Difference between revisions of "Team:XMU-China/Human Practices"

On September 30th 2018, we visited Mr. Su Mingluo, founder and chairman of Xiamen Kymem Membrane Technology Ltd, to have a deep discussion about information related to enterprises. He shared with us his experience of setting-up a new company, and rendered us prepared for drawing up business plans.

On September 30th 2018, we visited Mr. Su Mingluo, founder and chairman of Xiamen Kymem Membrane Technology Ltd, to have a deep discussion about information related to enterprises. He shared with us his experience of setting-up a new company, and rendered us prepared for drawing up business plans.

In the view of Mr. Su, having a sufficient knowledge about not only your company, but your competitors’ on the basis of market analysis was the premise of drawing up a good business plan. The business plan should involve some basic parts, namely the company’s technology introduction, market potential demand, market actual demand, team composition and competitive team analysis. We introduced our project, which joined competition this year, to Mr. Su, and highlighted the hardware products which we independently developed. Mr. Su expressed his expectation for the potential of our hardware equipment in the medical field. But on the other hand, given that starting a new business is tough, Mr. Su believed that our team would encounter five “new” threats in the initial stage, that is, new team, new cooperation partners, new capital, new technologies and new customers.

He also mentioned that at the initial period of our business, not only the company’s internal developing problems, but also the constrictions of the external environment would be the obstacle. Referring to his personal accumulated experience, Mr. Su further gave us some suggestions targeting on starting a new business: considering that China’s current intellectual property law system was still imperfect, innovative companies, like ours, who were known for their technological advantages must emphasize the patent-protection. What’s more, when our brand was still not so influential compared to those prestigious companies, we should put seeking target customers and broadening sales channels on the front burner, and carefully prevent our technology property right from being infringed. On top of that, he proposed that since our hardware product was based on the "government + grassroots + personal" operation mode, it was necessary to work hard in marketing, with clear targets and strategies.

We visited Mr. Jianghong Wu, president of Xiamen Wenda technology LTD. We had an enjoyed communication with Mr. Wu, which is about the communication mode between team hardware and software and the practicability of software design. We introduced the general situation of this year's team project to Mr. Wu, and emphasized the original intention of the team to solve the "aerial phenomenon" of the current "graded diagnosis and treatment" policy, as well as the difficulty in getting medical treatment in remote areas. Mr. Wu fully affirmed the innovative project and praised the team consciousness of social responsibility.

As for APP part he gave some professional advices and technical guidance, like how to accelerate our convolution neural network recognition speed with collected increasing number of images cases, and how to link block them up between chain technology public health service database to serve the public better. In addition, Mr. Wu gave us a lot of advice on product promotion and profit model.

● SYL3C-FAM (Sangon Biotech^®, see Appendices)
● EpCAM (ACROBiosystems^®)
● Binding Buffer (see Appendices)
● Fluorescence Spectrometer (Shimadzu^® RF-6000)
● Polarizer (from a pair of 3D glasses)

1. Make distributions in 1.5 mL centrifuge tubes:
1) Tube 1 (Aptamer Group): 1 μL 100 μM (100 pmol) SYL3C-FAM + 179 μL binding Buffer.
2) Tube 2 (Aptamer-EpCAM Group): 1 μL 100 μM (100 pmol) SYL3C-FAM + 179 μL binding Buffer.
2. Sample processing:
The above 2 tubes are simultaneously subjected to the following steps:
1) Denature at 95℃ for 10 minutes.
2) Remove the tubes, then put them at room temperature for about 20 minutes.
3) Add 20 μL of 100 μg/mL EpCAM protein to Tube 2.
4) Add 20 μL of binding Buffer to Tube 1.
5) Incubate all tubes in a shaker for 40 minutes (37℃, 200 rpm).
Note: Avoiding light in whole process is necessary.
3. Measure fluorescence intensity from vertical/parallel direction:
1) Turn on the instrument for 30 minutes before measuring.
2) Choose "Emission Mode". The excitation wave length is 495 nm, and the range of emission wave length is from 500 nm to 600 nm.
3) Before measuring, dilute the solution from step 2 to about 3 mL with binding Buffer in the fluorescence cuvette.
4) Attach a polarizer to the exit of the excitation light, and attach another polarizer to the receiving inlet of the emission light to make sure the polarization directions of the two polarizers are vertical/parallel.
5) Start measuring to obtain I_∥, _max (Aptamer), I_⊥, _max (Aptamer), I_∥, _max (Aptamer + EpCAM), I_⊥, _max (Aptamer + EpCAM).

