Revision as of 03:12, 2 November 2017

Team SUSTC-Shenzhen

Demonstrate

Project

In our project, we want to study the neural network activity and behavioral response of Caenorhabditis elegans with orthogonal optogenetic stimulation of two neurons.

So far, we have achieved almost all of our project objectives, building a platform to study the learning behavior of Caenorhabditis elegans. The whole project is demonstrated in the following parts:

Choosing the suitable excitation wavelength of light-sensitive proteins
Genetic integration of synthetic circuits into C.elegans strains
Examining the functions of AWA and AWB neurons
Using Microfluidic device to examine the behavioral change of individual worms
Training C.elegans to be addicted to alcohol

Choosing the suitable excitation wavelength

Expression of two types of channelrhodopsins in two pairs of receptor neurons enables worm to be attracted by blue light and be repelled by red light. At the same time, we plan to use GEM-GECO as calcium indicator to examining the neuron activation, and perform feedback control in the future.

To avoid potential crosstalk, we need to separate the excitation wavelengths of CoChR activation and GEM-GECO imaging. In our project, we chose 395nm and 470nm to activate CoChR and GEM-GECO.

Before the experiment in worms, we conducted a validation experiment on CHO-K1 cell line expressing CoChR to identify a suitable excitation wavelength for the light-sensitive ion channel.

[[File: |6000px|frameless]]

Fig.1 Results of experiment for CoChR spectrum determination. In the experiment ,we expressed CoChR in the CHO-K1 cell line and R-GECO is used to detect the calcium influx into the cell. A) This figure shows that R-GECO in cells are activated after the 200mM KCl inducing. We take this group as the positive control group since we consider that the indicators were full-activated at this state. This is because 200mM KCl can cause plenty of calcium influx into the cell through calcium channel in cells membrane. B) This figure shows the light induced R-GECO activation, the light can cause the conformational change of CoChR, then cause calcium influx into the cell. The sharp peak on this curve proves that the light inducing is much more sensitive than the chemical signal.

Based on the result, we determine to use 470nm to activate the CoChR and 395nm for the excitation of GEM-GECO[There is no light activation data of CoCHR in figure 1!!!].

Genetic integration of synthetic circuits into C.elegans strains

We used the miniMos system to integrate the gene circuits into C.elegans genome. After injection and selection (Fig.2), we successfully obtained the inserted worms (Odr-10::CoChR::GEM-GECO::mCherry and str-1::Chrimson::GEM-GECO::GFP) .

[[File: |6000px|frameless]]

Fig.2 F1 Negative selection. peel-1 is a negative selection marker used to select against animals carrying extra- chromosomal arrays and assist with identifying animals that have endogenous locus insertions. This marker was co-injected with the target gene along with the heatshock promoter, after heatshock the worms with multiple extra- chromosomal arrays will all die (A) while the worms with endogenous locus insertion will survive (B). After 34°C,4h heatshock, these alive worms will be picked out for fluorescence checking.

[[File: |6000px|frameless]]

Fig.3 Confocal Image of Odr-10::CoCHR::GEM-GECO::mCherry worm’s head Through the red fluorescence of mCherry, we could determine the location of AWA neuron <B>(A). Compared to the 3D neuron model (B), this result demonstrates that our circuit was successfully inserted and expressed in the C.elegans.</B>

[[File: |6000px|frameless]]

Fig.4 Confocal Image of str-1::Chrimson::GEM-GECO::GFP worm’s head Through the green fluorescence of GFP, we could determine the location of AWB neuron (A). Compared to the 3D neuron model (B), this result demonstrates that our

[[File: |6000px|frameless]]

Fig 5. The mapping results of circuit Odr-10::CoCHR::GEM-GECO::mCherry after BLAST on wormbase.org. The black line represents location of the gene we insert. The comparison result showed that our gene inserted in the chromosome I and started at 14851599.

After getting the genetically inserted worms, we successfully got the hybrid by mating 2 strains.

