Part:BBa_K309007:Experience

User Reviews

UNIQ4eb5798fa39cd3d6-partinfo-00000000-QINU UNIQ4eb5798fa39cd3d6-partinfo-00000001-QINU

User name: 2015 China_Tongji

Author: 2015 China_Tongji

Since our project also focus on Optogenetics, we want to express channelrhodopsin in C.elegans. There are many kinds of channelrhodopsin now, but we want to start with the most traditional one, ChR1 or ChR2. Then we search in the iGEM database and we find this part submitted by iGEM10_Queens-Canada. The iGEM10_Queens-Canada focus on establishing some basic parts for later users to use in C.elegans. And we decide to make use of this part, not only establish our own project but also test this part and characterize it. However, we find it being defined as “Sample It's complicated”by iGEM which means that it has been sequenced with a result of Single Base Error, Partially Confirmed, Inconsistent, or Bad Sequencing. What’s more, there are no samples of that part are available in distributions. So we choose to have the part synthesized according to the sequence submitted by iGEM10_Queens-Canada. And we have YFP in our lab in Tongji University, so we use overlapping PCR to link the sequence of YFP and ChR2, thus constructing a fusion protein, ChR2-YFP.

We ligates pmyo2-ChR2-YFP and pmyo3-ChR2-YFP into PPD95.75. Then we micro-inject the PPD95.75-pmyo2-ChR2-YFP and PPD95.75-pmyo3-ChR2-YFP into the C.elegans. The result of our testing on C.elegans( pmyo2-ChR2-YFP) and C,elegans( pmyo2-ChR2-YFP) are displayed below.

Test 1

We test these worms by using 5W LED blue light (470nm) with 1000mA LED driver.

Control group

1. Lite1 worm without any modified

(1) Video

Please pay attention to the top right corner of the video, the appearance of blue point represents the change from white light to 5W LED blue light (470nm)

(2) Track

Figure1. The track of lite1 worm given blue light (470nm, 5W, 1000mA)

[ Red points represents the track under white light. ]

[ Blue points represents the track under blue light (470nm) ]

(3) This group is lite1 worm which is not sensitive to the lights, while other types of worms make have response to the light. So we choose lite1 worm to be our experimental subject to avoid unnecessary factors.

2. Pmyo2-ChR2-YFP worm without ATR

(1) Video

Please pay attention to the top right corner of the video, the appearance of blue point represents the change from white light to 5W LED blue light (470nm)

(2) Track

Figure2. The track of myo3-ChR2-YFP with blue light (470nm, 5W, 1000mA, fostered without ATR)

[ Red points represents the track under white light. ]

[ Blue points represents the track under blue light (470nm) ]

(3) This group we use the mediums without ATR to foster the pmyo3-ChR2-YFP worms. We use this group to find out the effect of ATR. As a result, we find out that the ATR is necessary to our project. Only being fostered in the mediums which have ATR do the worms have response to the lights we give.In our project, we also set up other control groups to each strain, and they all have the same phenomenon. We choose pmyo3-ChR2-YFP worms as an example for this kind of worm have the highest efficiency.

3. Functional worms with green light

(1) Video

Please pay attention to the top right corner of the video, the appearance of blue point represents the change from white light to 5W LED blue light (470nm)

(2) Track

Figure3. The track of pmyo3-ChR2-YFP worm given green light (490nm, 3W, 1000mA)

[ Red points represents the track under white light. ]

[ Blue points represents the track under blue light (470nm) ]

(3) This group we use green light to stimulate the pmyo3-ChR2-YFP worm. By doing this, we try to find out if the worm have response to all kinds of lights. At last, we find out that our worms would only have response to blue light (470nm, 5W, 1000mA). In our project, we also set up other control groups to each strain, and they all have the same phenomenon. We choose pmyo3-ChR2-YFP worms as an example for this kind of worm have the highest efficiency.

pmyo2-ChR2-YFP

(1) Video

Please pay attention to the top right corner of the video, the appearance of blue point represents the change from white light to 5W LED blue light (470nm)

(2) Track

Figure4. pmyo2-ChR2-YFP worm given blue light (470nm, 5W, 1000mA)

[ Red points represents the track under white light. ]

[ Blue points represents the track under blue light (470nm) ]

(3) Reactions:

We test this kind of worms by using 5W LED blue light (470nm) with 1000mA LED driver. When we give light to this kind of worms, we can find some obvious responses. First, after we give light, the worm would change their direction in about 2 seconds in average. Their reactions are always step back. Secondly if we focus on the movement of their heads, we can find the turning angles change a lot during this time. It means the blue light can stimulate the muscle of their heads and as a result the worm will change the direction. But their behavior will turn to normal at the moment we turn off the light instantaneously. The speed of worm doesn’t have some apparent changes.

