Part:BBa_K1634004

pmyo3-ChR2-YFP ( C.elegans )

pmyo3-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]

“myo-3 encodes MHC A, the minor isoform of MHC (myosin heavy chain) that is essential for thick filament formation, and for viability, movement, and embryonic elongation; expressed in body muscle, the somatic sheath cell covering the hermaphrodite gonad, and also expressed in enteric muscle, vulval muscles of the hermaphrodite and the diagonal muscles of the male tail. “(from Wormatlas) Since we want to control the C.elegans' movement.We decided to use pmyo-3 (promoter of myo-3)(BBa_K1634002) to construct a plasmid which can drive the expression in the worm’s body muscle.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 Pmyo3,specific promoter in the muscle of whole body, 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,changging the condition of the muscle. Then 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.

We construct pSB1C3-pmyo3-ChR2-YFP and PPD95.75-pmyo3-ChR2-YFP. The pSB1C3-pmyo3-ChR2-YFP is for submission. Then we micro-inject the PPD95.75-pmyo3-ChR2-YFP into the C.elegans. The result of our testing on C.elegans(pmyo3-ChR2-YFP) is displayed below.

Expression Pattern

Figure 1: Express areas of pmyo3 on C.elegans

Test 1

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:　Pmyo3-chR2-YFP C.elegans movement track

[ The red points represents the track under white light, and the blue points 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.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.

4. Modeling

Figure 3: modeling of the track of pmyo3-chR2-YFPC.elegans

[ The red points represents the track under white light, and the blue points represents the track under blue light (470nm) ]

Test 2

We try to figure out relation between the Light intensity and the response index.

We pick up thoseC.elegans which have some response under the light with the intensity of 1000mA. We test 10 of them every time we add 50 mA to the prior current.

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

Chart 1: Relation between the Light intensity and the pmyo3-chR2-YFPresponse index

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

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 1068
  Illegal XhoI site found at 1106
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 1747
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 2556
  Illegal SapI site found at 1471
  Illegal SapI.rc site found at 259
  

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.