Difference between revisions of "Part:BBa K2492004"

Latest revision as of 03:36, 2 November 2017

pODR-10: the promoter of coding odorant diacetyl receptor protein.

Part BBa_K2492004 contains odr-10. It is a section of genomic DNA acts as a promoter. In C.elegans, ODR-10 encodes a large family of seven transmembrane olfactory receptor genes[1]which is specifically expressed in neuron AWA and affect chemotaxis to the volatile diacety. POrd-10 is a section of genemoic DNA in front of Ord-10 genes promoting expression of Ord-10 genes[2].

AWA is a member of the olfactory neurons [2]. It responses to attractive volatile odorants. Since we expect to express the functional channelrhodopsins and indicator in specific neuron(AWA for instance), we use pODR-10 to construct plasmid.

If CoCHR is expressed in AWA, we are capable of activating it to manipulate the state of AWA. In our project, we integrated ord-10::Chrimson::GEM-GECO::mCherry transgene to C.elegans by microinjection. Thus, CoCHR (Part BBa_K2492001:CoCHR) promoted by ord-10 is expressed in AWA. We can enhance the attraction of C.elegans through the control of AWA.

[1]:Sengupta P., Chou J.H., Bargmann C.I.Cell 84:899-909(1996)

[2]:Emily R. et al. (1997). Reprogramming Chemotaxis Responses: Sensory Neurons Define Olfactory Preferences in C. elegans, Cell, Vol. 91, 161–169.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
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:

1. Determine that transgenic worms are sensitive with 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).

2.Test 2: 480nm blue light induction

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 this part, 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 the promoter worked well, the constructed plasmid would successfully expressed well in AWA.

Since after shedding 480nm light, the transgenic worms were chasing the light, thus the light manipulated AWA successfully. The successful manipulation of AWA through blue light represented that the cell- specific promoter worked well. Since if the promoter not worked, none of the ligated proteins could be expressed.

Hence, we successfully demonstrated that we successfully expressed this part and this part worked well.

@@ Line 13: / Line 13: @@
 [2]:Emily R. et al. (1997). Reprogramming Chemotaxis Responses: Sensory Neurons Define Olfactory Preferences in C. elegans, Cell, Vol. 91, 161–169.
-<!-- Add more about the biology of this part here
-===Usage and Biology===
 <!-- -->
@@ Line 20: / Line 19: @@
 <partinfo>BBa_K2492004 SequenceAndFeatures</partinfo>
-[[File:T--SUSTech Shenzhen--cycle.gif|800px|thumb|left|GFP expressed in AWB| Control the C. elegans by the light: We used the optical fiber to form a light spot and the worm can follow the spot.]]
+Materials:
-[[File:T--SUSTech Shenzhen--getup.gif|800px|thumb|left|GFP expressed in AWB| 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).]]
+Odr10::CoChR::GEM-GECO::mCherry transgenic starving worms
+NGM plate with ATR
+Mercury lamp and optical fiber (provides blue light)
+Methods:
+.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.
+.Induce the Odr10::CoChR::GEM-GECO::mCherry successfully transgenic worms to draw a cycle on the NGM plate through blue light at 480nm.
+ Result:
+. Determine that transgenic worms are sensitive with which wavelength of light
+[[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
+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>
+<div class="row">
+<div class="col-md-1"></div>
+<div class="col-md-10">
+<video width="600px" controls="controls" autoplay="autoplay">
+  <source src="https://static.igem.org/mediawiki/2017/9/9f/T--SUSTech_Shenzhen--Microfuildics--tracking.mp4" type="video/ogg" />
+</video>
+<p ><B>Fig. 1 Control the <i>C. elegans</i> by the light.</B> We used the optical fiber to form a light spot and the worm can follow the spot.</p>
+</div>
+<div class="col-md-1"></div>
+</div>
+<div class="row">
+</html>
+Conclusion:
+In order to demonstrate this part, 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 the promoter worked well, the constructed plasmid would successfully expressed well in AWA.
+Since after shedding 480nm light, the transgenic worms were chasing the light, thus the light manipulated AWA successfully. The successful manipulation of AWA through blue light represented that the cell- specific promoter worked well. Since if the promoter not worked, none of the ligated proteins could be expressed.
+Hence, we successfully demonstrated that we successfully expressed this part and this part worked well.
@@ Line 28: / Line 130: @@
 <partinfo>BBa_K2492004 parameters</partinfo>
 <!-- -->
+<!-- Add more about the biology of this part here
+===Usage and Biology===