Pmyo3-chR2- YFP

(1) Video

Please pay attention to the top right corner of the video, the appearance of blue point represents the change from white light to 5W LED blue light (470nm)

(2) Track

Figure5. pmyo3-ChR2-YFP worm given blue light (470nm, 5W, 1000mA)

[ Red points represents the track under white light. ]

[ Blue points represents the track under blue light (470nm) ]

(3) Reactions:

We test this kind of worms by using 5W LED blue light (470nm) with 1000mA LED driver.This kind of worms is one of the highest expression worms among all the strains we have, and its reaction is very obvious. First when the light is on, we can find the whole body of this worm contract, which means the blue light lead to the muscle contraction of the worm. Secondly the worms will not move until we turn of the lights. It means that the blue light prevent the worm from moving. When giving the light, the worm will move. When the light is off the worm will keep moving. These means the speed of the worm is changing. It is very interesting that all these reactions are taken place instantaneous.

Phenotype summary

	Speed Change	Direction Change	Bending Degree	Muscle Contraction
Pmyo2-ChR2-YFP
Pmyo3-ChR2-YFP

Test 2

Turning angle measuring

As we know pmyo2 is express in pharyngeal of C.elegents, so the light will stimulate the head of the worms directly to the head. As a result, observing the movement of their heads is very significative. As we all know, the head of the worm is always shaking, so the turning angle (the angle of each shake) is a very useful data which reflect the response of the head. In this part, we use turning angle of their heads to evaluate the reaction of their head.

We choose pmyo2 worms as our experimental objects in this part. The results are showed below.

Figure6. The turning angle measuring of pmyo2-ChR2-YFP (using blue light, 470nm, 5W, 1000mA)

[ Red points represents the track under white light. ]

[ Blue points represents the track under blue light (470nm) ]

Figure7.The turning angle measuring of pmyo2-ChR2-EYFP (given no light)

[ Red points represents the track under white light. ]

[ Blue points represents the track under blue light (470nm) ]

Analysis:

From these figures we can get some useful information.

1. The pmyo2-ChR2-YFP worms have an obvious response. When we give the lights, we can see theamplitude of turning angles become larger. The change of the turning angle becomes drastic.

2. We use the pmyo2 worm given no lights as the control group. We test all the strains of pmyo2 worms, and they have the same reactions. We can see the fluctuation of turning angle is mild compare to those experimental groups.

Test 3

The relationship between light intensity and response index

By using DC2100 we can achieve the aim that we could control the current of LED accurately. For our LEDs, there is a direct proportion relationship between light intensity and the current which move across it. To test which value is the best to stimulate the C.elegents, we design this part to help us. Accord to the limitation of DC2100, the largest current we can use is 1000mA. So we pick some worms of all strains which have obvious reactions as our experimental material (using 1000mA to test the reactions before). Because 0mA is needn’t to be tested, we choose to start from 50mA. Every strain we pick up 10 worms to test it has reactions or not. After 50Ma has been tested, we test the 100mA and then 150mA and so on. Until we finish the test of 1000ma, we calculate the ratio of having reactions. Here is the graph we get due to the records.The results are showed below.

Figure8. The changing trend of response index with the change of light intensity (blue LED, 470nm, 5W, using pmyo2-ChR2-YFP worm)

Figure9. The changing trend of response index with the change of light intensity (blue LED, 470nm, 5W, using pmyo3-ChR2-YFP worm)

From these data, we can find out some conclusions.

1. For all these strains, the response index is getting larger with the increase of the current.

2. For pmyo2-ChR2-YFP and pmyo3-ChR2-YFP, when the current increase to about 600 we can see the worm can be totally activated.

3. For all the strains of worms, 1000mA is the most suitable current. So in our project we use 1000mA blue light (470nm, 5W) to test our worms.

Summary

ChR2 is a very functional channelrhopsin. After being with some specific promotor such as Pmyo2 or Pmyo3, it can be activated when giving the blue light (470nm) and change the activity of the muscle, thus changing the movement. and the fittest current(representing the fittest intensity of light in our testt) which could activate the ChR2 is between 600mA and 1000mA (using 5W LED, 470nm).

In one word, we strongly recommend you to start with this part if you want to set up your own project in Optogenetics.