Team:SUSTech Shenzhen/Results/Microfluidic

Team SUSTC-Shenzhen

Results

Project


Microfluidics were divide into three parts: the selective chip; the Gaussian chip; and the immobilization chip.

The Selection of the Caenorhabditis elegans

There are two plans of selecting worms. Why we need the C. elegans with the same stage The first one is using microfluidics. We designed the selective chip to select the Caenorhabditis elegans (C. elegans) with the appropriate size (Fig.1).


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Fig.1 The Selective Chip

We need a large number of the worms with the same stage to do the Gaussion chip. Why we need so many with the same stage 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 C. elegans have flexible body, some of the suitable size worms would still go through the second fences (Fig.2).


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Fig.2 The Problems of the Selective chip. 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.

The second plan was the C. elegans’ synchronization.[1](how to do the C. elegans’ 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 (\frac{the\, number\, of\, the\, worms\, at\, L4}{the\, number\, of\, all\, worms}*100%) could reach to about 80%.


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Fig.3 The Embryos of The Worms

Gaussian Chip

The Gaussian chip (Fig.4)[2] was designed to test if our exogenous genes would influence their olfactory receptor neuron pair (preference and repulsion to some chemical odors).


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Fig.4 The Gaussian Chip


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).


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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.


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Fig.6 The Experiment Process of the Gaussian Chip

The final result were not such a good Gaussian distribution like the Galton board because the C. elegans' choices were not absolutely normal. In order to adjust our results we built a model. https://2017.igem.org/Team:SUSTech_Shenzhen/Model3

Immobilization Chip

The immobilization chip was deigned to immobilize the C. elegans in worm traps or parallel channels for worm imaging and ethological experiments.[3]


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Fig.7 The Immobilization Chip

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).


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Fig.8 The Worms in Immobilization Chip.


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Fig.9 The mCherry of the AWA Neuro in the Odr10::CoChR::GEM-GECO::mCherry Worm.

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.


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Fig.10 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).


References

  1. Portadelariva, M., Fontrodona, L., Villanueva, A., & Cerón, J. (2012). Basic caenorhabditis elegans methods: synchronization and observation. Journal of Visualized Experiments Jove, 64(64), e4019.
  2. Albrecht, D. R. and C. I. Bargmann (2011). “High-content behavioral analysis of Caenorhabditis elegans in precise spatiotemporal chemical environments.” Nature Methods 8(7): 599-605.
  3. San-Miguel, A., & Lu, H. (2013). Microfluidics as a tool for c. elegans research. Wormbook the Online Review of C Elegans Biology, 1.

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