● Sequence of SYL3C-FAM:
5'-CACTACAGAGGTTGCGTCTGTCCCACGTTGTCATGGGGGGTTGGCCTG-FAM-3'
● Binding Buffer (pH 7.3~7.5) contains:
1×PBS Buffer with 5 mM MgCl₂

● SYL3C-TEG-biotin (Borui^TM, see Appendices)
● C3-FITC (Borui^TM, see Appendices)
● C4-FITC (Borui^TM, see Appendices)
● Binding Buffer (see Appendices)
● 50×TAE concentrate solution (Solarbio^®)
● Agarose (Biowest^®)
● DNA dye (TransGen^® GelStain)
● Distilled water
● Microwave oven
● 10×Loading Buffer (Takara^®)
● Electrophoresis instrument

Dilute 50×TAE concentrate solution to 1×TAE Buffer with distilled water.

1. Make distributions in 1.5 mL centrifuge tubes according to the following:
1) Tube 1 (Aptamer Group): 1 μL 100 μM (100 pmol) SYL3C-TEG-biotin + 19 μL binding Buffer.
2) Tube 2 (C4 Group): 1 μL 100 μM (100 pmol) C4-FITC + 19 μL binding Buffer.
3) Tube 3 (C3 Group): 1 μL 100 μM (100 pmol) C3-FITC + 19 μL binding Buffer.
4) Tube 4 (C4&Aptamer Group): 1 μL 100 μM (100 pmol) SYL3C-TEG-biotin +1 μL 100 μM (100 pmol) C4-FITC + 18 μL binding Buffer.
5) Tube 5 (C3&Aptamer Group): 1 μL 100 μM (100 pmol) SYL3C-TEG-biotin +1 μL 100 μM (100 pmol) C3-FITC + 18 μL binding Buffer.
Note: The Tube 2, 3, 4 and 5 should be coated by tin foil properly after distributing.
2. Sample processing:
The above 5 tubes are simultaneously subjected to the following steps:
1) Denature at 95℃ for 10 minutes.
2) Anneal to room temperature for 20 minutes.
3. Prepare 3% agarose gel:
1) Weigh 0.9 g of agarose in a 100 mL Erlenmeyer flask.
2) Add 30 mL of 1×TAE Buffer into the flask from 1).
3) Make agarose melt by microwave oven (medium-high heat, about 3 minutes).
4) Add 3 μL of GelStain, mix by shocking.
5) Assemble gel pouring apparatus by inserting gate into slots.
6) Pour agarose gel into the gel tray.
7) Cool for 40 minutes to solidify the DNA agarose gel.
8) Remove the pouring apparatus, put the gel into an electrophoresis instrument.
4. Electrophoresis:
1) Add 2 μL of 10×Loading Buffer to each tube, then pipette up and down to evenly mix.
2) Pipette all samples which have been mixed with loading Buffer into the slots.
3) Turn on the electrophoresis instrument, set the working electric current at 150 mA and the working electric voltage at 110 V.
4) Electrophoresis for 50 minutes.
5. Gel imaging:
Turn off the instrument, take the gel into the gel formatter to take and save photos.

● Sequence of SYL3C-TEG-biotin:
5'-CACTACAGAGGTTGCGTCTGTCCCACGTTGTCATGGGGGGTTGGCCTG-TEG-Biotin-3'
● Sequence of C3-FITC:
5'-CTCTGTAGTGTTTTTTTTTTTTTT-FITC-3'
● Sequence of C4-FITC:
5'-TCTGTAGTGTTTTTTTTTTTTTTT-FITC-3'
● Binding Buffer (pH 7.3~7.5)contains:
1×PBS Buffer with 5 mM MgCl₂

● SYL3C-TEG-biotin (Borui^TM, see Appendices)
● C3-FITC (Borui^TM, see Appendices)
● EpCAM (ACROBiosystems^®)
● Magnetic Beads (InvitrogenTM)
● Binding & Washing Buffer (see Appendices)
● Incubation Buffer (see Appendices)
● Vortex (IKA^® lab dancer)
● Fluorescence Spectrometer (Shimadzu^® RF-6000)

1.8 μL of 100 μM SYL3C-TEG-biotin is diluted by 1×B&W Buffer up to 75 μL, and 1.2 μL of C3-FITC is also diluted by 1×B&W up to 50 μL.
The tube of 100 μg/mL C3-FITC should be coated properly by tin foil in order to avoid being quenched.

