Latest revision as of 03:50, 2 November 2017

Team SUSTC-Shenzhen

Results

Project

Optical Experiments Results

Type of C. elegans	Fluorescence of mCherry / GFP	Fluorescence of GEM-GECO	CoChR work with GEM-GECO	CoChR work in behavioral Experiments
Odr10::CoChR::GEM-GECO::mCherry worms	mCherry is very bright and beautiful(Fig.1 & 2)	weak fluorescence in 497~527nm. Weak change after add diacetyl (Fig. 3).	Still testing	Successful.(See here)
Str1::Chrimson::GEM-GECO::GFP worms	GFP were observed in AWB neurons (Fig. 4)	weak fluorescence	Still testing	Exist response, but need more experiment to confirm.

mCherry expresses successfully in odr10::CoChR::GEM-GECO::mCherry worm in AWA neurons. The 3D video was captured by [http://luxendo.eu/ Luxendo Light-Sheet Microscope].

Fig. 1 The 3D reconstruction of the odr-10::CoChR::GEM-GECO::mCherry worms' mCherry in AWA neurons. Here are two worm in video. Each worm have two light point, which are pair of AWA neurons.

Fig. 2 Z project of confocal microscope sequences of mCherry in AWA.

Emission change at 497~527nm of GEM-GECO after adding diacetyl. It maybe was positive result, but need more control experiments to confirm it.

Fig. 3 Emission change at 497~527nm of GEM-GECO in odr-10::CoChR::GEM-GECO::mCherry after adding diacetyl.

GFP expresses successfully at AWB in str1::Chrimson::GEM-GECO::GFP worm.

T--SUSTech Shenzhen--C.elegans AWB GFP.png

Fig. 4 GFP expresses in AWB

Microfluidic Experiments

Here, we fixed the Caenorhabditis elegans in the Immobilization Chip to observe the Odr10::CoChR::GEM-GECO::mCherry worms under the fluorescence microscope and saw the neuronal activity successfully, which can confirm that the worms can express our target genes. We also put the Odr10::CoChR::GEM-GECO::mCherry worms into the chip, after a few minutes the worms would be inactive, then we can "wake up" the worms by the blue light.

T--SUSTech Shenzhen--Microfuildics--result00.png

Fig.1 A. The Immobilization Chip. B. The worms in the Immobilization Chip.

Then, we demonstrated that the insertion did not damage the olfactory receptor neuron pairs of the worms by testing their response to diacetyl and 2-nonanone in the Gaussian Plate.

T--SUSTech Shenzhen--Microfuildics--result011.png

Fig.2 A. The Gaussian Plate. B. The worms in the Gaussian Plate.

See Details

Behavioral Experiments

Here, we confirmed that the Odr10::CoChR::GEM-GECO::mCherry worms could sense the blue light by inducing the Odr10::CoChR::GEM-GECO::mCherry worms to crawl a cycle on NGM plate. The Odr10::CoChR::GEM-GECO::mCherry worms could follow the blue light spot just like the attract of the food.

T--SUSTech Shenzhen--Microfuildics--result012.png

Fig.3 A. The device for light inducing exoeriment made by mercury lamp and optical fiber. B. The worm under the microscope when doing inducing experiment.

Then, in order to study the worms' learning ability we put the worms in alcohol layer on the NGM plate and stimulated them by the blue light at the same time. After 2 hours' training we found that the worms could crawl towards to the alcohol.

T--SUSTech Shenzhen--Microfuildics--result013.png

Fig.4 A.The device made by mercury lamp and microscope for training the worms.(More details in Hardware B. Using the alcohol to induce the worms.

See Details

Plasmid Construction Results

According to the design of plasmid construction, we constructed Odr-10::CoCHR::GEM-geco::mCherry and str-1::Chrimson::GEM-GECO::GFP fusion genes in backbone pCFJ909 successfully. The fusion gene segments were all be sequenced.

We also amplify B series plasmid in miniMos system for microinjection. We integrated Odr-10::CoChR::GEM-GECO::mCherry and str-1::Chrimson::GEM-GECO::GFP fusion genes into C. elegans(Caenorhabditis elegans) by microinjection respectively.

T--SUSTech Shenzhen--Results-plasmid1.png

Fig.5 Odr-10::CoCHR::GEM-geco::mCherry

T--SUSTech Shenzhen--Results-plasmid2.png

Fig.6 str-1::Chrimson::GEM-GECO::GFP

Microinjection Results

In order to get C. elegans strains with the preference to blue lights and the aversion to red lights, we used miniMos injection to inject our plasmids in to worms for expression.

