Team:XMU-China/protocols

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Protocols

1 General protocols
1.1 Stock solution
50 mg/mL Kanamycin
- 0.5 g Kan, 10 mL water, filter sterilize with millipore express membrane, freeze in aliquots
100 mg/mL Ampicillin
-1 g Amp, 10 mL water, filter sterilize with millipore express membrane, freeze in aliquots.
50 mmol/L Arabinose
- 0.1876 g Arabinose, 25 mL water, filter sterilize with millipore express membrane.
0.25 mg/mL Anhydrotetracycline
- 0.1 mg Anhydrotetracycline, 0.4 mL PBS, filter sterilize with millipore express membrane.

 

1.2 Preparation of Competent BL21
Thaw an aliquot of cells (without any plasmid in them) on ice
- To 50 mL of sterile LB, add 100μL aliquot of the thawed cells: remember, this LB does not have any antibiotic in it, so work as aseptically as possible (i.e. autoclave all solutions and use sterile pipettes).
- Grow cells in the shaker at 37 ℃ and 200 rpm, until they reach an OD₆₀₀= 0.3-0.4. This usually takes 1.5-2 hours.
- Ice down the LB with growing cells for 10 min.
- Aliquot into sterile 1.5 mL tubes and spin down at 6000rpm for 10 min at 4 ℃; discard supernatant.
- Ice down sterile 100 mM CaCl₂ and 100mM MgCl₂ solutions during centrifugation.
- Gently resuspend each pellet with 400 μL 0.1 M MgCl₂ and 100 μL 0.1 M CaCl₂.
- Centrifuge 6000 rpm for 10 min and discard supernatant.
- Resuspend each pellet on ice in 100 μL 0.1 M ice cold CaCl₂ and combine into one tube

1.3 Transformation
- Add 10 μL of DNA. Swirl gently with pipette.
- Incubate tubes on ice for 20 min
- Heat pulse tubes in 42 ℃ water bath for 30 seconds.
- Incubate on ice for 2 min
- Add 790 μL of LB broth to each tube and incubate for an hour at 37 ℃ with shaking.
- Spread 100 μL and 50μL of each culture on an LB agar plate containing the appropriate antibiotics and incubate overnight at 37 ℃ (spread using beads).

1.4 Plasmid Purification
- Centrifuge sample in eppendorf tube approximately 1.5 mL at a time, draining off supernatant after each spin and adding more cell solution
-Resuspend the pelleted cells in 250 μL of the resuspension Solution (mixture with Solution I and RNasa A). The bacteria should be resuspended completely by vortexing or pipetting up and down until no cell clumps remain.
- Add 250 μL of the Lysis Solution (Solution II) and mix thoroughly and gently by inverting the tube 5-6 times, letting it stand for 1-2 min at room temperature until the solution becomes viscous and slightly clear.
- Add 350 μL of the Neutralization Solution (Solution III) and mix immediately and thoroughly by inverting the tube 5-6 times.
- Centrifuge for 10 min at 12,000 rpm to pellet cell debris.
- Apply the supernatant to the supplied spin column by decanting. Avoid disturbing or applying the white precipitate.
- Centrifuge for 1 min at 12,000 rpm. Discard flow-through and place the column back into the same collection tube.
- Add 500 μL of the Wash Buffer PB to the spin column. Centrifuge for 1min at 12,000 rpm and discard flow-through. Place the column back into the same collection tube.
- Add 500 μL of the Wash Buffer W to the spin column. Centrifuge for 1min at 12,000 rpm and discard the flow-through. Place the column back into the same collection tube.
- Repeat the step 9 again.
- Discard flow-through and centrifuge for an additional 3 min to remove residual Wash Solution.
- Place the spin column in a clean 1.5 mL centrifuge tube, and pipet 20 μL Elution Buffer TE (prewarm to 60 ℃) directly to the center of the column without touching the membrane. Let it stand for 2 min at room temperature and centrifuge for 1 min at 12,000 rpm.
- Discard the column and store the purified plasmid DNA at -20 °C.

1.5 Reaction system of restriction endonuclease

- System1、2、3 and 4 are used for Standard BioBrick Assembly .
- System 5 and 6 are used for restriction analysis. Digestion of sample: at least 500 ng DNA / 10 μL volume. Digest for 1 h at 37 °C, afterwards inactivated by adding 10× loading buffer and standing for 10 min at room temperature.

1.6 Standard BioBrick Assembly
- Digestion of insert: 2 μg~5 μg DNA / 100 μL volume, 10× H buffer, EcoR I, Spe I. Digestion and inactivation. Clean up the insert via gel electrophoresis. When cutting the insert out of the gel, try avoiding staining or exposure to ultraviolet light of the insert.
- Digestion of vector: 2 μg~5 μg DNA / 100 μL volume, 10× M buffer, EcoR I, Xba I. Digestion and inactivation. Clean up the insert via gel electrophoresis. When cutting the insert out of the gel, try to avoid staining or exposure to ultraviolet light of the insert.

