Team:BNU-China/Protocol

PROTOCOL

Protocol

This is our Protocol

Cloning

PCR

Reaction system:

Universal DNA polymerase TransGen
ddH2O	10x Taq Buffer	2.5mM dNTP	R+F-Primer	Template	Taq
2μL	5μL	1μL	10μL	10μL	2.5μL

TaKaRa TaqTM
ddH2O	5x Taq Buffer	2.5mM dNTP	R+F-Primer	Template	Taq
20μL	10μL	5μL	44μL	10μL	1μL

primeSTAR from TaKaRaTM
ddH2O	2x primeSTAR	R+F-Primer	Template
21μL	25ML	2μL	2μL

Process:

98°C 2min

\( \begin{equation} \left. \begin{array}{lcl} {98°C\ 10s} \\ {56°C\ 15s} \\{72°C\ 30s} \end{array} \right \} Cycle\ 35 \end{equation} \)

72°C 5min

4°C ---

98°C 2min

\(\begin{equation}\left. \begin{array}{lcl} {98°C\ 10s} \\ {55°C\ 5s} \\{72°C\ 8s} \end{array} \right\}Cycle\ 35\end{equation}\)

72°C 5min

15°C ---

Fusion PCR：

1. basic PCR
2. using the PCR product of step 1 as template does PCR
3. using the PCR product of step 2 as template does PCR, but first five cycles don’t add primer, after first five cycles, the sixth cycle adds primer and continue PCR.

The system of step 2:

\(H_2 O\ \ 21\mu\mathrm{L}\)

\(2\mathrm{x}\ \ primeSTAR\ \ 25m\mathrm{L}\)

\(R+F-Primer\ \ 2\mu\mathrm{L}\)

\(Template①\ \ 1\mu\mathrm{L}\)

\(Template②\ \ 1\mu\mathrm{L}\)

Electrophoresis---Gel Purification

Material:

Agarose gel: 1% agarose dissolved in 1 x TAE + gelstain

Protocol:

We used gelstain to stain the DNA and imaged it in a Transilluminator.

We used the gel extraction kit to get the objective fragment.

We used the DNA fragment purification kit to get the objective fragment.

Digestion

50μL reaction system
Reagent	10x \(\ \mathrm{H \ buffer}\)	\(Eco\mathrm{R}\ \mathrm{I}\)	\(Pat\ \mathrm{I}\)	\(\mathrm{Plasmid}\)	\(\mathrm{H_2 O}\)
Dosage	5μL	1.5μL	1.5μL	15μL	27μL

10μL reaction system
Reagent	10x \(\ \mathrm{H \ buffer}\)	\(Eco\mathrm{R}\ \mathrm{I}\)	\(Pat\ \mathrm{I}\)	\(\mathrm{Plasmid}\)	\(\mathrm{H_2 O}\)
Dosage	1μL	0.3μL	0.3μL	3μL	5.4μL

Ligation

Ligation reaction system
Reagent	DNA	Plasmid	T4 buffer	T4 ligase
Dosage	7μL	1μL	1μL	1μL

LR reaction

1. Entry linearization

β2-TOPO（plasmid concentration 117ng/μL） NotI 37°C enzyme digestion for the night

50μL Single enzyme system
10x BufferH	5μL
DNA	20μL
ddH2O	12.5μL
Enzyme	2.5μL
0.1%BSA	5μL
0.1%Triton X-100	5μL

2. LR system (\(4\mu\mathrm{L}\)):

100 ng/μL linear Entry: 0.5 μL

destination vector: 1 μL (pCambia1300-nluc / pCambia1300-cluceach one)

LR Clonase II enzyme mix: 1 μL

ddH₂O: 0.5 μL

mix slightly, water base for 5 h at 25°C 

transform, 4 μL, reactant transform 50 μL competent cells

Transformation

Material:

LB liquid medium
Reagent	Tryptone	Yeast extract powder	NaCl
Dosage	10 g/L	5 g/L	10 μL

Protocol:

Preparation of the competent cells

1μL ligation product + 50μL cells

Heatshock of Trans5α(42°C, 45s)

Put on ice(2min)

Add 500μL LB media and incubate for 1h(37°C, 150rpm)

Centrifuge at 4000rpm for 1min and remove 400μL supernatant

Resuspend the pellets using the left supernatant

Spread plates(with Kan;CHL)

Incubate for 12~16h(37°C)

