Difference between revisions of "Part:BBa K2492001"
 
(10 intermediate revisions by 2 users not shown)
Line 2: Line 2:
 
__NOTOC__
 
__NOTOC__
 
<partinfo>BBa_K2492001 short</partinfo>
 
<partinfo>BBa_K2492001 short</partinfo>
 +
 +
Introduction:
  
 
The central part of our project. Part BBa_K2492001 contains CoCHR(Chloromonas oogama channelrhodopsin) [1].
 
The central part of our project. Part BBa_K2492001 contains CoCHR(Chloromonas oogama channelrhodopsin) [1].
Line 18: Line 20:
 
<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_K2492001 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K2492001 SequenceAndFeatures</partinfo>
 +
 +
Materials:
 +
 +
Odr10::CoChR::GEM-GECO::mCherry transgenic starving worms
 +
NGM plate with ATR
 +
Mercury lamp and optical fiber (provides blue light)
 +
 +
Methods:
 +
 +
1.We injected the Odr10::CoChR::GEM-GECO::mCherry worms with ATR into the immobilization chip.
 +
After about 2 hours the Odr10::CoChR::GEM-GECO::mCherry worms were inactive, then we used lights with different wavelengths and intensities to stimulate them. We found only 480 nm from the projector could active them.
 +
2.Induce the Odr10::CoChR::GEM-GECO::mCherry successfully transgenic worms to draw a cycle on the NGM plate through blue light at 480nm.
 +
 +
Result:
 +
 +
Test 1: Determine that transgenic worms are sensitive with which wavelength of light
 +
 +
Compared to different wavelengths, worms are sensitive to wavelength of 480nm.
  
  
 
[[File:T--SUSTech Shenzhen--getup.gif|800px|thumb|left| The test of the CoChR using light with different wavelengths and intensity. The lights blue- and blue+ is from the projector whose wavelength is about 480 and the lights whose wavelengths are 395, 440, 470, 560 and 640 are from the LED of fluorescence microscope (Nikon eclipse Ti).]]
 
[[File:T--SUSTech Shenzhen--getup.gif|800px|thumb|left| The test of the CoChR using light with different wavelengths and intensity. The lights blue- and blue+ is from the projector whose wavelength is about 480 and the lights whose wavelengths are 395, 440, 470, 560 and 640 are from the LED of fluorescence microscope (Nikon eclipse Ti).]]
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
Test 2: 480nm blue light induction of CoCHR
 +
 +
While seeing the movie, pay attention to the track of light and worms, and the relations between the shedding light and chasing worms.
 +
 +
As we shedding light in cycle, worms followed the light in cycle.
 +
 +
 +
 +
 +
 +
 +
  
 
<html>
 
<html>
Line 38: Line 117:
 
<div class="row">
 
<div class="row">
 
</html>
 
</html>
 +
 +
Conclusion:
 +
 +
In order to demonstrate the function of CoCHR, we ligate Odr10::CoChR::GEM-GECO::mCherry plasmid and inject into worms. After confirmed that we have gained the stable transfected worms through location marker, we use blue light to induce the worm.
 +
 +
We induced the worm with 480nm blue light. The experiment results were as follows:
 +
 +
Worms could follow the blue light spot, which was similar to be attracted by food. We tried to shed light in cycle, and the worms seemed to chase the light.
 +
 +
Concluded from the results, CoCHR was successfully manipulated through blue light. CoCHR worked well. Moreover, after CoCHR was activated, we could indicate that the injected plasmid which contained cell specific promoter was well expressed. The activation of AWA specific promoter would lead to the whole activation of AWA. Thus the response shedding of blue light on worms was similar to food attraction. 
 +
 +
  
  

Latest revision as of 03:52, 2 November 2017


CoCHR Channelrhodopsin

Introduction:

