Difference between revisions of "Team:NPU-China/Protocols"

• Core-part
• System
• Pathway
• Product
• Conclusion

1. Core-part：the activity of rate limiting enzyme ceaS2 has been improved

1. Excise the agarose gel slice, transfer the gel slice into a 1.5ml microfuge tube.
2. Add a 3X sample volume of Buffer DE-A. the weight of gel is equivalent to a 100ul volume.
3. Resuspend the gel in Buffer DE-A by vortexing. Heat at 75℃ until the gel is completely dissolved(no more than 10 min)
4. Add 0.5X Buffer DE-A volume of Buffer DE-B, mix.
5. Place a miniprep colume into a 2ml microfuge tube. Transfer the solubilized agarose from step 4 into the column. Centrifuge at 12,000xg for 1 min.
6. Discard the filtrate. Add 500ul of Buffer W1. Centrifuge at 12,000xg for 30s.
7. Discard the filtrate. Add 700ul of Buffer W2. Centrifuge at 12,000xg for 30s.
8. Repeat wash with W2. Centrifuge at 12,000xg for 1min.
9. Discard the filtrate. Centrifuge at 12,000xg for 1min.
10. Transfer the miniprep column into a clean 1.5ml microfuge tube. Add 25-30ul of Eluent or deionized water to the center of the membrane. Let it stand for 1min at room temperature. Centrifuge at 12,000xg for 1min. (pre-warming the Eluent at 65℃)

2.System：S. cerevisiae is more suitable for chassis cells than E. coli

Agarose-Electrophoresis is used in order to see if the PCR product is correct and seperate DNA by the number of base pairs.
●marker was the 2 kb Plus DNA Ladder
●fill pockets with 5 µl DNA ladder or 10 µl sample volume with 6x loading buffer
●running conditions: 130 V for 30-40 minutes

3.Pathway：Successfully build a new acrylic acid synthesis pathway and increase acrylic acid production

1. Set up the reaction.
V(ul)=(0.02*bp)/concentration(ng/ul)
2. Add the fragments into Gibson system.
3. Incubate samples in a thermocycler at 50 °C for 1h.
4. Purify the product using DNA purification kit.
5. Transform the product into the competent cells of E.coli BL21 , following the transformation protocol.

 

 

 

4.Product：Multi - Conditional Optimization of Acrylic Cell Factory Catalytic Reaction Process

for acrylic acid

Samples have to be centrifuged at 12,000xg for 5 min in order to remove solids (cells/precipitates).

All instruments we used is ultra-high performance liquid chromatography mass spectrometry instrument ABSciex 5600 TripleTOF.
a) HPLC:
Colum: Waters SunFire C18(4.6×250 mm)
Detector wavelength: 210 nm
Flowing phase: 90% 10mM ammonium acetate/ 10% methanol
Colum temperature: 35℃
Flow rate: 1mL/min
b) MS detecting condition:
Anion mode
Ion Source Gas1: 55 psi
Ion Source Gas2: 55 psi
Curtain Gas: 35 psi
IonSpray Voltage Floating: 4500 v
Interface Heater Temperature: 550℃

5.Conclusion

1. Pre-chill 1.5ml and 2ml microcentrifuge tubes, deionized water, 10% glycerol. Add 1ml LB to 2ml microcentrifuge tubes.
2. When OD600=0.6, incubate competent cells on ice for 20 min.
3. Transfer the competent cells to 50 mL pre-chilled centrifuge tube. Centrifuge at 5,500r/min, 4 ℃ for 5 min.
4. Discard the filtrate. Add 30ml of pre-chilled deionized water, resuspend the cells gently.
5. Centrifuge at 5,500r/min, 4 ℃ for 5 min. Discard the filtrate. Repeat wash with deionized water.
6. Discard the filtrate. Add 30ml of pre-chilled 10% glycerol, resuspend the cells gently.
7. Centrifuge at 6,500r/min, 4 ℃ for 5 min. Discard the filtrate. Repeat wash with 10% glycerol.
8. Discard the filtrate and leave over 1ml 10% glycerol to resuspend the competent cells, pipet 80ul into each 1.5ml EP tube, add 5ul of DNA and carefully mix with the competent cells. Let it stand for 2min.
9. Add electrocompetent to DNA on ice. Move the mixture to the cuvette. Dry and shock the cells(2500V). Add 1ml of LB medium. Incubate at 30℃ for 4 hours shaking at 220rpm, pipet 100ul from each tube onto the appropriate plate, and spread the mixture evenly across the plate. Incubate at 30℃ overnight. Position the plates with the agar side at the top, and the lid at the bottom.