Protein expression

1. Inoculated 3 mL LB media including relevant antibiotics with the monoclonal colony of expression plasmid, incubate for 12~16h(37°C, 190rpm)
2. Inoculated 100 mL TM expression media including relevant antibiotics with the 1 mL bacteria liquid, incubate for 3h(37°C, 250rpm,OD600=0.6~0.8)
3. Add IPTG into it until its final concentration is 1mmol/L, incubate for 4~6h(37°C, 250rpm)
4. Centrifuge at 6000rpm for 10min and remove supernatant
5. Gather sediment, cryopreserve at -20°C

Material:

TM expression medium:1000 mL PH=7.4

Reagent	tryptone	Yeast extract powder	NaCl	glucose	glycerol
Dosage	1.2g	2.4g	1.0g	1.0g	0.6 mL

Autoclaving 115℃, 20min

Detection

SDS-PAGE

Materials

Gel	Tris-HCl	Acr/Bis 30%	SDS 10%	ddH2O	TEMED	AP 10%
Stacking Gel(4%)	pH=6.8 500μL	500 μL	25 μL	1350μL	2.5μL	12.5μL
Running Gel(12%)	pH=8.8 1250μL	2000 μL	50 μL	1675μL	2.5μL	25 μL
Running Gel(18%)	pH=8.8 1250μL	3000 μL	50 μL	675 μL	2.5μL	25 μL

Running Buffer
Reagent	Tris-HCl	Glycine	(w/v) SDS
Dosage	25 mmol/L	0.192 mol/L	0.1%

Protocol

The SDS polyacrylamide gels are prepared in the so-called PerfectBlue™ Twin Double Gel System.

After ensuring that the equipment is waterproof, the 12 % (or 18%) running gel is mixed and filled into the chamber. Pipetting about 1 ml of H₂O on top of the running gel to seal the gel.

After polymerization, the remaining H₂O is removed and the 12 % stacking gel is filled on top. Insert a comb to create sample pockets.

After the stacking gel also polymerized, 1 x running buffer is used to run the Double Gel System via the SDS gel.

After loading the generated pockets with the samples, the stacking gel is run at 100 V and then running gel at 120 V.

Western Blot

System

PBST:1000 mL(PH=7.4)
Reagent	NaCl(137mM)	KCl(2.7mM)	Na2HPO4(10mM)	K2HPO4(2mM)	Tween-20
Dosage	8g	0.2g	1.44g	0.24g	0.5 mL

Imprint buffer：2000 mL (PH=8.3) Transfer Buffer
Reagent	Tris	Gly	Methanol
Dosage	6.06g	28.8g	400 mL

Protocol

Transfer （Prepare transfer Buffer just before glue leaking, and precool at -20℃）.

1. Put the transfer Buffer and the black subface of transfer splint downword, and lay a sponge in it. Several filter paper(three pieces of filter paper), glue(except Stacking Gel).
2. Activation PVDF membrane in advance with anhydrous ethanol, and put it on the membrane.
3. Three layers of filter paper, sponge, Squeeze out of the bubbles, turn tight.
4. The black subface electric rotary groove stick to each other, put in ice.
5. 110V, 120min.
6. 5% skim milk powder (prepared by PBST), block for a night.
7. Dilute Primary antibody at the proportion of 1:2000 with 3% skim milk powder(add 0.02% sodium azide ), incubate 1h at the room temperature.
8. PBST elute, wash with shocking for 5min , three times.
9. Dilute Secondary antibody at the proportion of 1:2000 with 3% skim milk powder, incubate 1h at the room temperature.
10. PBST elute, wash with shocking for 5min, three times.
11. Color development.

Ni-beads protein purification

NPI-10 buffer（1L）pH=8.0 filtration sterilization
Reagent	NaH2PO4·H2O	NaCl	imidazole
Dosage	6.9	17.54	0.68

NPI-20 buffer（1L）pH=8.0 filtration sterilization
Reagent	NaH2PO4·H2O	NaCl	imidazole
Dosage （g）	6.9	17.54	1.36

NPI-250 buffer（1L）pH=8.0 filtration sterilization
Reagent	NaH2PO4·H2O	NaCl	imidazole
Dosage （g）	6.9	17.54	17.0

Protocol

1. Cut tips. Add 30μL Ni 6 fast flow Beads into 1.5 mL EP.
2. Add 1 mL NPI-10 buffer, mixing wash, sedimentate at low speed and wash 3 times.
3. Centrifuge and absorb supernatant into buffer. 4℃ binding 3h, rotate and mix.
4. After binding, Put on ice(5min), Centrifuge at 2000rpm for 1min.
5. Absorb 80μL supernatant as control and remove the other supernatant, add 1 mL NPI-20 washing, upside and down to mix, still standing, Centrifuge at 2000rpm for 1min(4℃), wash 3~5 times.
6. Add 500μL NPI-250 into Beads, rotate and mix for 15min, gather supernatant, add 500μL NPI-250 , rotate and mix for 15min, gather supernatant again.

