Cloning

PCR

Reaction system:

1.

\(H_2 O\ \ 2\mu L\)

\(10\mathrm{x}\ \ Taq \ \ buffer \ \ 5\mu\mathrm{L}\)

\(2.5mM \ \ dNTP \ \ 1\mu\mathrm{L}\)

\(R+F-Primer \ \ 10\mu\mathrm{L}\)

\(Template \ \ 10\mu\mathrm{L}\)

\(Taq \ \ 2.5\mu\mathrm{L}\)

\(Universal \ \ DNA \ \ polymerase \ \ TransGen\)

2.

\(H_2 O\ \ 20\mu\mathrm{L}\)

\(5\mathrm{x}\ \ Taq\ \ buffer\ \ 10\mu\mathrm{L}\)

\(2.5mM\ \ dNTP\ \ 5\mu\mathrm{L}\)

\(R+F-Primer\ \ 44\mu\mathrm{L}\)

\(Template\ \ 10\mu\mathrm{L}\)

\(Taq\ \ 1\mu\mathrm{L}\)

3.

\(primeSTAR\ \ from\ \ Takara\)

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

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

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

\(Template\ \ 2\mu\mathrm{L}\)

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

Ligation

LR reaction

1. Entry linearization

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

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

100 ng/ul linear Entry: 0.5 ul

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

LR Clonase II enzyme mix: 1 ul

ddH2O: 0.5 ul

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

transform, 4 ul, reactant transform 50 ul competent cells

Transformation

Material:

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 1mL 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:1000mL PH=7.4

Autoclaving 115℃，20min

Detetion

SDS-PAGE

Materials

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

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

Protocol

1. Cut tips. Add 30μL Ni 6 fast flow Beads into 1.5mL EP.
2. Add 1mL 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 1mL 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

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

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. Picked 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 ul 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.
27. Balance each tube and centrifuge at 100000 g for 1 h at 35℃. The pellet is fine purified product.

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.