Team:China Tongji/Achivement

Parts name	Type	Description	Length
BBa_K1634002	regulatory	pmyo2 ( a promoter in C.elegans )	694
BBa_K1634003	coding	ChR2-YFP	1809
BBa_K1634005	regulatory	pmyo3 (a promoter in C.elegans)	814
BBa_K1634006	Reporter	dsRed ( a reporter which is a red fluorescent protein)	678

Move your mouse on to the part line, you may see the sketch map below!

1.1.1 BBa_K1634002

Pmyo2 is a promoter which can drive the expression in the muscle of C.elegans especially the muscle in the head and pharynx. And we link the Pmyo2 with some channelrhodopsin which can be activated or suppressed by the light. Since we have learned that the choosing of direction in C.elegans depends on the muscle in the head, we can observe the obvious change in moving pattern of the C.elegans after we shed the light. In other words, we can use them to construct a light-sensed locomotion controlling system in C.elegans. And we achieve that goal by using this part to control the movement of the head, the movement of the whole C.elegans.

1.1.2 BBa_K1634003

ChR2 is a kind of channelrhodopsin. Channelrhodopsins are a subfamily of retinylidene proteins (rhodopsins) that function as light-gated ion channels.[1] They serve as sensory photoreceptors in unicellular green algae, controlling phototaxis: movement in response to light. When we express the channelrhodopsins in some specific cells in organisms and shed specific light on them, we can activate or suppress the specific ion channel to change the activity of the cell. And if the cell is a muscle cell or a neuron, this tiny change may trigger a great change in the organism's activity. To make it easier to confirm the location and expression level of the ChR2, we construct a fusion protein of ChR2 and YFP.

1.1.3 BBa_K1634005

"Myo-3 encodes MHC A, the minor isoform of MHC (myosin heavy chain) that is essential for thick filament formation, and for viability, movement, and embryonic elongation; expressed in body muscle, the somatic sheath cell covering the hermaphrodite gonad, and also expressed in enteric muscle, vulval muscles of the hermaphrodite and the diagonal muscles of the male tail." (from Wormatlas) Since we want to control the C.elegans' movement. We decided to use pmyo-3 (promoter of myo-3) to construct a plasmid which can drive the expression in the worm’s body muscle. When we express the channelrhodopsins in some specific cells in organisms and shed specific light on them, we can activate or suppress the specific ion channel to change the activity of the cell. According to this thought in optogenetics, we linked the pmyo3 with channelrhodopsin, then we use the light source assembled by ourselves to shed light of specific wave on the C.elegans, changing the condition of the muscle, thus changing the movement of the C.elegans.

1.1.4 BBa_K1634006

DsRed is a commonly used fluorescent protein, a reporter. If we wanted to express some special protein such as channelrhodopsins in those muscles, we need to inject the mixtures containing the plasmid we constructed into the sexual gland of the C.elegans. Then we can get the transgenic descendants we want. However, this doesn’t mean that we can get some successful transgenic descendants after every microinjection. So we need to pick up the several successful one from thousands of descendants. But sometimes the specific protein we express has not contained a reporter, and even though the ChR2 we express contains a YFP, the light is not strong enough for us to find the successful one from thousands of descendants. To solve this problem, we constructed this part, pmyo2-dsRed, which was called "the comarker"by us. We made mixture of pmyo2-dsRed and pmyo2-channelrhodopsin, then inject the mixture through microinjection. The possible red fluorescent in the descendants has 4 main functions. Firstly, it ensures that the microinjection process is successful. Secondly, it ensures that those plasmids have successfully passed to the descendants.Thirdly, since it has the same promoter as our target protein, we can assume that our target protein has also successfully expressed in the same position, and we can have a double-check if our protein\ has a reporter. Finally, it can also help us to learn the expression pattern and quantify the expression level roughly by observing the fluorescence intensity of dsRed.

1.2 Composite parts

Parts name	Type	Description	Length
BBa_K1634007	Composite	pmyo2-ChR2-YFP	2509
BBa_K1634008	Composite	Pmyo2-dsRed	1378
BBa_K1634004	Composite	pmyo3-ChR2-YFP ( C.elegans )	2629

Move your mouse on to the part line, you may see the sketch map below!