On July.7, we microinjected 20 worms with Odr-10::CoChR::GEM-GECO::mCherry and 20 worms with Str-1::Chrimson::GEM-GECO::GFP. After 3 days, for each kinds of worms, we obtained more than 6 free-moving F1 with fluorescences. Then, 10 days after microinjection, we did heat shock to screen stable inheritance worms. For worms with Odr-10::CoChR::GEM-GECO::mCherry, one plate successfully survived more than 30 worms without GFP(a selective marker), but none of worms with Str-1::Chrimson::GEM-GECO::GFP survived. In addition, we did mapping experiments and demonstrated that Odr-10::CoChR::GEM-GECO::mCherry had successfully inserted in chromosome 1.

Fig.7 heat shock

Fig.8 heatshock under fluorenscence microscope

Fig.9 CoChR under confocal microscope

On August 1st, we microinject worms using Str-1::Chrimson::GEM-GECO::GFP. This time we injected 20 worms and also observed F1 phenotype after 3 days, picking up free-moving worms with RFP and did heat shock 9 days later. After heat shock, on August 21th, we got about 10 worms expressed plasmids without arrays, meaning that we obtained stable inheritance worms expressing Str-1::Chrimson::GEM-GECO::GFP.

Fig.10 Chrimoson under confocal microscope

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-== Optical Experiments Resutls ==
+== Optical Experiments Results ==
-Under construction
+{|class="table table-striped"
+|-
+| Type of C. elegans
+| Fluorescence of mCherry / GFP
+| Fluorescence of GEM-GECO
+| CoChR work with GEM-GECO
+| CoChR work in behavioral Experiments
+|-
+| Odr10::CoChR::GEM-GECO::mCherry worms
+| <html><i class="ion-checkmark"></i></html> mCherry is very bright and beautiful(Fig.1 & 2)
+| <html><i class="ion-help"></i></html> weak fluorescence in 497~527nm. Weak change after add diacetyl (Fig. 3).
+| <html><i class="ion-load-a"></i></html> Still testing
+| <html><i class="ion-checkmark"></i></html> Successful.(See [https://2017.igem.org/Team:SUSTech_Shenzhen/Results#Behavioral_Experiments here])
+|-
+| Str1::Chrimson::GEM-GECO::GFP worms
+| <html><i class="ion-checkmark"></i></html> GFP were observed in AWB neurons (Fig. 4)
+| <html><i class="ion-help"></i></html>  weak fluorescence
+| <html><i class="ion-load-a"></i></html> Still testing
+| <html><i class="ion-loop"></i></html> Exist response, but need more experiment to confirm.
+|}
-| | First | Second |
-|--|------|---------|
-| 3 | 43   | 4   |
-== Microfluidic Experiments Resutls ==
+* mCherry expresses successfully in odr10::CoChR::GEM-GECO::mCherry worm in AWA neurons. The 3D video was captured by [http://luxendo.eu/ Luxendo Light-Sheet Microscope].
+{{SUSTech_Image_Center_8 | filename=T--SUSTech_Shenzhen--3dworm.gif|width=800px|caption=Fig. 1 The 3D reconstruction of the odr-10::CoChR::GEM-GECO::mCherry worms' mCherry in AWA neurons. Here are two worm in video. Each worm have two light point, which are pair of AWA neurons.}}
+{{SUSTech_Image_Center_8 | filename=T--SUSTech_Shenzhen--mCherry.jpg | caption=Fig. 2 Z project of confocal microscope sequences of mCherry in AWA.}}
-Microfluidics were divide into three parts: the selective chip; the Gaussian chip; and the immobilization chip.
+* Emission change at 497~527nm of GEM-GECO after adding diacetyl. It maybe was positive result, but need more control experiments to confirm it.
+{{SUSTech_Image_Center_8 | filename=T--SUSTech_Shenzhen--GEM_GECO.jpg|caption=Fig. 3 Emission change at 497~527nm of GEM-GECO in odr-10::CoChR::GEM-GECO::mCherry after adding diacetyl.}}
-=== The Selection of the <i>Caenorhabditis elegans</i> ===
+* GFP expresses successfully at AWB in str1::Chrimson::GEM-GECO::GFP worm.
+{{SUSTech_Image_Center_8 | filename=T--SUSTech_Shenzhen--C.elegans_AWB_GFP.png
+|caption=Fig. 4 GFP expresses in AWB}}
-There are two plans of selecting worms. <html><a href="https://2017.igem.org/Team:SUSTech_Shenzhen/Hardware">Why we need the C. elegans with the same stage</a></html> The first one is using microfluidics. We designed the selective chip to select the <i>Caenorhabditis elegans (C. elegans)</i> with the appropriate size (Fig.1).
+== Microfluidic Experiments ==
-{{SUSTech_Image_Center_8 | filename=T--SUSTech_Shenzhen--Microfuildics--selective.jpeg|width=600px|caption=<B>Fig.1 The Selective Chip</B>}}
