount of ovastacin required:

[We assume one active ovastacin cleaves one ZP2 glycoprotein present in the Zona pellucida matrix. To get the number of molecules of ovastacin needed, we calculated the number of ZP2 glycoproteins present on the surface of the ovum. For this, we reached out to studies that looked at the thickness of the ovum with and without the zona pellucida layer to find its overall thickness and the radius of the ovum[4].

Constants and calculations:

Assuming cuboid with all spheres of size same as ZP2 to get volume of one unit = 3.1 e-6 μm3

Volume occupied by whole ZP matrix = 12.4 e5 μm3

Calculated the number of ZP2 glycoproteins present in the ZP matrix = 3.92 x 1011 molecules

Assuming 1 ovastacin cleaves 1 ZP2

No. of ovastacin = No. of ZP2 = 12.4 e5 / 3.1 e-6 = 3.92 x 1011 molecules ]

In order to cause complete hardening __ number of molecules are required to reach the ovum. The next question that arises is how much is produced and how much of produced ovastacin will reach the ovum. So we have two major things left to look at:

2] Production and transport of ovastacin by GMO

The production of ovastacin is required for three days pre and post ovulation, please go to “Genetic Circuits” to learn details regarding the genetic circuit.

GENETIC CIRCUIT

Progesterone repressible system

JOURNEY OF OVASTACIN

From the papers, we know that the ovum is propelled by the ciliary motion away from the uterus and that means the ovum is in contact with the wall. Thesize ampulla region of the Fallopian tube (2.5 mm ≤ radius ≤ 5 mm ) is much larger than the radius of the ovum (61.7μm) A molecule under the influence of Brownian motion move according to the equation

• < r ² >= 4Dt (for 2-D)
• < r ² >= 6Dt (for 3-D)

Where,

• < r ² > → mean squared (radial) distance travelled by the molecule

using,

1. k_B = 1.38064852 ×10²³ m² kg^-2 s^-1 K [Boltzmann constant]
2. η = 0.799 Pa·s [Viscosity of Oviductal Fluid]
3. T = 35.5 + 273.15 = 308.65 K [Temperature of Oviduct]
4. r = 22kDa = 2.43 nm [Radius of ovastacin molecule]
5. a = 61.7 μm [Radius of the ovum]
6. s = 5 mm [Radius of the ampulla region of the oviduct]
7. N₀ = 3.92 ×10¹¹ molecules = 0.000650946529392 nanomoles [Number of ovastacin to react with all ZP2]

For, Ovastacin D=1.1644194699 ×10^-13 m² s^-1

• t → Time elapsed