Team:China Tongji/Project

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:

Method	Time
95℃ pre-denaturation	10min
95℃ denaturation	30s	35cycles
60℃ anneal	30s
72℃ extend	1min
4℃ save	end

4.3 Pfu PCR

(1) Put dNTP, primers, template,pfubuffer andpfuenzyme on ice;

(2) Prepare the mix liquid:

Experimental Material	Dose
Template	1ul
Primer-Front	2.5ul
Primer-Reverse	2.5ul
dNTPs	5ul
5*Loading Buffer	10ul
Pfu DNA polymerase	1ul
ddH2O	27ul
Total volume	50ul

(3) Mix solution well;

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

Method	Time
95℃ pre-denaturation	10min
95℃ denaturation	30s	35cycles
60℃ anneal	30s
72℃ extend	1min
4℃ save	end

4.4 AGE ( agarose gel electrophoresis )

(1) make of gel with 0.5g agarose and 50ml 10X TAE, add 2 drops of EB to dye the gel;

(2) mix the PCR sample with 10x loading buffer;

(3) 220V 30min;

(4)use UV light to view the result.

4.5 Gel extraction

(1) Excise the agarose gel slice containing the DNA fragment of interest with a clean, sharp scalpel under ultraviolet illumination. Briefly place the excised gel slice on absorbent toweling to remove residual buffer. Transfer the gel slice to a piece or plastic wrap or a weighing boat. Mince the gel into small pieces and weigh. In this application, the weight of gel is regarded as equivalent to the volume. For example, 100 mg of gel is equivalent to a 100 μl volume. Transfer the gel slice into a 1.5 ml microfuge tube.

(2) Add a 3x sample volume of Buffer DE-A.

(3) Resuspend the gel in Buffer DE-A by vortexing. Heat at 75°C until the gel is completely dissolved (typically, 6-8 minutes) Heat at 40°C if low-melt agarose gel is used. Intermittent vortexing (every 2-3 minutes) will accelerate gel solubilization.

(4) Add 0.5x Buffer DE-A volume of Buffer DE-B, mix. If the DNA fragment is less than 400 bp, supplement further with a 1x sample volume of isopropanol.

Example: For a 1% gel slice equivalent to 100 μl, add the following:

• 300 μl Buffer DE-A

• 150 μl Buffer DE-B

If the DNA fragment is %3c 400 bp, you would also add:

• 100 μl of isopropanol.

(5) Place a Miniprep column into a 2 ml microfuge tube (provided) Transfer the solubilized agarose from Step 4 into the column. Centrifuge at 12,000xg for 1 minute.

(6) Discard the filtrate from the 2 ml microfuge tube. Return the Miniprep column to the 2 ml microfuge tube and add 500 μl of Buffer W1. Centrifuge at 12,000xg for 30 seconds.

(7) Discard the filtrate from the 2 ml microfuge tube. Return the Miniprep column to the 2 ml microfuge tube and add 700 μl of Buffer W2. Centrifuge at 12,000xg for 30 seconds.

(8) Discard the filtrate from the 2 ml microfuge tube. Place the Miniprep column back into the 2 ml microfuge tube. Add a second 700 μl aliquot of Buffer W2 and centrifuge at 12,000xg for 1 minute.

(9) Discard the filtrate from the 2 ml microfuge tube. Place the Miniprep column back into the 2 ml microfuge tube. Centrifuge at 12,000xg for 1 minute.

(10) Transfer the Miniprep column into a clean 1.5 ml microfuge tube (provided) To elute the DNA, add 25-30 μl of Eluent or deionized water to the center of the membrane. Let it stand for 1 minute at room temperature. Centrifuge at 12,000xg for 1 minute.

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.