Part:BBa_K1634007

pmyo2-ChR2-YFP

pmyo2-ChR2-YFP
Use in	C.elegans
RFC standard	RFC 10 compatible
Backbone	pSB1C3
Submitted by	[http://2015.igem.org/Team:China_Tongji China_Tongji 2015]

Pmyo2 is a promoter which can drive the expression in the muscle of C.elegans especially the muscle in the head and pharynx. ChR2 is a kind of channelrhodopsin. Channelrhodopsins are a subfamily of retinylidene proteins (rhodopsins) that function as light-gated ion channels.[1] They serve as sensory photoreceptors in unicellular green algae, controlling phototaxis: movement in response to light.[2] When we express the channelrhodopsins in some specific cells in organisms and shed specific light on them, we can activate or supress the specific ion channel to change the activity of the cell. According to this thought in optogenetic, we linked the Pmyo2(BBa_K1634002), specific promoter in the muscle of head, with the ChR2-YFP(BBa_K1634003), then we use the light source assembled by ourselves to shed light of specific wave on the C.elegans,changing the condition of the muscle. Since we have learned that the chosing of direction in C.elegans depends on the muscle in the head, we can observe the obvious change in moving pattern of the C.elegans after we shed the light. In other words, we try to construct a light-sensed locomotion controlling system in C.elegans. And we achieve that goal by using this part to control the movement of the head, thus controlling the movement of the whole C.elegans.

We construct pSB1C3-pmyo2-ChR2-YFP and PPD95.75-pmyo2-ChR2-YFP. The pSB1C3-pmyo2-ChR2-YFP is for submission. Then we micro-inject the PPD95.75-pmyo2-ChR2-YFP into the C.elegans. The result of our testing on C.elegans(pmyo2-ChR2-YFP) is displayed below.

1. Express pattern of pmyo2

Figure 1: Express areas of pmyo2 on C.elegans

2. Response condition of pmyo2-chR2 in our test

Chart 1: the percentage of the worm which can response to the light

3. Test1

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

(1) Video

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

(2) We use real-time tracker to draw the moving track of the C.elegans.

Figure 2: Pmyo2-chR2-YFP C.elegans track

( The red point represents the track under white light,and the blue point represents the track under blue light (470nm). )

(3) Analysis:

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 stepping 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 return to normal at the moment we turn off the light instantaneously. The speed of worm doesn’t have some apparent changes.

(4) Modeling

Figure 3:　Modeling of Pmyo2-chR2-YFP C.elegans

4. Test 2

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.

Times	Turning angle (degree)
1	198.58
2	204.12
3	200.5
4	139.08
5	183.73
6	174.73
7	90.82
8	127.04
9	150.18
10	143.66
11	159.2
12	234.1
13	164.44
14	147.15
15	140.33
16	129.75

Chart2: pmyo2-chR2-YFP C.elegan’s head turning angel in different light situation

(The red ones are exposed under white light, and the blue ones are exposed under 470nm blue light)

Analysis:

chart3:　Turning angle of the head of pmyo2-chR2

(The red points means exposed under white light, and the blue points means exposed under 470nm blue LED light)

We can see that the pmyo2-chR2-YFP C.elegans have more obvious turning angel than the C.elegans exposed under white light. Since the pmyo-2 promoter will express at the neck of the worm, so the result we got just prove that chR2 actually works under the regulation of pmyo2. For this strain, the response index is getting larger with the increase of the current. And when the current increases to about 600mA, we can find that the worm can be totally activated.

5. Test 3

By using DC2100 we can achieve the goal 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.elegens, we design this part to help us. According 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). Using the worm which have reactions before is very important for this part. 0mA is needn’t to be tested, so we choose to start from 50mA. We pick up 10 worms from every strains to test whether it has response 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 are the graphs we get due to the records. The results are showed below.

Chart 4:Relation between the Light intensity and the pmyo2-chR2-YFPresponse index

(Response Index = The number of the worms which has response / 10)

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

(2) When the current increases to about 600mA, we can find that the worm can be totally activated.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 948
  Illegal XhoI site found at 986
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 288
  Illegal AgeI site found at 1627
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 2436
  Illegal SapI site found at 1351
  Illegal SapI.rc site found at 198
  

References

1. Nagel G, Ollig D, Fuhrmann M, Kateriya S, Musti AM, Bamberg E, Hegemann P (June 2002). "Channelrhodopsin-1: a light-gated proton channel in green algae". Science 296 (5577): 2395–8. doi:10.1126/science.1072068

2. Sineshchekov OA, Jung KH, Spudich JL (June 2002). "Two rhodopsins mediate phototaxis to low- and high-intensity light in Chlamydomonas reinhardtii". Proc. Natl. Acad. Sci. U.S.A. 99 (13): 8689–94. doi:10.1073/pnas.122243399.