1.2.1 BBa_K1634007

Pmyo2 is a promoter which can drive the expression in the muscle of C.elegans especially the muscle in the head and pharynx. ChR2 is a kind of channelrhodopsin. Channelrhodopsins are a subfamily of retinylidene proteins (rhodopsins) that function as light-gated ion channels.[1] They serve as sensory photoreceptors in unicellular green algae, controlling phototaxis: movement in response to light.[2] When we express the channelrhodopsins in some specific cells in organisms and shed specific light on them, we can activate or suppress the specific ion channel to change the activity of the cell. According to this thought in optogenetics, we linked the Pmyo2(BBa_K1634002), specific promoter in the muscle of head, with the ChR2-YFP(BBa_K1634003), then we use the light source assembled by ourselves to shed light of specific wave on the C.elegans, changing the condition of the muscle. Since we have learned that the choosing of direction in C.elegans depends on the muscle in the head, we can observe the obvious change in moving pattern of the C.elegans after we shed the light. In other words, we try to construct a light-sensed locomotion controlling system in C.elegans. And we achieve that goal by using this part to control the movement of the head, the movement of the whole C.elegans.

We construct pSB1C3-pmyo2-ChR2-YFP and PPD95.75-pmyo2-ChR2-YFP. The pSB1C3-pmyo2-ChR2-YFP is for submission. Then we micro-inject the PPD95.75-pmyo2-ChR2-YFP into the C.elegans. The result of our testing on C.elegans(pmyo2-ChR2-YFP) is displayed on the parts page and the result page.

1.2.2 BBa_K1634008

Pmyo2(BBa_K1634002) is a promoter which can drive the expression in the muscle of C.elegans especially the muscle in the head and pharynx. DsRed(BBa_K1634006) is a commonly used fluorescent protein,a reporter. If we wanted to express some special protein such as channelrhodopsins in those muscle,we need to inject the mixtures containing the plasmid we constructed into the sexual gland of the C.elegans. Then we can get the transgenic descendants we want. However, this doesn’t mean that we can get some successful transgenic descendants after every microinjection. So we need to pick up the several successful one from thousands of descendants. But sometimes the specific protein we express has not contained a reporter, and even though the ChR2 we express contains a YFP, the light is not strong enough for us to find the successful one from thousands of descendants. To solve this problem, we constructed this part, pmyo2-dsRed, which was called "the comarker"by us. We made mixture of pmyo2-dsRed and pmyo2-channelrhodopsin, then inject the mixture through microinjection. The possible red fluorescent in the descendants has 4 main functions. Firstly, it ensures that the microinjection process is successful. Secondly, it ensures that those plasmids has successfully passed to the descendants.Thirdly, since it has the same promoter as our target protein, we can assume that our target protein has also successfully expressed in the same position, and we can have a double-check if our protein\ has a reporter. Finally, it can also help us to learn the expression pattern and quantify the expression level roughly by observing the fluorescence intensity of dsRed.

1.2.3 BBa_K1634004

Myo-3 encodes MHC A, the minor isoform of MHC (myosin heavy chain) that is essential for thick filament formation, and for viability, movement, and embryonic elongation; expressed in body muscle, the somatic sheath cell covering the hermaphrodite gonad, and also expressed in enteric muscle, vulval muscles of the hermaphrodite and the diagonal muscles of the male tail. “(from Wormatlas) Since we want to control the C.elegans' movement. We decided to use pmyo-3 (promoter of myo-3)(BBa_K1634002) to construct a plasmid which can drive the expression in the worm’s body muscle.ChR2 is a kind of channelrhodopsin. Channelrhodopsins are a subfamily of retinylidene proteins (rhodopsins) that function as light-gated ion channels.[1] They serve as sensory photoreceptors in unicellular green algae, controlling phototaxis: movement in response to light.[2] When we express the channelrhodopsins in some specific cells in organisms and shed specific light on them, we can activate or suppress the specific ion channel to change the activity of the cell. According to this thought in optogenetics, we linked the Pmyo3,specific promoter in the muscle of whole body, with the ChR2-YFP(BBa_K1634003), then we use the light source assembled by ourselves to shed light of specific wave on the C.elegans, changing the condition of the muscle. Then we can observe the obvious change in moving pattern of the C.elegans after we shed the light. In other words, we try to construct a light-sensed locomotion controlling system in C.elegans.