Examining the function of AWA and AWB neurons

Firstly, the experiment in which inserted worms are strongly attracted by diacetyl shows that the insertion did not degrade normal neuron function (Fig.6)

We also test the result in the neuron level (Fig.7)

Fig. 6 C.elegans' locomotion in the Gaussian plate C.elegans can move forward freely in the channel, and then we will get its locomotion distribution just like the Galton Nail Plate model. However, after adding diacetyl in the side channel, the distribution is changed. C.elegans tend to move towards the right (or the left) due to their preference to diacetyl.

[[File: |6000px|frameless]]

Fig.7 Demonstrate the function of AWA neuron A) Confocal Image of worm’s head neuron cells of AWA neurons are circled at the photo (yellow) B) The fluorescence intensity of 497-527nm emission of GEM-GECO falls steeply after applying diacetyl.

Examining the behavioral change of individual worms

We designed two methods to observe inserted worms’ behavior change:

Inducing free-moving worms by certain wavelength (Fig.7)
Stimulating worms semi-fixed in microfluidic chips (Fig.8)

In the inducing experiments, Odr-10::CoCHR::GEM-GECO::mCherry worms shows a strong preference to blue light. In the later experiment, we will use this preference to train them to form specific behavior.

Unfortunately, str-1::Chrimson::GEM-GECO::GFP worms are not clearly repelled by the red light. The low sensitivity is probably due to the expression level of Chrimson under the control of str-1 promoter. An modified circuit containing amplification step is design, but the implementation cannot meet the deadline of this wiki.[The protein itself is expressed, demonstrated by the fluorescent protein expression ]

Fig. 7 Behavior experiments of C.elegans. These two figures show the worm have obvious response to the blue light which could activate channelrhodopsin CoChR. the first video shows the worm which expressed Odr-10::CoChR::GEM-GECO::mCherry circuit could run in circle to follow the blue light. The other figure shows the same type worm which looks “asleep” ( because of no food supply) be “waked up” also by blue light(preference).

T--SUSTech Shenzhen--Microfuildics--wake up.gif

Fig8. Observe individual worms in microfluidic chip. This PDMS channel is designed to fix worms and observe them under stereoscope. 4 channels with decreasing diameter is used to fix worm. Other 4 narrow channels allow worms to move in a line constraint.

Alcohol addiction

We trained the worms engineered with Odr-10::CoChR::GEM-GECO::mCherry to be “addicted to” alcohol successfully.

In this experiment, we added the alcohol on the NGM plate containing engineered worms. At the same time, blue light is turned on to stimulate worms continually.
After training for 2 hours, we washed the plate and recovered the worms in M9 buffer.
Then, we put the mixture contained worms and M9 buffer on one side of a new plate. After recovery of the worms, we added the alcohol on the other side of the same plate to see if the worms have the tropism to the alcohol.

The result shows that most of the worms transfected with Odr-10::CoChR::GEM-GECO::mCherry moved to the alcohol while the wild types could not (Fig.9).

What’s more, without the previous training by light, the worms transfected with Odr-10::CoChR::GEM-GECO::mCherry show no preference to alcohol, neither. (Fig.10).

6000px

Fig. 9 A.) The behaviors of Odr-10::CoChR::GEM-GECO::mCherry worms (all stages) after 2 hours' training. We circle the area of worms after training with blue light in red. B) The same worms in figure A would crawl towards alcohol inducing. The yellow circle area represent the alcohol. C) The wild type worms control. D) The wild types would not crawl towards the alcohol even thought training with blue light. Fig. 10 E) The buffer have no addition to worms transfected with Odr-10::CoChR::GEM-GECO::mCherry circuit. F) shows no preference to alcohol

This experiment showed that our genetically modified worms can learn new behavior(addiction to alcohol) after trained by blue light.

Future Plan

Our further work is to decipher the change of neuron connectivity due to the newly learned behavior.