Renaturation of the inclusion bodies

Binding buffer (1L) pH=8.0
Reagent	NaCl（500mmol/L）	Na3PO4·12H2O(20mmol/L)	imidazole (20mmol/L)
Dosage（g）	29.22	7.6	1.36

After cell disruption, sediment dissolves in binding buffer(8mol/L urea)

Washing buffer（1L）
Reagent	Tris-HCl(50mmol/L)	EDTA(5mmol/L)	NaCl(100mmol/L)	Triton X-100(1%)
Dosage	100 mL	1.8612g	5.844g	10 mL

1. Induced expression
2. Collect sediment after ultrasonication, use washing buffer including 2, 3mol/L urea to wash sediment in turn. Finally, use washing buffer including 8mol/L urea to dissolve. Centrifuge and absorb supernatant, measure protein concentration and make it keep about 1mg/mL. Dialyze these supernatant using binding buffer including concentration gradient urea(6,5,4,3,2,1,0.5 and 0mol/L).

Extraction tubulin from porcine brains

1. Pick up 20 porcine brains from Beijing No.5 Meat Processing. For tubulin extraction experiment, the brains should be as fresh as possible. Take an ice box to store the brains. Avoid contact between the brains.
2. While getting the brains, another student should stay in the lab and prepared the centrifuge (set one at 4℃ and another at 37℃, also pre-warm the rotor). Put electronic balance, grinder, a graduated cylinder in refrigerator and pre-warm the glycerol and PEM in 37℃. prepare the fresh ATP and GTP buffer in the morning.
3. Clean the brain by tearing off the meninges and blood clots using kim wipes or by hand.
4. After cleaning, weigh the brains, put the brains in the blender, then add the same volume buffer PEM(with 1 mM DTT) in it accordingly.
5. Homogenate the brain for 3 s, 10 times, time interval between two homogenate is 5 s, in order to avoid destroy the tubulin because of high thermos.
6. Pour the homogenate into a flask, incubate in 4℃ for 30 min to depolymerize microtubules.
7. Pour the homogenate into tubes for Type 45Ti rotor and balance each tube.
8. Centrifuge at 8000 rpm for 40 min at 4 ℃. Filter the supernatant with 4 gauzes. Then centrifuge the filtrate at 40000 g for 40 min at 4℃.
9. Add 1/2 volume of warmed glycerol drop-wise with continuous shaking, mix gently but thoroughly. Add GTP (final concentration 0.1 mmol/L), MgCl₂ (final concentration 3 mmol/L), and EGTA (final concentration 1 mmol/L). Incubate in a 37℃ water bath for 1 h, shake gently and occasionally.
10. Balance each tube and centrifuge at 100000 g for 40 min at 35℃.Discard supernatant, the pellet is crude extracts. Split charge them into 50 ml centrifuge tubes, each tube contains 5 g. Snap freeze the tubulin in 15 μL aliquots in liquid nitrogen and further stored at -80 ℃.
11. When going to do refined depuration, melt the freezing crude extract at 4 ℃ refrigerator on ice over night. Pop out 1 g of the pellet out of the tubes with a spatula. Put the pellets in the Dounce grinder, then add cold PEM in the tube to wash off residual pellets.
12. Re-suspend the pellets with grinder, keep the grinder on ice, and grinding occasionally. After 30 min, pour out the solution and rinse the grinder with cold PEM. Total re-suspended volume is 5 ml.
13. Add GTP (final concentration 0.1 mmol/L). Place it on ice for 1 h to depolymerize. Shake it occasionally.
14. Centrifuge the depolymerized tubulin at 100000 g for 40 min at 4 ℃.
15. Recover the supernatant and pour it into a flask. Add equal volume of warmed PIPES( pH =6.9 ), DMSO(final concentration 10%), GTP (final concentration 0.1 mmol/L), MgCl₂ (final concentration 1 mmol/L) and EGTA (final concentration 1 mmol/L). Mix gently but thoroughly.
16. Incubate in a 37℃ water bath for 1 h. the solution would look cloudy.
17. Balance each tube and centrifuge at 100000 g for 1 h at 35℃.
18. Discard the supernatant. Risen the pellet briefly with cold PEM, add GTP (final concentration 0.1 mmol/L). Place it on ice for 1 h to depolymerize. Shake it occasionally.
19. Centrifuge the depolymerized tubulin at 100000 g for 40 min at 4 ℃.
20. Recover the supernatant and pour it into a flask. Add equal volume of warmed PIPES ( pH= 6.9 ), DMSO(final concentration 10%), GTP (final concentration 0.1 mmol/L), MgCl₂ (final concentration 1 mmol/L) and EGTA (final concentration 1 mmol/L). Mix gently but thoroughly.
21. Incubate in a 37℃ water bath for 1 h. the solution would look cloudy.
22. Balance each tube and centrifuge at 100000 g for 1 h at 35℃.
23. Discard the supernatant. Risen the pellet briefly with cold PEM, add GTP (final concentration 0.1 mmol/L). Place it on ice for 1 h to depolymerize. Shake it occasionally.
24. Centrifuge the depolymerized tubulin at 100000 g for 40 min at 4℃.
25. Recover the supernatant, add equal volume polymerize buffer (containing 100 mmol/L PIPES-KOH, 2 mmol/L EGTA, 2 mmol/L MgCl₂, 2 mmol/L GTP and 60% glycerol).
26. Incubate in a 37℃ water bath for 1 h. Adding a series concentrations of taxol.
27. Balance each tube and centrifuge at 100000 g for 1 h at 35℃. The pellet is fine purified product.