+Here, we fixed the <i>Caenorhabditis elegans</i> in <i>the Immobilization Chip</i> to observe the Odr10::CoChR::GEM-GECO::mCherry worms under the fluorescence microscope and saw the neuronal activity successfully, which can confirm that the worms can express our target genes. We also put the Odr10::CoChR::GEM-GECO::mCherry worms into the chip, after a few minutes the worms would be inactive, then we can "wake up" the worms by the blue light.
-We need a large number of the worms with the same stage to do the Gaussion chip. <html><a href="https://2017.igem.org/Team:SUSTech_Shenzhen/Model3">Why we need so many with the same stage</a></html> However, we found that the chip only has 12 fences (Fig.2) The efficiency of the selective chip was very low because of such a small number of the fences. In addition, the <i>C. elegans</i> have flexible body, some of the suitable size worms would still go through the second fences (Fig.2).
+{{SUSTech_Image_Center_fill-width |  filename=T--SUSTech_Shenzhen--Microfuildics--result00.png|width=800px|caption=<B>Fig.1 A. <i>The Immobilization Chip</i>. B. The worms in <i>the Immobilization Chip</i>.</B>}}
-{{SUSTech_Image_Center_8 | filename=T--SUSTech_Shenzhen--Microfuildics--selective1.png|width=600px|caption=<B>Fig.2 The Problems of the Selective chip.</B> The efficiency of the chip was very low because of the small amount of the fences. Besides, the worms will also escape from the second fences just like the red one.}}
+Then, we demonstrated that the insertion did not damage the olfactory receptor neuron pairs of the worms by testing their response to diacetyl and 2-nonanone in <i>the Gaussian Plate</i>.
-The second plan was the <i>C. elegans’</i> synchronization.(how to do the <i>C. elegans’</i> synchronization 链接到 protocl) We got the embryos (Fig.3) from the old worms so that the worms would be at the same stage because of the hatches of the embryos were at the same time. We selected several conditions of the synchronization, finally, we could get the worms at the same stage. The synchronous rate ({{SUSTech_Shenzhen/math|equ=<nowiki>\frac{the\, number\, of\, the\, worms\, at\, L4}{the\, number\, of\, all\, worms}</nowiki>}}*100%) could reach to about 80%.
+{{SUSTech_Image_Center_fill-width | filename=T--SUSTech_Shenzhen--Microfuildics--result011.png|width=800px|caption=<B>Fig.2 A. <i>The Gaussian Plate</i>. B. The worms in <i>the Gaussian Plate</i>.</B>}}
-{{SUSTech_Image_Center_8 | filename=T--SUSTech_Shenzhen--2.png|width=600px|caption=<B>Fig.3 The Embryos of The Worms</B>}}
+<html><a target="_blank" href="https://2017.igem.org/Team:SUSTech_Shenzhen/Results/Microfluidic" class="btn btn-default"><i class="ion-arrow-right-c"></i> See Details</a></html>
-=== Gaussian Chip ===
+== Behavioral Experiments ==
-The Gaussian chip (Fig.4) was designed to test if our exogenous genes would influence their olfactory receptor neuron pair (preference and repulsion to some chemical odors).
+Here, we confirmed that the Odr10::CoChR::GEM-GECO::mCherry worms could sense the blue light by inducing the Odr10::CoChR::GEM-GECO::mCherry worms to crawl a cycle on NGM plate. The Odr10::CoChR::GEM-GECO::mCherry worms could follow the blue light spot just like the attract of the food.
-{{SUSTech_Image_Center_8 | filename=T--SUSTech_Shenzhen--Microfuildics--gstd.png|width=600px|caption=<B>Fig.4 The Gaussian Chip</B>}}
+{{SUSTech_Image_Center_fill-width | filename=T--SUSTech_Shenzhen--Microfuildics--result012.png|width=800px|caption=<B>Fig.3 A. The device for light inducing exoeriment made by mercury lamp and optical fiber. B. The worm under the microscope when doing inducing experiment.</B>}}
+Then, in order to study the worms' learning ability we put the worms in alcohol layer on the NGM plate and stimulated them by the blue light at the same time. After 2 hours' training we found that the worms could crawl towards to the alcohol.
-We got the worms’ distributions(Fig.5) after several experiments for the wild type worms and our experimental worms with or without the chemicals (Fig.6).