We construct pSB1C3-pmyo3-ChR2-YFP and PPD95.75-pmyo3-ChR2-YFP. The pSB1C3-pmyo3-ChR2-YFP is for submission. Then we micro-inject the PPD95.75-pmyo3-ChR2-YFP into the C.elegans. The result of our testing on C.elegans(pmyo3-ChR2-YFP) is displayed on the parts page and the result page.

1.3 Plasmids we use in worm

Please click here to see more details!

1.4 Plasmid we improved

Please click here to see more details!

1.5 Reference

[1] Nagel G, Ollig D, Fuhrmann M, Kateriya S, Musti AM, Bamberg E, Hegemann P (June 2002). "Channelrhodopsin-1: a light-gated proton channel in green algae". Science 296 (5577): 2395–8. doi:10.1126/science.1072068.

[2] Sineshchekov OA, Jung KH, Spudich JL (June 2002). "Two rhodopsins mediate phototaxis to low- and high-intensity light in Chlamydomonasreinhardtii". Proc. Natl. Acad. Sci. U.S.A. 99 (13): 8689–94. doi:10.1073/pnas.122243399.

2. Equipment--Install our LED light Source

2.1 Introduction

ChR2 needs a high power light to let it work on C.elegan. However, in our lab, we don’t have the light source which is powerful enough to let us use it for optogenetics. So we bought some parts from THORLABS, and prepare to build our own LED light source by ourselves.

Here are all the parts we need to build LED light source:

ACL2520-A

B1CM

B4CM

C4W

DC2100

DMLP550R

FFM1

FL460-10

FL532-10

FL560-10

FL635-10

LEDD1B

M470L3

M530L3

M590L3

M625L3

SM1A14

SM1CP2

SM1L03

SM1T2

SM1V05

TPS001

2.2 The installation of light source

(1) Put the proper light filter into SM1L03 and fix it by SM1RR, and then fix the SM1L03 containing its filter to its light source. After that fix ACL2520-A inside of SM1V05, then fix them in front of SM1L03. Link this packaged light source to the C4W cube by using SM1T2.

(2) Fix FFM1 to the B4C/M by using the prepared screws, then use FFM1 to nip the DMLP550R (please pay attention to the position which the filter located, the light whose wavelength is larger than 550nm will pass this filter, otherwise the light will be reflexed. When choosing the location of light sources, this matter should be considered as well). Then fixed the B4C/M to the cube carefully.

(3) The surface which fixed with B4C/M is the underside of the cube. Use B1C/M to seal up the top surface of the cube. Fix two proper light sources to the surfaces which lights come in of C4W. The surface which light comes out will links to the microscope by using SM1T2 and SM1A14. The remaining surface will blocked by SM1CP2.

(4) The installation has been completed.

Figure 2-1: The LED light source
Figure 2-2: We are trying to assemble C4W box of our light source
Figure 2-3: We are figuring out how to assemble FFM1 into C4W box

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2.3 The adjustment of light path

(1) The light path will be collimated by changing the length between light source and convex lens. When the length is changing, pay attention to the light spot on the optical screen. If the spot is clear and convergent, it means the light path has been collimated.

(2) After join the light sources to the C4W cube, the light spot can be changed by rotating B4C/M and the screws on B4C/M.

(3) The adjustment has been completed.

Figure 2-4: We are adjusting the light path, the spot is clear and convergent.

2.4 The refit of the light sources

After we build our LED light source, we found that it’s still not powerful enough for our experiment, so we decided to change the light source to 5W. we bought the 5W LED light from internet and replaced the old one.

(1) Take down the LED of the light sources by using welding gun. Replace them by the high power LED. Make sure the surfaces of the substrate and the radiator are parallel.

(2) The refit has been completed.

Figure 2-5: The 5W red and green LED source which we bought from internet
Figure 2-6: The 5W blue and yellow light source that we bought from internet
Figure 2-7: We are trying to replace the old LED source with the 5W new one.

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3. Modeling

3.1 Real-time tracing and mathematical analysis

We can analyze the reaction of the worms by watching the video we make when we test them. But the video is visual and dynamic. If we can get the track of those worms, it could benefit us a lot. Comparing to video results, the track is static and directly. So we decide to analyze the track of the worms.