Preparing STM samples

1. Use 200 # copper mesh to prepare the samples. This procedure was done in Institute of Biophysics, Chinese Academy of Science. Pretreat the copper mesh with Varian Plasma Cleaner PDC-32G.
2. Add protein solution on copper mesh. Keep still for 1 min.
3. Add 3 drops of uranyl acetate on parafilm. Mix the protein binding side with uranyl acetate thoroughly.
4. Store the sample carefully. Observe by transmission electron microscopy in Beijing Normal University.

The absorbance value determination of tubulin at 350 nm

1. Take out the tubulin monomer solution after precision purification from -80℃, and melt on the ice.
2. Prepare 10 um tubulin monomer solution with the same concentration, and add isopyknic polymerization buffer (the formula is the same as the polymerization buffer used in precision purification). This step finished on the ice in order to avoid the influence of the Spontaneous tubulin polymerization because of the high room temperature.
3. Prepare a series concentration gradient of taxol solution and add the tubulin polymerization system.
4. Incubate for 1 hour at 37℃.
5. Use Nanodrop UV-IS to determinate absorbance value, and set measurement parameters 280 nm, 350 nm and 750 nm, but we only read at 350nm.
6. Use isopyknic mixed solution of polymerization buffer and PEM buffer to calibrate baseline, each group of samples detect three times in parallel.

Improvement

Culture and collection

1. Use LB medium to preculture transformed media 5 ml for 12 hrs, 200 rpm/ 37℃.
2. Culture 5 mL preculture media into 100 mL TB medium for about 3 hrs until OD=0.4～0.6, 200 rpm/ 37℃.
3. Add arabinose or turn to 42℃ to induce P(3HB) expression for 72 hrs, 220rpm.
4. Collect cells and centrifuge for 3 min, 5,000 rpm.
5. Remove supernatant and suspend with pure water.
6. Centrifuge again for 3 min, 5,000 rpm and remove its supernatant.

Extract PHB¹

• Centrifuge settings: 4000RPM, 10mins.
• Scale as appropriate.
• After each centrifuge step the supernatant should be poured off.

1. Resuspend precipitation in 10 mL Triton X-100(1% v/v in PBS) for 30mins at room temp.
2. Centrifuge, resuspend in 10 mL PBS.
3. Centrifuge, add 10 mL sodium hyperchlorite solution and incubate at 30˚C for 1 hour.
4. Centrifuge, wash with 10 mL 70% EtOH.
5. Allow powder to dry.

[1]: Shahryar Shakeri, Comparison of intracellular polyhydroxybutyrate granules formation between different bacterial cell subpopulations by flow cytometry, Jundishapur Journal of Microbiology 2011.

Agents formula

TB medium (1L)
yeast extract	tryptone	K2HPO4	KH2PO4	Glycerol	Glucose	ddH2O
24 g	12 g	72 mM	17 mM	8 mL	2%	add to 1 L

PBS (1L)
NaCl	KCl	Na2HPO4·12H2O	KH2PO4·3H2O	pH	ddH2O
8 g	0.2 g	3.63 g	0.31g	adjust to 7.4	add to 1 L