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

1. Overview

2. Background

• 2.1 Challenges
• 2.2 Solution

3. Design

4. Protocol

• 4.1 Introduction
• 4.2 Taq PCR
• 4.3 Pfu PCR
• 4.4 AGE(agarose gel electrophoresis)
• 4.5 Gel extraction
• 4.6 Digestion & ligation
• 4.7 Seamless cloning
• 4.8 Transformation
• 4.9 Plasmid Extraction
• 4.10 Microinjection

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:

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 < 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.

4.6 Digestion & ligation

4.6.1 Digestion

(1) Add enzyme A 1ul and enzyme B 1ul;

(2) Add plasmid 4ul or gene 10ul;

(3) Add buffer 2ul;

(4) Add enough water;

(5) 37℃ 2h;

(6) Do agarose gel electrophoresis;

(7) Gel extraction.

4.6.2 Ligase reaction

(1) Add 1ul ligase;

(2) Add 2ul ligase buffer;

(3) Add 10ul gene that have digested;

(4) Add 3ul digested plasmid;

(5) Add water;

(6) 12℃ 8h.

4.7 Seamless cloning

The design of primers of PCR amplification for cloning of your sequence of interest is based on the same principles as the design of PCR primers for any sequence. The only difference is that simply add the 14-18 bases of vector sequence to the 5’end of your sequence-specific PCR primers when designing primers. After PCR clean up, the resulting PCR- amplified insert is ready for Fast Seamless Cloning.

(1) Digest the vector with two enzymes;

(2) Set up fast seamless gene cloning reaction: 7.5ul seamless cloning enzyme mix with 1ul linearizedvector and 1.5ul gene;

(3) 42℃ 30min;

(4) Transformation.

4.8 Transformation

(1) Get competence E.coli from -80C fridge;

(2) Add 15ul plasmid liquid into competence E.coli, put it in ice water for 15-30min. then give it a 42℃ heat shock for 90 sec. finally put it out of the 42℃ water bath as quick as possible. Put it into ice water for 5 min;

(3) Add 500ul LB into competence E.coli;

(4) At 37℃ we train them for 1h;

(5) Add 100ul into a LB plate which has already added ampicillin. Put them in the 37C incubator for 14h-16h.

4.9 Plasmid Extraction

(1) Put the bacterium liquid in the EP tube, Centrifuge at 12100rpm for 1 minute at room temperature;

(2) Pour out the supernatant as clean as possible;

(3) Add 150 ul P1 to the bacteria sediment, suspend the bacteria in P1 buffer;

(4) Add 150 ul P2, and shake the tube gentle 6-8 times until the liquid become in clear purple color;

(5) Add 350 ul P5, and shake it quickly for 12-20 times;

(6) Centrifuge at 12100rpm for 2 minutes at room temperature;

(7) Transfer 700 ul of the mixture(from Step 6) into a clean DNA Mini Column assembled in a 2ml collection tube(provided) Centrifuge at 12100rpm for 2 min at room temperature to pass solution through column;

(8) Pour out the liquid;

(9) Add 300ul PWT in the column, Centrifuge at 12100rpm for 2 min at room temperature to pass solution through column;

(10) Pour out the liquid and centrifuge again at 12100rpm for 1 min at room temperature;

(11) Place column into a new clean 1.5ml micro-centrifuge tube. Add 50ul 50℃ ddH2O directly onto the column matrix and centrifuge at 12100rpm for 2min to elute DNA;

(12) Exam the OD.

4.10 Microinjection

4.10.1 Equipment

(1) Injection table;

(2) Inverted DIC microscope;( Zeiss Observe.A1)

(3) Micromanipulator;(Zeiss)

(4) Pressurized injection system with needle holder;

(5) Needle puller. Sutter instruments MODEL P-1000 micropipette pullers.

4.10.2 Materials

(1) Microinjection needles;

(2) Injection pads.(Bring 2% agarose in water to a boil, mix well, and place in a heat block. Using a broken Pasteur pipette or a cut-off P200 tip, place a drop (~100ul) of hot agarose onto a #1, 50X22-mm glass coverslip. Quickly place a second coverslip on the drop and lightly tap it. Alternatively, place several drops on the first coverslip, which should merge and mostly cover the surface after adding the second coverslip;)

(3) Injection oil. Series 700 Halocarbon oil;

(4) Worm pick;

(5) M9 buffer;

(6) Worms; (Well-fed, young to middle-aged (≥1day old) gravid hermaphrodites with a full but single row of eggs.)