1. Make distributions in 1.5 mL centrifuge tubes according to the following:
Tube 1 (Blank Control): 100 μL incubation Buffer.
Tube 2 (Negative Control): 25 μL diluted SYL3C-TEG-biotin + 25 μL 1×B&W Buffer.
Tube 3 (Competition Group): 25 μL diluted SYL3C-TEG-biotin + 25 μL diluted C3-FITC.
Tube 4 (Competition Group without EpCAM): 25 μL diluted SYL3C-TEG-biotin + 25 μL diluted C3-FITC.
Tube 5 (Positive Control): 0.6 μL 100 μM C3-FITC + 100 μL incubation Buffer.
Note: The Tube 3, 4 and 5 should be coated by tin foil properly after distributing.
2. Denature, anneal and renature:
The Tube 2, 3, and 4 are denatured at 95°C for 5 minutes, then anneal to room temperature.
3. Wash magnetic beads:
1) Resuspend the beads and vortex for 10 seconds to make them even.
2) Transfer 75 μL of 10 mg / mL beads to 3 200 μL centrifuge tubes (ie 25 μL per tube).
3) Add 25 μL of 2×B&W Buffer to each tube.
4) Vortex for 5 seconds to resuspend.
5) Place the tubes in a magnetic field for 1 minute, then aspirate the supernatant.
6) Remove the tubes from the magnetic field and add 50 μL of 1×B&W Buffer to each tube.
7) Repeat steps 4) to 5).
8) Add 50 μL of 1×B&W Buffer to each tube.
4. Bind to magnetic beads:
1) Add 50 μL of solution of Tube 2,3 and 4 from step 2 to 50 μL of washed beads, respectively.
2) Vortex for 5 seconds to resuspend.
3) Incubate at room temperature for 30 minutes in the dark.
4) Vortex for 5 seconds to resuspend.
5) Place the tubes in a magnetic field for 2 minute, then aspirate the supernatant.
6) Remove the tubes from the magnetic field and add 100 μL of 1×B&W Buffer to each tube.
7) Repeat steps 4) to 5).
8) Add 100 μL of incubation Buffer to Tube2, 3 and 4, respectively.
5. Compete:
1) Add 17 μL of diluted EpCAM to Tube 2 and 3. And add 17 μL of incubation Buffer to the last each tube.
2) Incubate at room temperature for 40 minutes in the dark.
3) Place the tubes in a magnetic field for 2~3 minutes, then pipette 100 μL of the supernatant into a new 200 μL centrifuge tube and mark it.
4) Measure the fluorescence intensity of Tube 1, 5 and the supernatant from 3).
Note: Before measuring, the solution of each tube will be further diluted by adding 3, 000 μL of incubation Buffer into it. The excitation wave length is 495 nm, and the range of emission wave length is from 500 nm to 600 nm.

● Sequence of SYL3C-TEG-biotin:
5'-CACTACAGAGGTTGCGTCTGTCCCACGTTGTCATGGGGGGTTGGCCTG-TEG-Biotin-3'
● Sequence of C3-FITC:
5'-CTCTGTAGTGTTTTTTTTTTTTTT-FITC-3'
● 2×Binding & Washing Buffer (pH 7.5) contains:
10 mM Tris 1 mM EDTA 2 M NaCl
● Incubation Buffer (pH 7.5) contains:
20 mM Tris 140 mM NaCl 5 mM KCl 1 mM MgCl₂

● Cas12a (Alt-R^® CRISPR-Cas12a (Cpf1) System)
● crRNA (IDT^®)
● TE Buffer (Sangon Biotech^®)
● DNaseAlert^TM Substrate Nuclease Detection System (IDT^®, see Appendices)
● 21nt ssDNA (Borui^TM, see Appendices)
● Black 96-well plate (Corning^®)
● Microplate reader (Tecan Infinite^® M200 Pro)

● Add 26.7 μL of filtered TE Buffer into the tube of crRNA to make a 75 μM stock.
● Add 88.6 μL of filtered TE Buffer into the tube of ssDNA to make a 100 μM stock.