We have selected 2 promoters(cho-1 and unc-8) to express optogenetic circuits on the neurons connected to AWA and AWB, such as AIA, AIY, VB, ASH, AVB, VB, AVA.See details The next step will be using optofluidics platform above to analyze the activation of downstream neurons in the new-formed or modulated neural circuit, with or without the aforementioned training.

@@ Line 7: / Line 7: @@
 {{:Team:SUSTech_Shenzhen/main-content-begin}}
 <!--------往下直接写内容----------------->
-==Overview ==
-So far, our project has been almost completed. The whole project is demonstrated in the following
-parts:
-.Optogenetic results of spectrum determination
+In our project, we want to study the neural network activity and behavioral response of Caenorhabditis elegans with orthogonal optogenetic stimulation of two neurons.
-.Microinjection results
+So far, we have achieved almost all of our project objectives, building a platform to study the learning behavior of Caenorhabditis elegans. The whole project is demonstrated in the following parts:
+#Choosing the suitable excitation wavelength of light-sensitive proteins
+#Genetic integration of synthetic circuits  into C.elegans strains
+#Examining the functions of AWA and AWB neurons
+#Using Microfluidic device to examine the behavioral change of individual worms
+#Training C.elegans to be addicted to alcohol
-.Neuron activation
+------
-.Behavior experiment (including single worm and population)
+==Choosing the suitable excitation wavelength ==
-.Addiction inducing results
+Expression of two types of channelrhodopsins in two pairs of receptor neurons enables worm to be attracted by blue light and be repelled by red light. At the same time, we plan to use GEM-GECO as calcium indicator to examining the neuron activation, and perform feedback control in the future.
-------
-==Spectrum determination==
+To avoid potential crosstalk, we need to separate the excitation wavelengths of CoChR activation and GEM-GECO imaging. In our project, we chose 395nm and 470nm to activate CoChR and GEM-GECO.
-The channelrhodopsins we chose in our project were evolved from the algae, so if we applied these light sensitive channel to the <i>Caenorhabditis elegans</i> as the receptors, we need to check its optical parameters before our behavior experiment. The spectrum of Chrimson and CoCHR have been already measured by other scientists. With the spectrums of two channelrhodopsins, we need to figure out the another two questions :crosstalk and indicator spectrum.
+<html><!-----------------------------图？------------------------------></html>
-The first question is that whether the channelrhodopsin CoCHR will have the wavelength crosstalk with the indicator protein—GEM-GECO , the excitation wavelength of CoCHR is crossed with the emission wavelength of activated GEM-GECO combined with calcium, which means that we can not activate CoCHR and receive GEM-GECO emission fluorescence in the same time with same wavelengths because of the filter. So we have to do the experiment based on the cell to select another suitable excitation wavelength for CoCHR.
+Before the experiment in worms, we conducted a validation experiment on CHO-K1 cell line  expressing CoChR to identify a suitable excitation wavelength for the light-sensitive ion channel.
-{{SUSTech_Image_Center_fill-width| filename=T--SUSTech_Shenzhen--demonstrate-1.png|width=6000px|caption=<B>Fig.1 The experiment result have been showed in figure 1a and 1b. In this experiment ,we have expressed CoCHR combined with another indicator R-GECO to avoid the crosstalk. The figure 1a showed that the indicator have been all activated because the KCl had induced plenty of calcium influx inside cells through calcium channel. Compared with the full-state ,we choose the relatively high excitation wavelength 470nm which can activate almost 60% of the indicator protein. Also the sharp and acute peak in this figure proves that the light inducing is much more sensitive than the chemical signal.</B>}}