+{{SUSTech_Image_Center_fill-width | filename=T--SUSTech_Shenzhen--Microfuildics--result013.png|width=800px|caption=<B>Fig.4 A.The device made by mercury lamp and microscope for training the worms.(More details in [https://2017.igem.org/Team:SUSTech_Shenzhen/Hardware#Light_Modulator Hardware] B. Using the alcohol to induce the worms.</B>}}
-{{SUSTech_Image_Center_8 | filename=T--SUSTech_Shenzhen--Microfuildics--gsdata.png|width=600px|caption=<B>Fig.5 The distribution of the worms. A) The distribution of the worms without diacetyl. B) The distribution of the worms with diacetyl in channel A.</B>}}
+<html><a target="_blank" href="https://2017.igem.org/Team:SUSTech_Shenzhen/Results/Behavior" class="btn btn-default"><i class="ion-arrow-right-c"></i> See Details</a></html>
-{{SUSTech_Image_Center_8 | filename=T--SUSTech_Shenzhen--3.gif | caption=<B>Fig.6 The Experiment Process of the Gaussian Chip</B>}}
+==Plasmid Construction Results==
-The final result were not such a good Gaussian distribution like the Galton board because the <i>C. elegans'</i> choices were not absolutely normal. In order to adjust our results we built a model. https://2017.igem.org/Team:SUSTech_Shenzhen/Model3
+According to the design of plasmid construction, we constructed Odr-10::CoCHR::GEM-geco::mCherry and str-1::Chrimson::GEM-GECO::GFP fusion genes in backbone pCFJ909 successfully. The fusion gene segments were all be sequenced.
-=== Immobilization Chip ===
+We also amplify B series plasmid in miniMos system for microinjection.
+We integrated Odr-10::CoChR::GEM-GECO::mCherry and str-1::Chrimson::GEM-GECO::GFP fusion genes into <i>C. elegans(Caenorhabditis elegans)</i> by microinjection respectively.
-The immobilization chip was deigned to immobilize the <i>C. elegans</i> in worm traps or parallel channels for worm imaging and ethological experiments.
+{{SUSTech_Image_Center_8 | filename=T--SUSTech_Shenzhen--Results-plasmid1.png|width=800px|caption=<B>Fig.5 Odr-10::CoCHR::GEM-geco::mCherry</B>}}
-{{SUSTech_Image_Center_8 | filename=T--SUSTech_Shenzhen--Microfluidics--fig8.png|width=600px|caption=<B>Fig.7 The Immobilization Chip</B>}}
+{{SUSTech_Image_Center_8 | filename=T--SUSTech_Shenzhen--Results-plasmid2.png|width=800px|caption=<B>Fig.6 str-1::Chrimson::GEM-GECO::GFP</B>}}
-We could immobilize the worms in the worm traps (Fig.8) and watch the neuronal activity (Fig.9) successfully using fluorescence microscope (Nikon eclipse Ti).
+==Microinjection Results==
-{{SUSTech_Image_Center_8 | filename=T--SUSTech_Shenzhen--Microfuildics--guding1.jpeg|width=600px|caption=<B>Fig.8 The Worms in Immobilization Chip.</B>}}
+In order to get <i>C. elegans</i> strains with the preference to blue lights and the aversion to red lights, we used miniMos injection to inject our plasmids in to worms for expression.
-{{SUSTech_Image_Center_8 | filename=T--SUSTech_Shenzhen--Microfuildics--mcherry.png|width=600px|caption=<B>Fig.9 The mCherry of the AWA Neuro in the Odr10::CoChR::GEM-GECO::mCherry Worm.</B>}}
+On July.7, we microinjected 20 worms with Odr-10::CoChR::GEM-GECO::mCherry and 20 worms with Str-1::Chrimson::GEM-GECO::GFP. After 3 days, for each kinds of worms, we obtained more than 6 free-moving F1 with fluorescences. Then, 10 days after microinjection, we did heat shock to screen stable inheritance worms. For worms with Odr-10::CoChR::GEM-GECO::mCherry, one plate successfully survived more than 30 worms without GFP(a selective marker), but none of worms with Str-1::Chrimson::GEM-GECO::GFP survived. In addition, we did mapping experiments and demonstrated that Odr-10::CoChR::GEM-GECO::mCherry had successfully inserted in chromosome 1.
-In addition, we could stimulate the Odr10::CoChR::GEM-GECO::mCherry worms to be active from the low state (Fig.10). On the other hand, we could also get the excitation wavelength of CoChR. The result showed that the light from the projector without filter (OD8) and the lights with 395 and 440 wavelengths from the LED of fluorescence microscope (Nikon eclipse Ti) could influence the C. elegans the other lights cannot. The lights with 395 and 440 wavelengths are closed to the ultraviolet which would hurt the C. elegans, so we got that the lights from the projector without the filter could active the CoChR. (参数啥的连接到光学) Unfortunately, we cannot see the clear neuro activity in str1::Chrimson::GEM-GECO::GFP worms using fluorescence microscope but we can use confocal microscope to observe the neuronal successfully.
