Team:Xiamen city/Experiment
Experiment
● LB Kanamycin Culture
LB Kanamycin Culture(liquid)
200ml double distilled H2O (ddH2O)
2g Tryptone
2g NaCl
1g Yeast Extract
0.2ml Kanamycin 50mg/ml
LB Kanamycin Culture (solid)
200ml ddH2O
2g Tryptone
2g NaCl
1g Yeast Extract
0.2ml Kanamycin 50mg/ml
3g Agar
● PCR Extraction for the gene fragments of SOD and CAT
SOD/CAT Extraction
Material 25ᶙL ddH2O
3ᶙL 25MM MgSO4
5ᶙL 2MM dNTPs
5ᶙL pmol/ᶙL Primer Front
5ᶙL pmol/ᶙL Primer Rear
1ᶙL Template
5ᶙL 10*PCR Buffer
1ᶙL KOD-Plus-Neo (polymerase)
Procedure
For pre-denaturation--95℃ 5min
For denaturation--95℃ 30s
For annealer--55℃ 30s
For extension--68℃ 4min
Repeat above step for 35 times
For complete extension--68℃ 5min
For preservation-- 4℃
● Gel test
Motive: detect whether the genes of SOD and CAD multiply successfully
Material: 1*TAE buffer
6*TAE buffer
Agarose
DNAgreen
SOD gene fragments
CAT gene fragments
Loading marker
Procedure:
1. Produce gel.
A. pour 1*TAE buffer in a conical flask. Weight matched mass of agarose.
B. Cover the mouth of flask with plastic wrap. Stick some tiny holes on plastic wrap. Heat the conical flask with microwave oven until agarose completely dissolving.
C. Cool flask down to 50-60℃, and then add 1/10000 DNAgreen. Blend the gel.
D. Pour gel into mould and insert comb. Wait until solidification.
2. Blend SOD fragments with 6*TAE buffer (1:5). Blend CAT fragments with 6*TAE buffer (1:5)
● Gel Extraction
Material: Buffer DE-A
Buffer DE-B
Washing Buffer 1
Washing Buffer 2
Spin column
Target gel
1. Cut gel containing target DNA fragment and transfer target gel into a 1.5ml Eppendorf (EP tube)
2. Inject Buffer DE-A (Volume of Buffer: Volume of gel = 3:1). Heat EP tube until gel completely melting.
3. Inject Buffer DE-B (Volume of Buffer DE-B : Volume of Buffer DE-A = 2:1)
4. A. Transfer mixed liquid into 2ml EP tube equipped with spin column. Centrifuge tube at the centrifugal force of 12000g for 1min. Pour out the filtrate.
B. Inject 500ᶙl Buffer W1. Centrifuge tube for 30s. Pour out the filtrate.
C. Inject 700ᶙl Buffer W2. Centrifuge tube for 30s. Pour out the filtrate. Repeat the step C again.
Centrifuge tube at the speed of 12000rmp for 1 min.
5. Transfer spin column into a new tube. Inject 25-30ᶙl ddH2O. Wait for 1min and then centrifuge tube. Collect the filtrate containing target DNA fragments.
● Digestion
Digestion DNA fragment of SOD
1ᶙg DNA
1ᶙl Nhel
5ᶙl 10*NE Buffer
44ᶙl ddH2O
Digestion DNA fragment of CAT
1ᶙg DNA
1ᶙl XhoI
5ᶙl 10*NE Buffer
44ᶙl ddH2O
Digestion DNA fragmentof Plasmid
1ᶙg plasmid
1ᶙl Nhel
1ᶙl XhoI
10ᶙl 10*NE Buffer
38ᶙl ddH2O
● Ligation
After cleavage, blend plasmids with SOD, and plasmids with CAT together (Cb : Cb = 1:1). Then blend blended groups together. All the step needs involvement of T4 DNA ligase.
● DNA sequencing
Confer simple of CAT and SOD to sequencing center (Use T7 common primers).
● Transform and multiplication E. coli(DH5α and BL21DH3)
For melting-1. Take out the four Eppendorfs (two of them contain DH5α competent cells and the other contain BL21DH3 competent cells from -80℃ condition)，and insert these four tubes into ice, wait 5 min.
For plasmids attaching to E. coli membrane-2. Inject recombinant plasmids (SOD and CAT) into four tubes (DH5α with SOD, DH5α with CAT, BL21DH3 with SOD, BL21DH3 with CAT), and wait 25 min
For accepting plasmids-3. Put these tubes in 42℃ condition for 45s, and then bring back to the ice immediately.
For growing cell wall-4. Inject 700ᶙl LB liquid culture (free of antibiotic). Shake tubes at the speed of 200rpm in Shaking Incubator for 60 min
For concentrating-5. Centrifuge tubes at the speed of 5000rmp for 1 min. Extract the upper transparent liquid until 100ᶙl and use pipette to blend upper transparent liquid and E. coli in the bottom.
Apply each tubes of liquid evenly on the different LB cultures (contain Kanamycin). Make signatures to each LB culture.
For propagating-6. Inverse petrie dish in 37℃ condition for 12h.
