Difference between revisions of "Team:Korea U Seoul/Protocol"

1. E.coli TSS competent cells

Day1 :
1. Prepare 20ml of TSS buffer and 200ml of LB (Both must be autoclaved)
2. Autoclave centrifuge pellets
3. Chill the centrifuge pellet and TSS buffer in 4C refrigerator..
4. Grow your desired strain of E.coli in 3ml LB overnight.

Day2 :
1. Dilute 2ml of the culture in 200ml LB and grow till the OD600 reaches 0.4-0.5.
2. Incubate the cells on ice for few hours.
3. Centrifuge the cells at 3500rpm, 4C for 15minutes.
4. Remove supernatant and resuspend the cells in chilled 20ml TSS buffer.
5. Aliquot the 120 ul of cells in pre-chilled 1.5ml tubes and freeze the tube in liquid nitrogen.
6. Store at -80C

2. TSS Buffer

3. Chemical transformation

1. Thaw competent cells on ice.
2. Add 1ul to 5ul of DNA into 50ul of competent cells and gently mix.
3. Incubate on ice for 10 to 20 minutes.
4. Heat shock at 42C for 1 minute.
5. Incubate on ice for 5 minutes.
6. Add 200ul of LB and mix thorougly.
7. Grow in 37C shaking incubator for 1hour.
8. Plate the cells on a agar plate with appropriate antibiotics.
9. Incubate plates at 37C overnight.

4. Diaphorase assay

1. Prepare autoclaved D.W, 10mM NADH, 20mM DCPIP
2. Prepare your sample and controls. Purified enzymes are recommended as the sample since crude bacteria cell extract could already have reducing agents that can reduce DCPIP.

3. Purified enzymes	Xul (Depends on the concentration of your purified enzymes)
   20mM DCPIP	1ul
   10mM NADH	2ul
   D.W	17-X
   Total	20ul

4. Check whether the colour changes.

5. Induction (IPTG. L-Arabionose)

Day 1 :
1. Prepare 200ml of LB and autoclave.
2. Grow your desired strain of E.coli in 3ml LB overnight.

Day 2 :
1. Dilute 2ml of the culture in 200ml LB and grow till the OD600 reaches 0.5~0.8.
2. Put your desired inducing agent into the 200ml culture. (Final concentration : IPTG - 0.5mM, L-arabionose - 0.5%)
3. Incubate the 200ml culture in 20C overnight (12hours)

6. Enzyme purification (Mini scale)

We followed the protocols of NI-NTA Spin Kit handbook.

(https://www.qiagen.com/cn/resources/resourcedetail?id=3fc8c76d-6d21-4887-9bf8-f35f78fcc2f2&lang=en)

Protocols for battery device

1. Battery device design

① Cut 15mL tube into enough length(about 8cm).
② Make a hole at 50ｍL tube's cap about ①'s diameter. Make 2 of it.
③ Pierce a very small hole for wire next to ②'s hole.
④ Connect electrode and wire with silicon waterproof adhesive.
　(We used 2cm*5cm carbon paper with coated back)
⑤ Attach ①, ③, ④ with silicon adhesive. You should paste it very well.
　Two 50mL tube's cap should be arranged opposite. 50mL tubes will be linked from 　outside.
⑥ Prepare catholyte and anolyte at 50mL tube each.
⑦ Add 175mM Sodium Chloride and Agar(15g/L) in DW, and autoclave it.
　Next, put a lid on ①'s 15mL tube and fill it with salt bridge solution.
⑧When salt bridge solidified in enough time, connect ⑤(body of device) and ⑥(catholyte and anolyte).
⑨ Link ⑧ into voltage measuring equipment.
⑩ After using the device, wash it softly and autoclave it.

2. Prepare catholyte and anolyte

① Make a pure culture of bacteria(MR-1, BW25113, BL21, etc) in LB solid medium 　with antibiotics. You can use ampicillin for culturing Shewanella oneidensis MR-1.
② Make a seed culture with 4mL LB culture medium from single colony, add 　antibiotics, and culture 12 hours.
③ Insert 1mL of seed culture, antibiotics in 100mL LB. Culture 24 Hours.
④ Put the cell down using centrifuge(3,000 RPM, 20 minutes, and 4°C).
⑤ If you need cell disruption, use sonicator(ultrasonic processor). Turn on 2 second, 　turn off 10 second for 1 minute. Total time is 4 minutes.
⑥ Add mediator(30μM methylene blue) and substrate. It is convenient if you prepare 　mediator as a stock solution.
⑦ Put 30mM Sodium ferricyanide(electron acceptor) in anolyte.

3. Electricity analysis

① We measured voltage every single minute by an electric measuring circuit using 　potentiometer(1,000 ohm) and Keithley Digital Multimeter
② Therefore, we can calculate current with Ohm's law(V=IR).
③ Now we can get electric power with P=VI.
④ So we can draw voltage, current, and electric power graph each.