+{{SUSTech_Image_Center_fill-width | filename=|width=6000px|caption=<B> Fig.1 Results of experiment for CoChR spectrum determination. In the experiment ,we expressed CoChR in the CHO-K1 cell line and R-GECO is used to detect the calcium influx into the cell. A) This figure shows that R-GECO in cells are activated after the 200mM KCl inducing. We take this group as the positive control group since we consider that the indicators were full-activated at this state. This is because 200mM KCl can cause plenty of calcium influx into the cell through calcium channel in cells membrane. B) This figure shows the light induced R-GECO activation, the light can cause the conformational change of CoChR, then cause calcium influx into the cell. The sharp peak on this curve proves that the light inducing is much more sensitive than the chemical signal. </B>}}
-{{SUSTech_Image_Center_8 | filename=T--SUSTech_Shenzhen--demonstrate-1.c.png|width=6000px|caption=<B>Fig 1-c</B>}}
+Based on the result, we determine to use 470nm to activate the CoChR and 395nm for the excitation of GEM-GECO[There is no light activation data of CoCHR in figure 1!!!].
-The excitation and emission wavelength are the most important parameters for all kinds of optical protein, so if we want to know what happened inside the worms’ neuron after light induing, the spectrum of indicators expressed in the <i>C.elegans</i> must be figured out. So we use the sensitive detector to receive  the emission wavelength from the <i>C.elegans</i> AWA neuron after activated by diacetyl inducing. Then we analyze the emission data and select the best two wavelength area for the indicator emission wavelength with and without calcium existing. This part will be shown in the neuron experiment.
+----------------
-----------
+==Genetic integration of synthetic circuits into C.elegans strains==
-==System construction in <i>C.elegans</i>==
-In order to select the worms stable transacted with the system we have constructed, we design a series selection experiments for verification. These figures showed that the result of the selection process using the fluorescence marker selection ,the rescue selection and the negative poison selection. We then do the mapping for the genome of the worms to check the insertion of the system. Finally we use confocal microscope to check the location marker to test the expression of the whole system.
+We used the miniMos system to integrate the gene circuits into C.elegans genome. After injection and selection (Fig.2), we successfully obtained  the inserted worms (Odr-10::CoChR::GEM-GECO::mCherry and str-1::Chrimson::GEM-GECO::GFP) .
-{{SUSTech_Image_Center_fill-width | filename=T--SUSTech_Shenzhen--demonstrate-3-change.png|width=6000px|caption=<B>Fig.2 F1 Negative selection, a poisonous gene which were co-injected with the target gene along with the heatshock promoter, after heatshock the worms with multiple arrays will all died. After 34°C,4h heatshock, these stable transfected worms which were survived with freemoving will be picked out for fluorescence checking.</B>}}
+{{SUSTech_Image_Center_fill-width | filename=|width=6000px|caption=<B>Fig.2  F1 Negative selection.</B> peel-1 is a negative selection marker used to select against animals carrying extra- chromosomal arrays and assist with identifying animals that have endogenous locus insertions. This marker was co-injected with the target gene along with the heatshock promoter, after heatshock the worms with multiple extra- chromosomal arrays will all die <B>(A)</B> while the worms with endogenous locus insertion will survive <B>(B)</B>. After 34°C,4h heatshock, these alive worms will be picked out for fluorescence checking.}}
-{{SUSTech_Image_Center_fill-width | filename=T--SUSTech_Shenzhen--demonstrate-4-change.png|width=6000px|caption=<B>Fig.3 Confocal Image for Odr-10::CoCHR::GEM-GECO::mCherry, mCherry is the location marker of AWA neuron (compared with picture from database). This figure can show our circuit both insertion and expression in the <i>C.elegans</i> we selected.</B>}}
+{{SUSTech_Image_Center_fill-width | filename=|width=6000px|caption=<B>Fig.3 Confocal Image of Odr-10::CoCHR::GEM-GECO::mCherry worm’s head Through the red fluorescence of mCherry, we could determine the location of AWA neuron <B>(A)</B>. Compared to the 3D neuron model <B>(B)</B>, this result demonstrates that our circuit was successfully inserted and expressed in the C.elegans.</B>}}