● Preliminary screening
1. Select 10 single E. coli colonies of four LB cultures, and sign 1-10 to these 10 colonies in order to select again. Put colonies into 1.5ml EP tubes
2. Blend each E. coli colony with PCR system for extraction.
PCR system: 9ᶙl mix (including dNTPs, polymerase, and PCR buffer)
9ᶙl ddH2O
1ᶙl T7 primer
1ᶙl template (E. coli colonies)
3. Take the step of gel test and gel extraction to every colony in order to preliminary screening out target colonies.
● DNA sequencing for secondary screening
1. Confer simple of target colonies to sequencing center (Use T7 common primers).
2. Propagate target colonies in BL liquid culture.
●Purification of proteins
1. Pour target E. coli liquid culture in 50 ml EP tubes.
2. Centrifuge EP tubes and collect bottom E. coli.
3. Use ultrasonic wave to disrupt the E. coli,
4. Centrifuge the disruption of E. coli at the centrifugal force of 12000g. Collect the upper
transparent liquid. Filter upper transparent liquid through nickel column.
5. Wash through nickel column by different molar concentration of imidazole. (0mM, 50mM, 100mM, 250mM, 500mM), collect and sign each filtrate.
The imidazole systems:
0mM imidazole: 15ml loading buffer
50mM imidazole: 0.15ml 5M imidazole and 14.85ml loading buffer
100mM imidazole: 0.3ml 5M imidazole and 14.7ml loading buffer
250mM imidazole: 0.75ml 5M imidazole and 14.25ml loading buffer
500mM imidazole: 1.5ml 5M imidazole and 13.5ml loading buffer
6. Blend filtrate with gel buffer system for protein gel test.
7. The result indicates that filtrate with 100mM imidazole contains SOD/CAT.
●Test SOD enzyme activity
Material: SOD testing buffer（buffer）
nitroblue tetrazolium (NBT)
Xanthine Oxidase (XO) solution
40*Primer fluid
SOD solution
1. Preparation
A. Make working solution. Blend XO solution with NBT and SOD testing buffer together (1:1:158)
B. Blend 40*primer fluid with SOD testing buffer (1:39)
C. Dilute SOD solution to five ranks (20U/ml, 10U/ml, 5U/ml, 2U/ml, 1U/ml, 0.5U/ml)
2. A. Set up 8 groups in 96-hole plate.
Samples: 20ᶙl SOD solution (20U/ml, 10U/ml, 5U/ml, 2U/ml, 1U/ml, 0.5U/ml)+160ᶙl working solution+20ᶙl primer fluid
Blank1: 20ᶙl buffer+160ᶙl working solution+20ᶙl primer fluid
Blank2: 40ᶙl buffer+160ᶙl working solution
Blank3: 20ᶙl SOD solution+20ᶙl buffer+160ᶙl working solution
B. Incubate the groups for 30min in 37℃.
C. Test absorbance in 560nm.
3. Calculate SOD enzyme activity
Inhibition percentage=[(Ablank1-Ablank2) - (Asample-Ablank3)]/(Ablank1-Ablank2) * 100%
IP (SOD solution:0.5U/ml) = [(0.236-0.041) - (0.101-0.043)]/(0.236-0.041)*100%
=0.137/0.197*100%
= 69.543%(30%<IP<70%)
Enzyme activity percentage=inhibition percentage / (1-inhibition percentage) units
=69.543% / (1-69.543%) units
= 2.283units (SOD solution:0.5U/ml)
●Test CAT enzyme activity
Material: CAT testing buffer
H2O2(1M) solution
CAT termination solution
Chromogenic substrate
Peroxidase
CAT
1. Preparation
A. Dilute H2O2 solution to 5mM and 250mM.
B. Make 4ml chromogenic working solution on the ice (peroxidase : chromogenic substrate=1:1000).
2. determination of standard curve
A. Inject 0, 12.5, 25, 50, 75ᶙl 5mM H2O2 solution in 1.5ml EP tubes. Inject CAT testing buffer to each tube to 100ᶙl.
B. Transfer 4ᶙl liquid in each tube into 96-hole plate. Incubate plate for 20min in 25℃ and test the absorbance in A520.
C. Calculate standard curve.
3. determination of sample
A. Set up 5 samples and blank in EP tubes, incubate EP tubes for 5min in 25℃.
S1: 0.125ᶙl CAT + 39.5ᶙl buffer + 10ᶙl 250mM H2O2
S2: 0.25ᶙl CAT + 39ᶙl buffer + 10ᶙl 250mM H2O2
S3: 0.5ᶙl CAT + 38ᶙl buffer + 10ᶙl 250mM H2O2
S4: 1ᶙl CAT + 36ᶙl buffer + 10ᶙl 250mM H2O2
S5: 2ᶙl CAT + 32ᶙl buffer + 10ᶙl 250mM H2O2
Blank: 40ᶙl buffer + 10ᶙl 250mM H2O2
B. Inject 450ᶙl termination solution. Bland the tubes gently.
C. Inject 40ᶙl buffer in 6 new EP tubes. Inject 10ᶙl of aforementioned six systems separately in new tubes. Bland the tubes gently.
D. Transform 10ᶙl solution from six tubes to 96-hole plat. Inject 200ᶙl chromogenic working solution in 6 holes.
E. Incubate plate for 15min in 25℃. Test the absorbance in A520.
4. Calculation
Standard curve: A520=84.976[mM H202] + 0.0605
The rest H2O2 in sample= (Asample-Ablank)/k
CAT enzyme activity= (H2O2 Moles consumed × Dilution times) / (Time×Volume×Concentration of protein) units
Takiishi, T. et al. Reversal of Diabetes in NOD Mice by Clinical-Grade Proinsulin and IL-10-Secreting Lactococcus lactis in Combination With Low-Dose Anti-CD3 Depends on the Induction of Foxp3-Positive T Cells. Diabetes 66, 448-459, doi:10.2337/db15-1625 (2017).
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