We draw up the trace of all these strains of worms by using real-time tracking technology. In our project, we keep tracking the head of the worm to draw up the trace. Due to the video we made, we generate a point at the location of the worm head at every 100ms. As a result, we can get a picture which stands for the trace of our worms.

But the information is still not enough for us, so we try to give a coordinate system to each trace, which means that we can get the coordinate of each point. So we define the first point as the original point, and the first two points on the X axis or Y axis. Then we get the trace with coordinate system.

Having the track of worms doesn’t mean we can get useful information from them. Only standardize the video can we make comparison to the different type of worms. To evaluate the reaction of these gene modified worms, we find some different aspects to observe their trace, speed and analyse the turning angle when the C.elegans makes a turn. So standardizing the video is very important for us to analyze the speed and the trace.

So we use 5-10-10 routine to make the video of the worms, so that it can benefit our analyzation later.The 5-10-10 routine means that the first 5 seconds leave the worm in white light, after that give it a 10 second of LED light, and at last leave it in white light for about 10 seconds or more. The 5-10-10 routine is better for us to analyze the speed of those worms. And the first 5 seconds' white light is use to observe the normal behavior of the worms which can make comparison to the following period. The third period is use to observe how long the worm can return to normal condition.

Here are the tracks we build with coordinate system.

Figure 3-1: The trace of pmyo2-ChR2-YFP (with coordinate)

[ Red points represents the track under white light. ]

[ Blue points represents the track under blue light (470nm) ]

We test this kind of worms by using 5W LED blue light (470nm) with 1000mA LED driver. When we give light to this kind of worms, we can find some obvious responses. First, after we giving light, the worm would change their direction in about 2 seconds in average. Their reactions are always step back. Secondly if we focus on the movement of their heads, we can find the turning angles change a lot during this time. It means the blue light can stimulate the muscle of their heads and as a result the worm will change the direction. But their behavior will turn to normal at the moment we turn off the light instantaneously. The speed of worm doesn’t have some apparent changes.

Figure 3-2: The trace of pmyo2-ChETA-EYFP (with coordinate)

[ Red points represents the track under white light. ]

[ Blue points represents the track under blue light (470nm) ]

We test this kind of worms by using 5W LED blue light (470nm) with 1000mA LED driver.When giving the light, this kind of worms has little responses. But you can see the behavior of the head have changed a lot. It means the blue light can still infect the muscle near the head. When the light is on, the behaviors of the worms become stiff compare to the normal worms. At the same time, after the light is given, you can find the speed of the worm obviously slow down. It is very interested that the worm will stop or even recede when the light is turned off. It means it will take some time for the worm to turn to normal.

Figure 3-3: The trace of pmyo2-iC1C2-EYFP (with coordinate)

[ Red points represents the track under white light. ]

[ Blue points represents the track under blue light (470nm) ]

We test this kind of worms by using 5W LED blue light (470nm) with 100mA LED driver. This kind of worms also has obvious reaction under the blue light. Firstly, you can see an apparent direction change when giving the light. Their reactions are always step back when they are about to changing directions, but the changes are not taken place instantaneously. It means it will be 5-7 seconds later when the worm changes direction. Secondly, you can see they twist their body when stimulated by the light. The turning angles of their head have changed a lot comparing to the normal worms.

Figure 3-4: The trace of pmyo3-ChR2-YFP (with coordinate)

[ Red points represents the track under white light. ]

[ Blue points represents the track under blue light (470nm) ]

We test this kind of worms by using 5W LED blue light (470nm) with 1000mA LED driver.This kind of worms is one of the highest expression worms among all the strains we have, and its reaction is very obvious. First when the light is on, we can find the whole body of this worm contract, which means the blue light lead to the muscle contraction of the worm. Secondly the worms will stay still until we turn oof the lights. It means that the blue light prevent the worm from moving. When giving the light, the worm will stop move. When the light is off the worm will start move again. These mean taht the speed of the worm is changing. It is very interesting that all these reactions are taken place instantaneous.

Figure 3-5: The trace of pmyo3-chETA-EYFP (with coordinate)

[ Red points represents the track under white light. ]

[ Blue points represents the track under blue light (470nm) ]

We test this kind of worms by using 5W LED blue light (470nm) with 1000mA LED driver.This kind of worm has apparent response to the blue light. Firstly, when we give it blue light we found their bodies seem to be loss of control. It is remarkable that the blue light affect the muscles of the worm a lot. After the light turn off, it still need about 10 to 20 seconds for the worms to turn to be normal. But there seem to be no obvious changes when we analyze the direction and speed.