-{{SUSTech_Image_Center_fill-width | filename=T--SUSTech_Shenzhen--demonstrate-8-change.png|width=6000px|caption=<B>Fig.4 The left figure is fluorescence image for Str-1::Crimson::GEM-GECO::GFP in AWB neuron. Another figure is TM4063 (wild type) originally with body fluorescence, so the fluorescence noise on above noise should be subtracted.</B>}}
+{{SUSTech_Image_Center_fill-width | filename=|width=6000px|caption=<B>Fig.4 Confocal Image of str-1::Chrimson::GEM-GECO::GFP worm’s head Through the green fluorescence of GFP, we could determine the location of AWB neuron (A). Compared to the 3D neuron model (B), this result demonstrates that our </B>}}
-----------
-==Activation of <i>C.elegans</i> head neurons==
-Since the whole system has expressed in the <i>C.elegans</i>, we need to check the function of each part in our project. So we have done many experiments on the head neuron of the worms, we first check the function of the GEM-GECO.
+{{SUSTech_Image_Center_fill-width | filename=|width=6000px|caption=<B>Fig 5. The mapping results of circuit Odr-10::CoCHR::GEM-GECO::mCherry after BLAST on wormbase.org. The black line represents location of the gene we insert. The comparison result showed that our gene inserted in the chromosome I and started at 14851599. </B>}}
-These figures showed the position and the reaction of AWA neuron which expressed GEM-GECO after diacetyl inducing.
+After getting the genetically inserted worms, we successfully got the hybrid by mating 2 strains.
-==Microfluidics ==
+==Examining the function of AWA and AWB neurons==
-In order to study locomotion of <i>C.elegans</i> populations, we design the Gaussian Chip, a pillar-filled area, where the pillars were designed such that they allow crawling-like behaviors of <i>C.elegans</i>  even though worms are immersed in liquid environment.
+Firstly, the experiment in which inserted worms are strongly attracted by diacetyl shows that the insertion did not degrade normal neuron function (Fig.6)
-{{SUSTech_Image_Center_8 | filename=T--SUSTech_Shenzhen--3.gif|width=6000px|caption=<B>Fig. 5 Gaussian plate. C.elegans can move award freely in our channel without any difficulties, and then we would get a similar Gaussian distribution ideally. Next, after we injected a kind of chemical (diacetyl or 2- nonanone) in the right (or left) lateral channel, the distribution of worms will be changed, in other words, the peak of this distribution will move towards the right (or the left)</B>}}
+We also test the result in the neuron level (Fig.7)
-{{SUSTech_Image_Center_8 | filename=T--SUSTech_Shenzhen--demonstrate-5.png|width=6000px|caption=<B>Fig. 6 Immobilized channel. In order to observe a neuronal response in an individual worm, we design four subuliform channels to fix its head in case of the influence of worms’ drastic movement. In addition, we also want to research whether <i>C.elegans</i> will prefer blue light (like diacetyl), and repulse red light (like 2-nonanone). Therefore, we design another four narrow channels that <i>C.elegans</i> can just move forwards and back.</B>}}
+{{SUSTech_Image_Center_8 | filename=T--SUSTech_Shenzhen--3.gif|width=6000px|caption=<B>Fig. 6 C.elegans' locomotion in the Gaussian plate</B> C.elegans can move forward freely in the channel, and then we will get its locomotion distribution just like the Galton Nail Plate model. However, after adding diacetyl in the side channel, the distribution is changed. C.elegans tend to move towards the right (or the left) due to their preference to diacetyl.}}
---------
-==Response to light (Individual)==
-To verify the response to specific wavelength light inducing, we picked out a single worm to observe the behavior change.
+{{SUSTech_Image_Center_fill-width | filename=|width=6000px|caption=<B>Fig.7 Demonstrate the function of AWA neuron  A) Confocal Image of  worm’s head</B> neuron cells of AWA neurons are circled at the photo (yellow) <B>B) The fluorescence intensity of 497-527nm emission of GEM-GECO falls steeply after applying diacetyl.</B>}}
+==Examining the behavioral change of individual worms ==
