Difference between revisions of "Part:BBa K2492001"

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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

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 which wavelength of light

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).

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. If CoCHR worked well, the AWA would be induced due to CoCHR activation (principles of CoCHR are stated above). 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. After CoCHR was activated, AWA which contained transgenic CoCHR part, would be activated. Thus the shedding of blue light on worms was similar to food attraction.