(7) Needle-loading pipettes.

4.10.3 Method

(1) Fill a needle-loading pipette by capillary action with ≥ 1 ul of DNA injection mix;

(2) Insert the pipette tip in through the back of the injection needle, and expel injection mix onto the needle's internal filament;

(3) Place a loaded needle into the needle holder and mount on the manipulator;

(4) Position the needle so that the tip is in the center of the microscope's field of view using the 5X objective;

(5) Place a drop of oil on an injection pad and place under a dissecting microscope on top of a small Petri plate cover;

(6) Scoop one to several worms from a bacteria-free region of an NGM plate with a naked pick and transfer to the oil drop. Avoid contact with the worm's head. Alternatively, first touch the worm pick to the oil, and use the oil droplet to pick up the animals from a bacteria-free region. The idea is to minimize transfer of bacteria to the pad;

(7) Flame then cool the worm pick and use it to position the worms in the oil drop, and to gently push them down onto the pad. Orient the worms in rows with their ventral sides facing the same direction (opposite the needle direction) If the worms fail to adhere to the pad, move to a new location or rub the bodies with the pick to remove water or bacteria droplets. If adherence is still a problem re-bake the pads or use thicker or higher concentration agarose pads (see above)

(8) Transfer the slide face-up onto the microscope stage. Center the first worm to be injected and focus using the 5X objective. Move the needle down and in close proximity to the dorsal surface of the first animal. Switch to the 40X objective and focus on the worm;

(9) First make sure the needle is flowing;

(10) Insert the needle into the worm;

(11) Inject the DNA solution;

(12) Recover the worms: (Return the coverslip to the dissecting scope, and add a drop (~20 ul) of recovery buffer on the worms.)

4.10.4 Preparation of NGM plates

4.10.4.1 Equipment and Reagents

• NaCl

• Agar

• Peptone

• 5 mg/ml cholesterol in ethanol (Do not autoclave!)

• 1 M KPO4 buffer pH 6.0 (108.3 g KH2PO4, 35.6 g K2HPO4, H2O to 1 litre)

• 1M MgSO4

• Petri plates

• Peristaltic pump

4.10.4.2 Methods

(1) Mix 3 g NaCl, 17 g agar, and 2.5 g peptone in a 2 litre Erlenmeyer flask. Add 975 ml H2O. Cover mouth of flask with aluminium foil. Autoclave for 50 min;

(2) Cool flask in 55°C water bath for 15 min;

(3) Add 1 ml 1 M CaCl2, 1 ml 5 mg/ml cholesterol in ethanol, 1 ml 1 M MgSO4 and 25 ml 1 M KPO4 buffer.Swirl to mix well;

(4) Using sterile procedures, dispense the NGM solution into petri plates using a peristaltic pump. Fill plates 2/3 full of agar;

(5) Leave plates at room temperature for 2-3 days before use to allow for detection of contaminants, and to allow excess moisture to evaporate. Plates stored in an air-tight container at room temperature will be usable for several weeks.

4.10.4.3 Seeding NGM plates

Using sterile technique, apply approximately 0.05 ml of E. coli OP50 liquid culture to small or medium NGM plates or 0.1 ml to large NGM plates using a pipet. If desired, the drop can be spread using the pipet tip or a glass rod. Spreading will create a larger lawn, which can aid in visualizing the worms. Take care not to spread the lawn all the way to the edges of the plate; keep the lawn in the center. The worms tend to spend most of the time in the bacteria. If the lawn extends to the edges of the plate the worms may crawl up the sides of the plate, dry out and die. Allow the E. coli OP50 lawn to grow overnight at room temperature or at 37°C for 8 hours (cool plates to room temperature before adding worms) Seeded plates stored in an air-tight container will remain usable for 2-3 weeks.

4.10.5 Seeding ATR NGM plates

Add 1.75ul 5uM ATR into 1 ml E.coli OP50 liquid.1ml liquid can seed 10 small NGM plates.

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.