The central part of our project. Part BBa_K2492001 contains CoCHR(Chloromonas oogama channelrhodopsin) [1]. In our project, CoCHR is a cation-selective light sensor, which promptly generates a large permeability of divalent cations after absorbing photons from our excitation light source. In our project, we hope to investigate how olfactory messages are transmitted by the neuron systems. In order to decrease delay or response comparing to chemical stimulus, we choose optogenetic techniques. Since C.elegans do not have sense to light, we express channelrodopsins in pairs of neuron as light receptors. CoCHR is the channelrodopsin in transgenic AWA. In order to express CoCHR in AWA specifically, we express CoCHR through the linkage to the AWA cell-specific promoter odr-10. Thus, when we shed ~470nm excitation light to transgenic C.elegans, the activated AWA is able to cause obvious response such as body swimming towards. While wild-type C.elegans not behave abnormally under the same stimulation. Whether the certain neuron is activated or not is indicated by GEM-GECO, a calcium indicator. In short, we established a light-sensed AWA controlling system, so as to utilize this part to manipulate the activation of AWA, establish the response to input stimulation. The merit of CoCHR is its sensitivity to light which is five-times than formal ChR2. Thus, the low intensity requirement prevents the cross-activation of Crimson(Chlamydomonas noctigama channelrhodopsin). Chrimson is another integrated channelrhodopsin in AWB. (Moreover, in order to indicate where the target neurons are. We also construct a fusion reporter protein called mCherry. At the same time, the fusion protein is allowed to estimate the expression level of CoCHR.)

[1]Schild, L. C., & Glauser, D. A. (2015). Dual color neural activation and behavior control with chrimson and cochr in caenorhabditis elegans. Genetics, 200(4), 1029-34.

[2] 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.


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Unknown
  • 12
    INCOMPATIBLE WITH RFC[12]
    Unknown
  • 21
    INCOMPATIBLE WITH RFC[21]
    Unknown
  • 23
    INCOMPATIBLE WITH RFC[23]
    Unknown
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]

Materials:

Odr10::CoChR::GEM-GECO::mCherry transgenic starving worms NGM plate with ATR Mercury lamp and optical fiber (provides blue light)

Methods:

1.We injected the Odr10::CoChR::GEM-GECO::mCherry worms with ATR into the immobilization chip. After about 2 hours the Odr10::CoChR::GEM-GECO::mCherry worms were inactive, then we used lights with different wavelengths and intensities to stimulate them. We found only 480 nm from the projector could active them. 2.Induce the Odr10::CoChR::GEM-GECO::mCherry successfully transgenic worms to draw a cycle on the NGM plate through blue light at 480nm.

Result:

Test 1: Determine that transgenic worms are sensitive with which wavelength of light

Compared to different wavelengths, worms are sensitive to wavelength of 480nm.


The test of the CoChR using light with different wavelengths and intensity. The lights blue- and blue+ is from the projector whose wavelength is about 480 and the lights whose wavelengths are 395, 440, 470, 560 and 640 are from the LED of fluorescence microscope (Nikon eclipse Ti).
























Test 2: 480nm blue light induction of CoCHR

While seeing the movie, pay attention to the track of light and worms, and the relations between the shedding light and chasing worms.

As we shedding light in cycle, worms followed the light in cycle.





Fig. 1 Control the C. elegans by the light. We used the optical fiber to form a light spot and the worm can follow the spot.

Conclusion:

In order to demonstrate the function of CoCHR, we ligate Odr10::CoChR::GEM-GECO::mCherry plasmid and inject into worms. After confirmed that we have gained the stable transfected worms through location marker, we use blue light to induce the worm.

We induced the worm with 480nm blue light. The experiment results were as follows:

Worms could follow the blue light spot, which was similar to be attracted by food. We tried to shed light in cycle, and the worms seemed to chase the light.

Concluded from the results, CoCHR was successfully manipulated through blue light. CoCHR worked well. Moreover, after CoCHR was activated, we could indicate that the injected plasmid which contained cell specific promoter was well expressed. The activation of AWA specific promoter would lead to the whole activation of AWA. Thus the response shedding of blue light on worms was similar to food attraction.