Team:NPU-China/Protocols

• DNA gel extraction
• Electrophoresis
• Gibson assembly
• HPLC
• Knock out genes of Ecoli
• Crispr-cas9
• Plasmid preparation
• Plasmid transformation
• Point mutation
• Reagents
• Whole cell catalysis
• the LiAc SS carrier DNA PEG method
• Measure protein concentration
• References

DNA gel extraction

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℃)

Electrophoresis

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

Gibson assembly

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.

 

 

 

HPLC

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℃

Knock out genes of E ▪ coli (MG1655)

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.

Knock out the genes of Saccharomyces cerevisiae with Crispr-Cas9

 

2. Purify the PCR product with a DNA purification kit.
3. Add the appropriate amount of DMT enzyme, hold for one hour at 37 ° C.
4. transform the DNA into competent cells.
50ul competent cell + 15ul purified DNA，incubate on ice for 30min，heat shock 45s，incubate on ice for 2min，add LB medium and incubate for 1h.
5. Pipet 100ul from each tube onto the plate with resistance, and spread the mixture evenly across the plate. Incubate for 12h. Position the plates with the agar side at the top, and the lid at the bottom.
6. use a sterile pipet tip to pick Saccharomyces cerevisiae from plates，throw the tip into the tubes of 5 ml of LB + antibiotics，incubate in a rotary shaker. Prepare plasmid with kit for sequencing.
7. Transfer plasmid and fragment into Saccharomyces cerevisiae using the LiAc SS carrier DNA PEG method.

8. Prepare the template: use a sterile toothpick to pick Saccharomyces cerevisiae from plates，put the toothpick into 100ul 20mMNaOH and mix，99° C boiling for 30min.

 

 

plasmid preparation

Tiangen mini plasmid kit

Plasmid transformation

1. Pipette 50µl of competent cells and 2µl of plasmid into 1.5ml tube
2. Heat shock tubes at 42°C for 30s
3. Incubate on ice for 2min
4. Pipette 250µl LB media to each transformation
5. Incubate at 37°C for 1h
6. Plating
7. Pick single colonies
Reference: http://parts.igem.org/Help:Protocols/Transformation

Point mutation

 

2. Purify the PCR product with a DNA purification kit.
3. Add the appropriate amount of DMT enzyme, hold for one hour at 37 ° C.
4. Transform 5μl digested DNA into competent cells DH5α, incubate on ice for 30min.
42° C heat shock, 45s. Incubate on ice for 2min.
add 200μl of LB. incubate at 37 °C for 1 h, 220rpm/min.
5. pipet 200μl from each tube onto the plate with appropriate resistance, and spread the mixture evenly across the plate. Incubate at 37℃ overnight. Position the plates with the agar side at the top, and the lid at the bottom.
6. Select single colonies for sequencing.

Reagents

1. LB medium（lysogeny broth）
The recipe for 1l LB media is as follows:
Tryptone 10g/L
Yeast extract 5g/L
NaCl 10g/L
2. 0.1mM Kanamycin
MW of Kanamycin：582.58
Store at -20℃
3. LB plate
The recipe for 1l LB plate is as follows:
Tryptone 10g/L
Yeast extract 5g/L
NaCl 10g/L
15g Agar
Add appropriate amount of resistance.
4. 2YT medium
The recipe for 1l LB plate is as follows:
Tryptone 16g/L
Yeast extract 10g/L
NaCl 5g/L
5. 0.5mM IPTG
MW of IPTG：238.30
Store at -20℃
6. 50mM PBS buffer,PH8.0
A：0.05mol/L Na2HPO4
B：0.05mol/L KH2P04
137mMNaCl，2.7mMKCl，10mMNa2HPO4,2mMKH2PO4 for 1L.

