Team:Shenzhen SFLS/Protocol

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• Judging Form

• 1. Transformation
• 2. Select monoclonal colonies of BL21 cells
• 3. Inducing the E.coli to produce protein
• 4. Preserve seed cells
• 5. Purification of proteins
• 6. The preparation of SDS-Page gel
• 7. Bacteria/plasmid PCR

Transformation

Materials and equipment:

50 ul BL21 competent cell, 1 ul plasmid, ice, 42℃ water, 300 ul non-antibiotic LB nutrient solution, LB plates with kanamycin (K+), shaker, pipettes, pipette tips, EP tubes, palette knife, alcohol burner.

Steps:

• 1) Take competence and plasmid out and put them on ice, and wait for melting.
• 2) Add 1μL of plasmid and 50μL of competence into an EP tube.
• 3) Put it into ice for 30 minutes.
• 4) Put it into water with 42℃ for 90 seconds.
• 5) Put it into ice for 5 minutes.
• 6) Add 300μL of nonreactive LB fluid medium.
• 7) Put the tube in to shaker, with 37℃, 200 rpm, for 40 minutes to 1 hour.
• 8) In the benchtop, light the burner and put the palette knife on it to sterilize. Then take 200μL of bacteria solution out and drop it on one side of the medium. After the knife cool down, use it to spread evenly. After the liquid is dry, you can do the next one.
• 9) Put the medium into incubator for 12-16 hours.

Notice:

• ● Both liquid and solid mediums should be preserved at 4℃
• ● Take out plasmid first because competence is easy to unfreeze
• ● Do not shake or blow the bacteria solution

Select monoclonal colonies of BL21 cells

Materials and equipment:

LB nutrient solution with kanamycin (K+), overnight cultured plates, shaker, pipettes, pipette tips, alcohol burner.

Steps:

• 1) Choose monoclonal colonies that looks like small, white, rounded dot
• 2) In the benchtop, use pipette tips to dig out the monoclonal bacteria slightly
• 3) Add 5mL of LB fluid medium with kana in each 15mL shaking tube.
• 4) Add the bacteria in each tube
• 5) Put the tube into shaker, with 200rpm, 37℃.

Notice:

• ● Do not dig too much of medium
• ● Steps 2- 4 should be accomplished in benchtop.
• ● Remember to use the burner to hit the pipette tips after you use them to dig bacteria.

Inducing the E.coli to produce protein

• 1) Add 1ml of the E.coli solution of 5ml system into a conical flask, add 99ml LB with K+ to make into 100ml inducing system (2ml for 200ml system)
• 2) Culturing in the concentrator, 37°C for 1-1.5h to OD0.6-0.8
• 3) Add 100 ul IPTG into the solution till the final concentration to 0.5M, express proteins in the concentrator, 37°C, 4h

Preserve seed cells

• 1) Take 300ul solution from step 2 into a clean EP tube
• 2) Add 300ul 50% glycerol in to the solution
• 3) Well mixed, freeze in -80°C

Purification of Proteins

Bacteria SDS-PAGE

• 1) Take 400 ul bacteria solution, 14000rpm centrifugation, eliminate the supernate
• 2) Add 40 ul Non-denatured Lysis Buffer
• 3) Add 10 ul protein loading buffer 5X
• 4) Well mixed, boiling, 98°C, 20min
• 5) Take 8 ul of the solution for electrophoresis

Dye SDS-PAGE Gel

• 1) Wash the gel gently
• 2) Add Coomassie Brilliant Blue Quick Dye, covering the gel
• 3) Dye for 1h
• 4) Recycle the dye and wash the gel gently, removing residual dye
• 5) Steep the gel in clean water, place on the horizontal rotators, fade for 1h
• 6) Change the water each 20-30min during the step 5

Dye SDS-PAGE Gel

• 1) Wash the gel gently
• 2) Add Coomassie Brilliant Blue Quick Dye, covering the gel
• 3) Dye for 1h
• 4) Recycle the dye and wash the gel gently, removing residual dye
• 5) Steep the gel in clean water, place on the horizontal rotators, fade for 1h
• 6) Change the water each 20-30min during the step 5

His-tag purification

• 1) Collect the product of induction.
• 2) Resespend the product by cell lysis in the ratio of 1g to 4ml.
• 3) Add His-tag lysozyme and GuHCl to make a resultant concentration of 1mg/ml and 8M respectively
• 4) Conduct Ultrasonic decomposition
• 5) Centrifuge under 4 celsius degrees, 15000rpm for 25mins
• 6) Load the purification tube with Ni-NTA purification resin
• 7) Collect the supernatant from step 5 and run it through the purification tube
• 8) Wash the tube for 5 times using 1ml washing buffer
• 9) Elute the protein for 8 times using 1ml elution buffer.
• 10) Collect the samples from each step

The preparation of SDS-Page gel

• 1) Find a beaker and wash it to be clean.
• 2) Produce the resolving gel solution first
• 3) Decide concentration (in this case 15%) according to protein molecular weight and add corresponded volume of reagent into the beaker.

Resolving gel

Acrylamide range 13.5-27%	27%	24%	20%	17.5%	15%	13.5%
44.4% Acrylamide/1.2% Bis	12.16 mls	10.81 mls	9.01 mls	7.88 mls	6.76 mls	6.08 mls
1M Tris/HCI pH=8.8	7.5 mls	7.5 mls	7.5 mls	7.5 mls	7.5 mls	7.5 mls
Distilled water	0.03 mls	1.38 mls	3.18 mls	4.31 mls	5.43 mls	6.11 mls
10% SDS	200 uls	200 uls	200 uls	200 uls	200 uls	200 uls
10% Ammonium Persulfate	100 uls	100 uls	100 uls	100 uls	100 uls	100 uls
TEMED	10 uls	10 uls	10 uls	10 uls	10 uls	10 uls

Stacking gel

22.2% Acrylamide/Bisacrylamide	2 mls
Distilled water	6.6 mls
1M Tris/HCI pH=6.8	1.25 mls
10% SDS	100 uls
10% Ammonium Persulfate	50 uls
TEMED	5 uls

• 1) Mix the solution evenly quickly
• 2) Use pipette to transfer the solution into the SDS glass pane up to 4cm below the top and fill the left with water to prevent oxygen getting in and inhibiting polymerisation.
• 3) After 20mins, when a boundary line forms between the gel and the water, pour the water directly.
• 4) Then producing the stacking gel solution and transfuse and fill the glass pane with it. And insert the comb gently immediately into the glass pane.
• 5) After 30 mins, the gel polymerise and can be used for electrophoresis later.
• 6) Add loading buffer (10ul protein mixed with 2.5ul loading buffer) to the preliminary protein solution and heat it with water bath at 100℃ for about 10 mins.
• 7) Fill the first grid of the gel with 4ul marker and others with 10ul protein solution from step 9 for gel electrophoresis, 120V, 1h

Bacteria/Plasmid PCR

We conducted an extra plasmid PCR and electrophoresis to ensure our design is correct. The PCR is conducted in the system as follows:

reagents	plasmid	Q5 master mix 2x	F primer	N primer	ddH2O
Amount(concentration)	1ul (100ng/ml in ddH2O)	15ul	1.5ul	1.5ul	11ul

The PCR program is as follow

98°C	55
98°C	15s
55°C	15s
72°C	30s/kb
72°C	3min
12°C	∞