+{{SUSTech_Image_Center_8 | filename=T--SUSTech_Shenzhen--Results-MI1.png|width=800px|caption=<B>Fig.7 heat shock</B>}}
+{{SUSTech_Image_Center_8 | filename=T--SUSTech_Shenzhen--Results-MI2.png|width=800px|caption=<B>Fig.8 heatshock under fluorenscence microscope</B>}}
+{{SUSTech_Image_Center_8 | filename=T--SUSTech_Shenzhen--Results-MI3.png|width=800px|caption=<B>Fig.9 CoChR under confocal microscope</B>}}
-{{SUSTech_Image_Center_8 | filename=T--SUSTech_Shenzhen--Microfuildics--wake up.gif|width=600px|caption=<B>Fig.10 The test of the CoChR using light with different wavelengths and intensity. </B>The lights <i>blue-</i> and <i>blue+</i> 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).}}
+On August 1st, we microinject worms using Str-1::Chrimson::GEM-GECO::GFP. This time we injected 20 worms and also observed F1 phenotype after 3 days, picking up free-moving worms with RFP and did heat shock 9 days later. After heat shock, on August 21th, we got about 10 worms expressed plasmids without arrays, meaning that we obtained stable inheritance worms expressing Str-1::Chrimson::GEM-GECO::GFP.
-== Ethological Experiment Results ==
+{{SUSTech_Image_Center_8 | filename=T--SUSTech_Shenzhen--Results-MI4.png|width=800px|caption=<B>Fig.10 Chrimoson under confocal microscope</B>}}
-For the Odr10::CoChR::GEM-GECO::mCherry worms and str1::Chrimson::GEM-GECO::GFP worms we induced them to do some specific behaviors by the blue and red lights on the NGM plate. We could induce the Odr10::CoChR::GEM-GECO::mCherry worm to crawl a circle on the NGM plate by the blue light (Fig.11).
-{{SUSTech_Image_Center_8 | filename=T--SUSTech_Shenzhen--Microfuildics--cycle.gif|width=600px|caption=<B>Fig.11 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.}}
-Then, the Odr10::CoChR::GEM-GECO::mCherry worms were trained by the alcohol. We added the alcohol with different concentration on the NGM plate to form a liquid film and we stimulated the C. elegans in the plate at the same time. After 2 hours we washed the plate to recover the worms in M9 buffer and put the mixture on one side of a new plate. Finally, we put the alcohol on the other side to see if the worms have the tropism of the alcohol.
-Then we observed that some of the Odr10::CoChR::GEM-GECO::mCherry worms could swim to the alcohol but the wild type worms could not sense the alcohol (Fig.12). We could get that the Odr10::CoChR::GEM-GECO::mCherry worms (experimental worms) after training had the tropism of the alcohol (Fig.12 A).
-{{SUSTech_Image_Center_fill-width | filename=T--SUSTech_Shenzhen--Microfuildics--alcohol.png|width=600px|caption=<B>Fig.12 The results of the alcohol training.</B> A) The alcohol induction of the experimental worms after training. B) The alcohol induction of the wild type worms after training. C) The water induction of the experimental worms after training. D) The alcohol induction of the expermental worms without training. E) The worms after 8 hours of A). F) The worms under the microscope of E).}}
-We did 3 groups of the control experiments, we could prove that the experimental worms without training (Fig.12 B), the wild type worms after training (Fig.12 C) could not sense the alcohol and the experimental worms after training could not sense the water (Fig.12 D). Furthermore, we did the starvation treatment to our worms before the experiments, because of the hunger the worms would huddle together, however our experimental worms swam to the alcohol (Fig.12 A)) which means that they sensed something they prefer at that time. In our experiments, we could know that our training really made the experimental worms fill in love with the alcohol. After several hours they would still huddle together because the disappearance of alcohol order (Fig.12 E, F). In our experiments, we could know that our training really made the experimental worms fill in love with the alcohol.
-= References =
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