Whole-cell catalysis

Whole cell catalysis means using complete biological organisms (ie, whole cells, tissues or even individuals) as a catalyst. The essence is using enzymes in cells for catalysis. The method is a kind of biocatalytic technology between fermentation and extract enzyme for catalysis. The advantage of whole cell catalysis is that the intracellular complete multi-enzyme system can achieve the cascade reaction of enzyme, so as to make up the deficiency of cascade reaction in reaction which only use pure enzyme and improve the catalytic efficiency. While eliminating the complex process in enzyme purification, it is easier to carry out the reaction and lower production costs.
1. Prepare sterile tubes of 5 ml of 2YT+antibiotics. Use a sterile pipet tip to pick bacteria from plates. Throw the tip into the tubes. Incubate in a rotary shaker at37℃ for 3-4h.
2. Transfer 120µl of bacteria from a slant culture into an Erlenmeyer flask containing 60 mL LB medium with appropriate resistance, incubate at 37 °C.
3. When the OD600 reaches 0.6-0.8, the induction of IPTG (0.5 mM) should be carried out. Incubate at 30 °C on a rotary shaker incubator at 220 rpm for 14 h.
4. Harvest the bacteria(6000rpm/min 7min). Wash with 30ml PBS.
5. The biocatalytic reaction mixture contained 10% glycerol, E▪ coli and 50mM PBS. Reaction time gradient: 8h, 16h, 32h.
6. Use HPLC for further analysis.

Reference: Li N, He Y, Chen Y, et al. Production of cyclic adenosine-3′,5′-monophosphate by whole cell catalysis using recombinant Escherichia coli, overexpressing adenylate cyclase[J]. Korean Journal of Chemical Engineering, 2013, 30(4):913-917.

the LiAc SS carrier DNA PEG method

1.use a sterile pipet tip to pick Saccharomyces cerevisiae from plates，throw the tip into the tubes of appropriate medium，incubate in a rotary shaker for 12h.
2.measure OD600, transfer x(x=(50×0.2)/(OD600×dilution ratio)) ml Saccharomyces cerevisiae into 50ml YPAD.
3.incubate for 4-5h to make OD600 reaches 0.8-0.9.
4.boil ssDNA.
5.Centrifuge at 3000g for 5 min. Discard the filtrate. Repeat washes with 25ml deionized water twice.
6.Transfer the cells to 1.5 mL centrifuge tube. Add 1ml of deionized water, resuspend the cells gently.
7.Centrifuge at 13000rpm for 30s. Discard the filtrate.
8.Add 1ml of deionized water, resuspend the cells. Pipet 100ul into each 1.5 mL centrifuge tube.
9.Centrifuge using a Mini Centrifuge. Discard the filtrate.
10.System for transformation:

11.Incubate at 30℃ for 20min.
12.42℃ heat shock for 40min. pipet 100ul from each tube onto the appropriate plate, and spread the mixture evenly across the plate. Incubate at 30℃ for 2-3 days. Position the plates with the agar side at the top, and the lid at the bottom.
13.Prepare plasmid for sequencing.

Measure protein concentration

We used Pierce BCA Protein Assay Kit (Thermo Fisher Scientific) to measure protein concentration.
1. Measure OD at 280 nm to get rough protein concentration, and then diluted the protein to 0.5-1 mg / mL.
2. Prepare the reaction solution: reagent A and B in the BCA Protein Assay Kit are mixed in a 50: 1 ratio.
3. Pipette 200 uL of reaction solution in the coated wells
4. Pipette 25 uL of diluted protein, mixed it with the reaction solution. Hold at 37 ℃ for 30 min.
5. Measure OD at 562 nm. Protein concentration is measured according to the protein standard curve.

References

http://parts.igem.org/Help:Protocols/Transformation
https://2015.igem.org/Team:Aachen/Project/Overview
http://www.zymoresearch.com/category/all-products
http://www.corning.com/worldwide/en/products/life-sciences/resources/brands/axygen-brand-products.html
http://www.tsingke.net/shop/
http://www.cwbiotech.com/

---