Difference between revisions of "Team:SUSTech Shenzhen/Results"

 
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= Optical Experiments Resutls =
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== Optical Experiments Results ==
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Under construction
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{|class="table table-striped"
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|-
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| Type of C. elegans
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| Fluorescence of mCherry / GFP
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| Fluorescence of GEM-GECO
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| CoChR work with GEM-GECO
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| CoChR work in behavioral Experiments
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|-
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| Odr10::CoChR::GEM-GECO::mCherry worms
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| <html><i class="ion-checkmark"></i></html> mCherry is very bright and beautiful(Fig.1 & 2)
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| <html><i class="ion-help"></i></html> weak fluorescence in 497~527nm. Weak change after add diacetyl (Fig. 3).
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| <html><i class="ion-load-a"></i></html> Still testing
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| <html><i class="ion-checkmark"></i></html> Successful.(See [https://2017.igem.org/Team:SUSTech_Shenzhen/Results#Behavioral_Experiments here])
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|-
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| Str1::Chrimson::GEM-GECO::GFP worms
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| <html><i class="ion-checkmark"></i></html> GFP were observed in AWB neurons (Fig. 4)
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| <html><i class="ion-help"></i></html>  weak fluorescence
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| <html><i class="ion-load-a"></i></html> Still testing
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| <html><i class="ion-loop"></i></html> Exist response, but need more experiment to confirm.
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|}
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* 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].
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{{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.}}
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{{SUSTech_Image_Center_8 | filename=T--SUSTech_Shenzhen--mCherry.jpg | caption=Fig. 2 Z project of confocal microscope sequences of mCherry in AWA.}}
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* Emission change at 497~527nm of GEM-GECO after adding diacetyl. It maybe was positive result, but need more control experiments to confirm it.
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{{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.}}
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* GFP expresses successfully at AWB in str1::Chrimson::GEM-GECO::GFP worm.
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{{SUSTech_Image_Center_8 | filename=T--SUSTech_Shenzhen--C.elegans_AWB_GFP.png
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|caption=Fig. 4 GFP expresses in AWB}}
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== Microfluidic Experiments ==
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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.
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{{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>}}
  
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| | First | Second |
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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>.
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= Microfluidic Experiments Resutls =
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{{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>}}
  
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Microfluidics were divide into three parts: the selective chip; the Gaussian chip; and the immobilization chip.
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<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>
  
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== Selective Chip ==
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== Behavioral Experiments ==
  
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The selective chip was designed to select the <i>Caenorhabditis elegans</i> with the appropriate size (Fig.1).
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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.
  
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{{SUSTech_Image_Center_8 | filename=T--SUSTech_Shenzhen--Microfuildics--selective.jpeg|width=600px|caption=<B>Fig.1 The Selective Chip</B>}}
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{{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>}}
  
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We found that the chip only had 12 fences (Fig.2), but our next experiments needed a large number of the <i>C. elegans</i> which were at the same stage, so the efficiency of the selective chip was very low. 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).
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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.
  
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{{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.}}
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{{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>}}
  
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Then, the <i>C. elegans'</i> synchronization was utilized to get a large number of worms at the same stage. 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 ((The number of the worms at L4)/(All the worms)×100%) could reach to about 80%.
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<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>
  
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{{SUSTech_Image_Center_8 | filename=T--SUSTech_Shenzhen--2.png|width=600px|caption=<B>Fig.3 The Embryos of The Worms</B>}}
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==Plasmid Construction Results==
  
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== Gaussian Chip ==
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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.
  
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The Gaussian chip (Fig.4) was designed to test if our exogenous genes will influence their olfactory receptor neuron pair (preference and repulsion to some chemical odors).
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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.
  
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{{SUSTech_Image_Center_8 | filename=T--SUSTech_Shenzhen--Microfuildics--gstd.png|width=600px|caption=<B>Fig.4 The Gaussian Chip</B>}}
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{{SUSTech_Image_Center_8 | filename=T--SUSTech_Shenzhen--Results-plasmid1.png|width=800px|caption=<B>Fig.5 Odr-10::CoCHR::GEM-geco::mCherry</B>}}
  
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{{SUSTech_Image_Center_8 | filename=T--SUSTech_Shenzhen--Results-plasmid2.png|width=800px|caption=<B>Fig.6 str-1::Chrimson::GEM-GECO::GFP</B>}}
  
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We got the worms’ distributions(Fig.5) after several experiments for the wildtype worms and our experimental worms with or without the chemicals (Fig.6).
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==Microinjection Results==
  
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{{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>}}
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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.
  
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{{SUSTech_Image_Center_8 | filename=T--SUSTech_Shenzhen--3.gif | caption=<B>Fig.6 The Experiment Process of the Gaussian Chip</B>}}
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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.
  
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== Immobilization Chip ==
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{{SUSTech_Image_Center_8 | filename=T--SUSTech_Shenzhen--Results-MI1.png|width=800px|caption=<B>Fig.7 heat shock</B>}}
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{{SUSTech_Image_Center_8 | filename=T--SUSTech_Shenzhen--Results-MI2.png|width=800px|caption=<B>Fig.8 heatshock under fluorenscence microscope</B>}}
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{{SUSTech_Image_Center_8 | filename=T--SUSTech_Shenzhen--Results-MI3.png|width=800px|caption=<B>Fig.9 CoChR under confocal microscope</B>}}
  
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The immobilization chip was deigned to immobilize the <i>C. elegans</i> in worm traps or parallel channels for worm imaging and ethological experiments.  
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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.
  
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{{SUSTech_Image_Center_8 | filename=T--SUSTech_Shenzhen--9.png|width=600px|caption=<B>Fig.7 The Immobilization Chip</B>}}
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{{SUSTech_Image_Center_8 | filename=T--SUSTech_Shenzhen--Results-MI4.png|width=800px|caption=<B>Fig.10 Chrimoson under confocal microscope</B>}}
  
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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). In addition, we could stimulate the Odr10::CoChR::GEM-GECO::mCherry worms to be active from the low state (Video.1). Unfortunately, we cannot see the clear neuro images in str1::Chrimson::GEM-GECO::GFP worms using fluorescence microscope but we can use confocal microscope to observe the neuro successfully.
 
  
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{{SUSTech_Image_Center_8 | filename=T--SUSTech_Shenzhen--Microfuildics--guding1.jpeg|width=600px|caption=<B>Fig.8 The Worms in Immobilization Chip.</B>}}
 
  
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{{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>}}
 
  
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= References =
 
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<references />
 
 
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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].

T--SUSTech Shenzhen--3dworm.gif
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.

T--SUSTech Shenzhen--mCherry.jpg
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.

T--SUSTech Shenzhen--GEM GECO.jpg
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.


T--SUSTech Shenzhen--Results-MI1.png
Fig.7 heat shock

T--SUSTech Shenzhen--Results-MI2.png
Fig.8 heatshock under fluorenscence microscope

T--SUSTech Shenzhen--Results-MI3.png
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.


T--SUSTech Shenzhen--Results-MI4.png
Fig.10 Chrimoson under confocal microscope



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  • ©2017 SUSTech_Shenzhen