Figure 3-6: The trace of pmyo3-iC1C2-EYFP (with coordinate)

[ Red points represents the track under white light. ]

[ Blue points represents the track under blue light (470nm) ]

We test this kind of worms by using 5W LED blue light (470nm) with 1000mA LED driver.This strain has an obvious reaction to the blue light. When we give light to this kind of worm, we can see it step back in about 2 seconds in average. It means this reaction is fast and instantaneously. It is remarkable that when the light is off, the worm turn to normal rapidly. But the speed and the movement of the worms will not be affected by blue light.

Figure 3-7: The trace of lite1 control (with coordinate)

[ Red points represents the track under white light. ]

[ Blue points represents the track under blue light (470nm) ]

This group is lite1 worm which is not sensitive to the lights, while other types of worms make have response to the light. So we choose lite1 worm to be our experimental subject to avoid unnecessary factors.

Figure 3-8: The trace of no-ATR control (with coordinate)

[ Red points represents the track under white light. ]

[ Blue points represents the track under blue light (470nm) ]

This group we use the mediums without ATR to foster the pmyo3-ChR2-YFP worms. We use this group to find out the effect of ATR. As a result, we find out that the ATR is necessary to our project. Only being fostered in the mediums which have ATR do the worms have response to the lights we gave. In our project, we also set up other control groups to each strain, and they all have the same phenomenon. We choose pmyo3-ChR2-YFP worms as an example for this kind of worm have the highest efficiency.

Figure 3-9: The trace of green light control (with coordinate)

[ Red points represents the track under white light. ]

[ Blue points represents the track under blue light (470nm) ]

This group we use green light to stimulate the pmyo3-ChR2-YFP worm. By doing this, we try to find out if the worm have response to all kinds of lights or not. At last, we find out that our worms would only have response to blue light (470nm, 5W, 1000mA). In our project, we also set up other control groups to each strain, and they all have the same phenomenon. We choose pmyo3-ChR2-YFP worms as an example for this kind of worm have the highest efficiency.

Summary

So with these coordinates, we can change these tracks from image information to digital information, which benefit us a lot. By using these data, we find the curve which fit to the tracks. If the quantity of the samples is big enough, we can use these data to find the features hidden behand these images. We can even predict the movement of the C.elegens.

3.2 The relationship between light intensity and response index

By using DC2100 we can achieve the goal that we could control the current of LED accurately. For our LEDs, there is a direct proportion relationship between light intensity and the current which move across it. To test which value is the best to stimulate the C.elegens, we design this part to help us. According to the limitation of DC2100, the largest current we can use is 1000mA. So we pick some worms of all strains which have obvious reactions as our experimental material (using 1000mA to test the reactions before). Using the worm which have reactions before is very important for this part. 0mA is needn’t to be tested, so we choose to start from 50mA. We pick up 10 worms from every strains to test whether it has response or not. After 50Ma has been tested, we test the 100mA and then 150mA and so on. Until we finish the test of 1000ma, we calculate the ratio of having reactions. Here are the graphs we get due to the records.The results are showed below.

Figure 3-10: The changing trend of response index with the change of light intensity (blue LED, 470nm, 5W, using pmyo2-ChR2-YFP worm)

Figure 3-11: The changing trend of response index with the change of light intensity (blue LED, 470nm, 5W, using pmyo2-ChETA-EYFP worm)

Figure 3-12: The changing trend of response index with the change of light intensity (blue LED, 470nm, 5W, using pmyo2-iC1C2-EYFP worm)

Figure 3-13: The changing trend of response index with the change of light intensity (blue LED, 470nm, 5W, using pmyo3-ChR2-YFP worm)

Figure 3-14: The changing trend of response index with the change of light intensity (blue LED, 470nm, 5W, using pmyo3-ChETA-EYFP worm)

Figure 3-15: The changing trend of response index with the change of light intensity (blue LED, 470nm, 5W, using pmyo3-iC1C2-EYFP worm)

From these data, we can find out some conclusions.

(1) For all these strains, the response index is getting larger with the increase of the current.