+We designed two methods to observe inserted worms’ behavior change:
+#Inducing free-moving worms by certain wavelength (Fig.7)
+#Stimulating worms semi-fixed in microfluidic chips (Fig.8)
+In the inducing experiments, Odr-10::CoCHR::GEM-GECO::mCherry worms shows a strong preference to blue light. In the later experiment, we will use this preference to train them to form specific behavior.
+Unfortunately, str-1::Chrimson::GEM-GECO::GFP worms are not clearly repelled by the red light. The low sensitivity is probably due to the expression level of Chrimson under the control of str-1 promoter. An modified circuit containing amplification step is design, but the implementation cannot meet the deadline of this wiki.[The protein itself is expressed, demonstrated by the fluorescent protein expression ]
 <html>
@@ Line 70: / Line 77: @@
    <source src="https://static.igem.org/mediawiki/2017/9/9f/T--SUSTech_Shenzhen--Microfuildics--tracking.mp4" type="video/ogg" />
 </video>
-<p ><B>Fig.7 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>
+<p ><B>Fig. 7  Behavior experiments of C.elegans.</B> These two figures show the worm have obvious response to the blue light which could activate channelrhodopsin CoChR. the first video shows the worm which expressed Odr-10::CoChR::GEM-GECO::mCherry circuit could run in circle to follow the blue light. The other figure shows the same type worm which looks “asleep” ( because of no food supply) be “waked up” also by blue light(preference). </p>
 </div>
@@ Line 79: / Line 86: @@
 </html>
-{{SUSTech_Image_Center_8 | filename=T--SUSTech_Shenzhen--Microfuildics--wake up.gif|width=1000px|caption=<B>Fig8. The test of the CoChR using light with different wavelengths and intensity. 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).</B> }}
+{{SUSTech_Image_Center_8 | filename=T--SUSTech_Shenzhen--Microfuildics--wake up.gif|width=1000px|caption=<B>Fig8. Observe individual worms in microfluidic chip</B>. This PDMS channel is designed to fix worms and observe them under stereoscope. 4 channels with decreasing diameter is used to fix worm. Other 4 narrow channels allow worms to move in a line constraint. }}
 <html></div></br></br></br></html>
-==Alcohol addiction==
+==Alcohol addiction ==
-To verify the function of the system we constructed in the C.elegans and give our project a real word meaning, we design this experiment to train the C.elegans be addicted to alcohol.
-If our system worked and can control the worms prefer to alcohol after blue light (preference) trained, it can prove that all the function we purposed for this system has been already realized.
+We trained the worms engineered  with Odr-10::CoChR::GEM-GECO::mCherry to be “addicted to” alcohol successfully.
+#In this experiment, we added the alcohol on the NGM plate containing engineered worms. At the same time, blue light is turned on to stimulate worms continually.
+#After training for 2 hours, we washed the plate and recovered the worms in M9 buffer.
+#Then, we put the mixture contained worms and M9 buffer on one side of a new plate. After recovery of the worms, we added the alcohol on the other side of the same plate to see if the worms have the tropism to the alcohol.
-Here, we employ the Odr-10::CoChR::GEM-GECO::mCherry worms to measure the preference for the alcohol. We added the alcohol with different concentration on the NGM plate to form a liquid film to stimulate the C.elegans and give the worms blue light induced which is proved can make them show a preference. After 2 hours we washed the plate to recover the worms in M9 buffer and put the mixture (contained worms and M9 buffer) on one side of a new plate. After recovery of the worms, we put the alcohol on the other side of the same plate to find out if the worms have the tropism of the alcohol.
+The result shows that most of the worms transfected with Odr-10::CoChR::GEM-GECO::mCherry moved to the alcohol while the wild types could not (Fig.9).
-Then we observed that the Odr-10::CoChR::GEM-GECO::mCherry worms could move to the alcohol while the wild types could not (Fig.8). And both of the experimental worms and wild type worms show no response to the buffer after training.By the same time, the Odr-10::CoChR::GEM-GECO::mCherry worms show no response if we do not train the with the blue light.
