Difference between revisions of "Team:China Tongji/Project"

1. Overview

2. Background

• 2.1 Challenges
• 2.2 Solution

3. Design

4. Protocol

5. Summary and Result

1. Overview

Cells sense the environment, process information, and make response to stimuli. To make cells work well in complex natural environments, lots of processes have to be preset to react to various signals. However, when well-characterized modules are combined to construct higher order systems, unpredictable behaviors often occur because of the interplay between modules. Another significant problem is that complex integrated systems composed of numerous parts may cause cell overload.

Figure 1. Schematic demonstration of HIV

We proposed an elegant method to design higher order systems. Instead of merely combining different functional modules, we constructed one integrated processing module with fewer parts by utilizing the common structures between modules. The circuit we designed is a rewirable one and the topological structure of the processing module can be altered to adapt to environmental change. The basic idea is to rewire the connections between parts and devices to implement multiple functions with the help of the site-specific recombination systems.

Our design approach may lead to a revolutionary step towards system integration in synthetic biology. Potential fields of application include organism development, living therapeutics and environment improvement.

2. Background

Since its inception more than a decade ago, synthetic biology has undergone considerable development and has attained significant achievements with the help of the engineering slant. However, there are still obstacles to build a cell. Engineers try to abstract the DNA sequences into some standard functional parts and assemble them using some principles in electrical engineering. So far, the limited understanding of biological system prevents us to combine parts and modules to create larger scale systems. The complexity of synthetic systems didn’t increase rapidly as the Moore’s law (Purnick and Weiss, 2009).

2.1 Challenges

There are some common problems that make the circuits we designed not work as our expected. Many failure modes have been collated by Brophy and Voigy in their review (Brophy and Voigt, 2014). In our project, we mainly focus on two modes, crosstalk and host overload, that emerge especially when we create more sophisticated systems. More specifically, regulators may interact with each other’s targets leading to errors in the desired operation, and the synthetic circuits may compete with natural parts that maintain the normal cellular processes for limited resources.

2.2 Solution

We designed a time-sharing system that can process information according to the input signal. Cells rewire its synthetic circuit to alter the topological structure of regulatory pathway when they receive the corresponding stimuli. In this way, we reuse the existing synthetic module rather than add a new one to implement another function, which reduces the resource cost in running unnecessary function and prevents the interplay between parallel modules. After overcoming these two big problems, our engineered cells are more versatile and flexible in information processing.

3. Design

Cells sense the environment, process information, and make response to stimuli. To make cells work well in complex natural environments, lots of processes have to be preset to react to various signals. However, when well-characterized modules are combined to construct higher order systems, unpredictable behaviors often occur because of the interplay between modules. Another significant problem is that complex integrated systems composed of numerous parts may cause cell overload.

Figure 2. China Tongji logo

Our design approach may lead to a revolutionary step towards system integration in synthetic biology. Potential fields of application include organism development, living therapeutics and environment improvement.

4. Protocol

4.1 Introduction

Our project aims to control C.elegans’ movement by expressing chR2 in their muscle and neuron. In our plan, we will make over 20 parts of 3 kinds of channelrhodopsins with 5 different promoters.

First of all, we need to design the PCR primers with primer 5.Then we run taq PCR or pfu PCR to get our parts out of the C.elegans’ genome or plasmids.After that, we do the digestion of gene parts and vector pPD95.77. Use traditional method to do the ligation and transformation. Besides, we also use seamless cloning to deal with some difficult ligations. The last step in molecular construction is plasmid extraction.

Then we come to the C.elegans part, which includes microinjection, making NGM and ATR plates and seed plates. All the details will show below.

4.2 Taq PCR

(1) Put dNTP, primers, template, taqbuffer and taq enzyme on ice;

(2) Prepare the mix liquid:

Experimental Material	Dose
Template	1ul
Primer-Front	1ul
Primer-Reverse	1ul
dNTPs	4ul
Taq PCR buffer	5ul
taq enzyme	0.25ul
ddH2O	37.75ul
Total volume	50ul

(3) Mix solution well;

(4) Use the PCR machine and amplification the gene：

5. Summary and Result

Cells sense the environment, process information, and make response to stimuli. To make cells work well in complex natural environments, lots of processes have to be preset to react to various signals. However, when well-characterized modules are combined to construct higher order systems, unpredictable behaviors often occur because of the interplay between modules. Another significant problem is that complex integrated systems composed of numerous parts may cause cell overload.

Figure 2. China_Tongji_iGEM_logo

Our design approach may lead to a revolutionary step towards system integration in synthetic biology. Potential fields of application include organism development, living therapeutics and environment improvement.