(2) For pmyo2-ChR2-YFP and pmyo3-ChR2-YFP, when the current increases to about 600mA we can find that the worm can be totally activated.

(3) For pmyo2-iC1C2-EYFP and pmyo3-iC1C2-EYFP, when the current increases to about 800mA we can find that the worm can be totally activated.

(4) For pmyo2-ChETA-EYFP and pmyo3-ChETA-EYFP, when the current increases to about 900mA we can find that the worm can be totally activated.

(5) For all the strains of worms, 1000mA is the most suitable current. So in our project we use 1000mA blue light (470nm, 5W) to test our worms.

3.3 Turning angle measuring

As we know, pmyo2 is expressed in pharyngeal of C.elegents, so the light will stimulate the head of the worms directly. As a result, observing the movement of their heads is very significative. As we all know, the head of the worm is always shaking, so the turning angle (the angle of each shake) is a very useful data which reflect the response of the head. In this part, we use turning angle of their heads to evaluate the reaction of their head.

We choose pmyo2 worms as our experimental objects in this part. The results are showed below.

Figure 3-16: The turning angle measuring of pmyo2-ChR2-YFP (using blue light, 470nm, 5W, 1000mA)

[ Red points represents the track under white light. ]

[ Blue points represents the track under blue light (470nm) ]

Figure 3-17: The turning angle measuring of pmyo2-chETA-EYFP (using blue light, 470nm, 5W, 1000mA)

[ Red points represents the track under white light. ]

[ Blue points represents the track under blue light (470nm) ]

Figure 3-18: The turning angle measuring of pmyo2-iC1C2-EYFP (using blue light, 470nm, 5W, 1000mA)

[ Red points represents the track under white light. ]

[ Blue points represents the track under blue light (470nm) ]

Figure 3-19: The turning angle measuring of pmyo2-ChR2-EYFP (given no light)

[ Red points represents the track under white light. ]

[ Blue points represents the track under blue light (470nm) ]

Analysis:

From these figures we can get some useful information.

(1) The pmyo2-ChR2-YFP worms have an obvious response. When we give the lights, we can see theamplitude of turning angles become larger. The change of the turning angle becomes drastic.

(2) Compare to the pmyo2-ChR2-YFP worms, the other worm need a long time to be activated. And the time when the worm is activated has become longer.

(3) We use the pmyo2 worm given no lights as the control group. We test all the strains of pmyo2 worms, and they have the same reactions. We can see the fluctuation of turning angle is mild compare to those experimental groups.

4. Self Judgement

4.1 For a Bronze Medal

Register the team.

Successfully complete and submit this iGEM 2015 Judging form.

Create and share a Description of the team's project and document the team's parts.

Plan to present a Poster and Talk at the iGEM Jamboree.

Create a page on team wiki with clear attribution of each aspect of our project.

Document at least one new standard BioBrick Part or Device central to our project and submit this part to the iGEM Registry.

Part Number(s): BBa_K1634007 BBa_K1634002

4.2 For a Silver Medal

Experimentally validate that at least one new BioBrick Part or Device of our own design and construction works as expected.

Part Number(s): BBa_K1634003 BBa_K1634008 BBa_K1634004

Submit these new part to the iGEM Parts Registry.

Part Number(s): BBa_K1634003 BBa_K1634008 BBa_K1634004

Demonstrate how our team has identified, investigated and addressed one or more of these issues in the context of our project.

4.3 For a Gold Medal

(1) Expand on our silver medal Human Practices activity by demonstrating how we have integrated the investigated issues into the design and/or execution of our project.

(2) Demonstrate an innovative Human Practices activity that relates to our project.

Help the another registered iGEM team study their parts and test their software.

Improve the function OR characterization of a previously existing BioBrick Part and enter this information in the part's page on the Registry.

Part Number(s): BBa_K309007

Demonstrate a functional prototype of our project. Our prototype can derive from a previous project by our team or by another team. Show this system working under real-world conditions that we simulate in the lab.

4.4 For iGEM special Prizes

Integrated Human Practices

Education and Public Engagement

Modeling

Applied Design

4.5 For Team Parts Prizes

Best New Basic Part

Part Number(s): BBa_K1634003

Best New Composite Part

Part Number(s): BBa_K1634007

5. Judging Form

Click and link to the Team:China_Tongji/Judging Form!