1. Form the RNP complex:
1) Prepare 3 200 μL tubes. Add 1 μL of Cas12a and 1 μL of diluted crRNA into each nuclease-free 200 μL tube.
2) Incubate Cas12a with crRNA at 37℃ for 30 minutes to form the RNP complex.
2. Pretreatment of substrate:
1) Prepare 3 DNaseAlert^TM Substrate single-use tubes. Add 5 μL of Nuclease-Free Water to each tube.
2) Add 5 μL of 10×DNaseAlert^TM Buffer to each tube.
3. Make distributions:
1) Cas12a Group: DNaseAlert^TM Substrate (from step 2) + 2 μL RNP complex (from step 1) + 1 μL diluted ssDNA + 37 μL filtered TE Buffer + 40 μL Nuclease-Free Water.
2) Negative Control: DNaseAlertTM Substrate (from step 2) + 40 μL Nuclease-Free Water + 40 μL Nuclease-Free Water.
3) Positive Control: DNaseAlertTM Substrate (from step 2) + 1 μL DNase I + 39 μL filtered TE Buffer + 40 μL Nuclease-Free Water.
4. Measure fluorescence intensity by microplate reader:
1) Incubate the reaction system at 37℃ for 30 minutes, then transform the reaction system into the black 96-well plate.
2) Before measure, add 12 μL of TE Buffer into each tube.
3) Set temperature: 37℃, choose plate type: Black. Set excitation wavelength: 536 nm and emission wavelength: 556 nm. Choose mode: Optimal.
4) Measure fluorescence intensity at 30 min, 40 min, 50 min, 60 min, 70 min, 90 min, 110 min (Timing is from the beginning of incubation).

● DNaseAlert^TM Substrate Nuclease Detection System contains:
1) DNaseAlert^TM Substrate (25 single-use tubes (50 pmol per tube) or 2 tubes bulk substrate (2 nmol per tube))
2) DNaseAlert^TM Buffer (250 μL)
3) Nuclease-Free Water (2 mL)
4) DNase I Enzyme (25 μL)
5) Nuclease Decontamination Solution (50 mL)
● Sequence of 21nt ssDNA:
5'-CAGGCCAACCCCCCATGACAA-3'

● TE Buffer (Sangon Biotech^®)
● DNaseAlert^TM Substrate Nuclease Detection System (IDT^®, see Appendices)
● Fluorescence spectrometer (Shimadzu^® RF-6000)

1. Make distributions:
Add reagent into 2 DNaseAlert^TM Substrate single-use tubes according to following instruction. Avoiding light is necessary during the whole process.
1) Negative Control: 5 μL Nuclease-Free Water + 5 μL 10×DNaseAlert Buffer + 40 μL Nuclease-Free Water + 40 μL Nuclease-Free Water.
2) Enzyme Group: 5 μL Nuclease-Free Water + 5 μL 10×DNaseAlert Buffer + 39 μL Nuclease-Free Water + 40 μL Nuclease-Free Water.
Place the two tubes at room temperature for 30 minutes to enough dissolve fluorescent substance.
2. Make distributions further according to the time gradient:
1) Prepare 6 200μL-PCR tubes in advance, then divide into 2 groups (3 tubes per group). Mark "W" and "I" on the tubes of two groups, respectively. Further mark the tubes according to the time gradient of 3 min, 15 min and 30 min (e.g. "W 3", "I 15").
2) Add 1 μL of DNase I into the DNaseAlert^TM Substrate single-use tube of Enzyme Group.
3) Mix immediately, then transfer 30 μL of reaction liquid from each single-use tube to each PCR tube marked before. Place all PCR tubes in 37℃ water bath. Then start timing.
4) Each tube: React for corresponding time, then measure florescence intensity immediately.
3. Measure fluorescence intensity:
1) Turn on the instrument for 30 minutes before measuring.
2) Choose "Emission Mode". The excitation wave length is 536 nm, and the range of emission wave length is from 540 nm to 580 nm.
3) Before measuring, dilute the reaction liquid to about 3 mL with TE Buffer in the fluorescence cuvette.

● DNaseAlert^TM Substrate Nuclease Detection System contains:
1) DNaseAlert^TM Substrate (25 single-use tubes (50 pmol per tube) or 2 tubes bulk substrate (2 nmol per tube))
2) DNaseAlert^TM Buffer (250 μL)
3) Nuclease-Free Water (2 mL)
4) DNase I Enzyme (25 μL)
5) Nuclease Decontamination Solution (50 mL)