+What’s more, without the previous training by light, the worms transfected with Odr-10::CoChR::GEM-GECO::mCherry show no preference to alcohol, neither. (Fig.10).
-{{SUSTech_Image_Center_fill-width | filename=T--SUSTech_Shenzhen--Demonstrate8.png|width=6000px|caption=<B>Fig.9  A. The Odr-10::CoChR::GEM-GECO::mCherry worms after 2 hours' training. We cycle the area of worms after training in red. B. The worms in A after alcohol inducing. The yellow cycled area represents the alcohol and the worms could crawl towards the alcohol. C. The wild type worms control. The wild types could not crawl towards the alcohol although trained with blue light.</B>}}
+{{SUSTech_Image_Center_fill-width | filename=T|width=6000px|caption=<B>Fig. 9  A.)</B> The behaviors of Odr-10::CoChR::GEM-GECO::mCherry worms (all stages) after 2 hours' training. We circle the area of worms after training with blue light in red. <B>B)</B> The same worms in figure A would crawl towards alcohol inducing. The yellow circle area represent the alcohol. <B>C)</B> The wild type worms control. <B>D)</B> The wild types would not crawl towards the alcohol even thought training with blue light.
+<B>Fig. 10 E)</B> The buffer have no addition to worms transfected with Odr-10::CoChR::GEM-GECO::mCherry circuit. <B>F)</B> shows no preference to alcohol }}
-{{SUSTech_Image_Center_fill-width | filename=T--SUSTech_Shenzhen--Demonstrate9.png|width=6000px|caption=<B>Fig.10  A. The Odr-10::CoChR::GEM-GECO::mCherry worms after training and alcohol induing (the same as (Fig.8 B). The Odr-10::CoChR::GEM-GECO::mCherry worms after training, then use buffer to induce. The yellow cycled area represents the buffer and the worms could not crawl towards the alcohol. C. The Odr-10::CoChR::GEM-GECO::mCherry worms without training, then use the alcohol to induce them, the worms show no response.</B>}}
+This experiment showed that our genetically modified worms can learn new behavior(addiction to alcohol) after trained by blue light.
-This experiment showed that our worms which constructed the circuit (specific promoter::channelrhodopsin::Calcium indicator) can form the habit we purposed after trained with the light (decided by the channelrhodopsin). In brief, we have proved that our experimental worms have the learning ability inducing by light. Thus, the goal of our experiment has been almost achieved. We get the worms controlled by the light and the neuron activity can also be traced by observing the change of the indicator.
+==Future Plan==
-===Future Plan===
+Our further work is to decipher the change of neuron connectivity due to the newly learned behavior.
-In the future, we will track the neuron activity and do the alcohol inducing at the same time on the Odr-10::CoChR::GEM-GECO::mCherry worms after 2 hours training. By the time, we will build a model to select the downstream neuron of the AWA/AWB neurons and then using the specific promoters of these neurons fused with indicator to verify the relationship between them.Then finally we will figure out the whole neuron network of the learning ability, we can do the simulation on wherever we like to test the network, and we can know the logical order of the neurons when a new ability formed.
+We have selected 2 promoters(cho-1 and unc-8) to express optogenetic circuits on the neurons connected to AWA and AWB, such as AIA, AIY, VB, ASH, AVB, VB, AVA.<html><a href="https://2017.igem.org/Team:SUSTech_Shenzhen/Neuron_Network_Model">See details</a></html> The next step will be using optofluidics platform above to analyze the activation of downstream neurons in the new-formed or modulated neural circuit, with or without the aforementioned training.

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

Revision as of 03:12, 2 November 2017

Demonstrate

Contents

Choosing the suitable excitation wavelength

Genetic integration of synthetic circuits into C.elegans strains

Examining the function of AWA and AWB neurons

Examining the behavioral change of individual worms

Alcohol addiction

Future Plan