1.7 Suffix Insertion
- Digestion of insert: 2 μg~5 μg DNA / 100 μL volume, 10× M buffer, Xba I, Pst I. Digestion and inactivation. Clean up the insert.
- Digestion of vector : 2 μg~5 μg DNA / 100 μL volume, 10× H buffer, Spe I, Pst I. Digestion and inactivation. Clean up the vector.

1.8 Ligation
- After digestion and clean-up, the next step is ligation. ligation at 16 ℃ for 4 h or at 4 ℃ for 16 h. Table 2 is the system of ligation.
Table 2  Ligation system

 

1.9 Restriction analysis
- Pick one colony with a sterile tip and cultivation in 20 mL LB for overnight at 37 ℃
- Isolation of Plasmid
- Digest BioBrick，the system of Restriction analysis refer to table1
- Gel electrophoresis：add 2.2 μL loading buffer to digestion mixture. An agarose concentration is 1 %.

1.10 Gel Extraction
- Weigh a 1.5 mL centrifuge tube for each DNA fragment to be isolated and record the weight.
- Excise gel slice containing the DNA fragment using a clean scalpel or razor blade. Cut as close as possible to the DNA to minimize the gel volume. Place the gel slice into a pre-weighed 1.5 mL tube and weigh. Record the weight of the gel slice.
- Add Bing Buffer BD at a ratio of 100 μL of solution per 100 mg of agarose gel slices.
- Incubate the gel mixture at 55-65 ℃ for 7-10 min or until the gel slice is completely dissolved. Mix the tube by inversion every few minutes to facilitate the melting process. Ensure that the gel is completely dissolved.
- After the dissolved gel mixture cool down, transfer it to the Spin Columns assembly and incubate for 2 min at room temperature.
- Centrifuge the Spin Columns assembly in a microcentrifuge at 12,000 rpm for 1 min, and discard the flow-through.
- Wash the columns by adding 500 μL of Wash Buffer PE to the Columns. Centrifuge the columns assembly for 1 min at 12,000 rpm, and discard the flow-through.
- Repeat step 7 again.
- Centrifuge the Columns for an additional 3 min to completely remove residual wash buffer.
- Empty the Collection Tube and recentrifuge the column assembly for 1 min with the microcentrifuge lid open (or off) to allow evaporation of any residual ethanol.
- Place the spin column in a clean 1.5 mL microcentrifuge tube, and pipet 20 μL deionized water (pH is 8.0-8.5 and prewarm to 60 ℃)directly to the center of the column without touching the membrane. Incubate at room temperature for 2 min.
- Centrifuge for 1 min at 12,000 rpm. Discard the columns and store the microcentrifuge tube containing the eluted DNA at–20 ℃ .

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2 Characterization
2.1 Fluorescence Measurements
- The samples to be tested are cultured from plates in 20 mL of the Basal Minimal Medium with appropriate antibiotics and incubated overnight at 37 ℃ at 200 rpm.
- The culture is checked for OD₆₀₀ next day and then subculture by the same medium with antibiotics at 37 ℃ shaking for 2 hours.
- Add corresponding inducer at concentration gradients into the above-mentioned culture and keep on incubating. During the time incubating, every 15 min, take 1 mL bacteria liquid, then centrifuge the cells( 6000 rpm, 10 min ) and resuspend them in 1 mL PBS. At last, pipette to a 96 well plate.
- The plate reader made by Molecular Device then read.
- The program does the following:
- In endpoint reads, following measurements are taken in a time interval of 15 min: absorbance (600 nm filter) and fluorescence (485 nm and 520 nm for GFP).
- The results then transfer to excel sheet and interpret.

 

2.2 Protein electrophoresis
2.2.1 Preparation of Samples
- The samples to be tested are cultured in the Basal Medium with appropriate antibiotics, and take 200 μL bacteria liquid to determine its OD₆₀₀ at appropriate time.
- Dilute or concentrate the next 200 μL bacteria liquid in order to let the OD₆₀₀ equals to 4.0 while the computational formula is the actual OD₆₀₀ * 200=2.0 * X, and X presents the total volume of the bacteria liquid after being diluted or concentrated while its unit is μL as well.
- Add 30 μL diluted or concentrated liquid into corresponding 1.5 mL centrifugal tubes, then mix up them with 10 μL loading buffer.
- Put these centrifugal tubes into metal bath and heat them in 100 ℃ in around 5 to 8 min, then centrifuge them at the speed of 13000 rpm for 5 min, the supernatant is what we need.

2.2.2 Manufacture Albumen Gel
- Prepare a clear centrifuge tube in the capacity of 50 mL, and make running gel, high concentration one, following the formula below. Then mix up them and pour the mixture into a glass pane.

- Add some absolute alcohol to planish the top of gel. There will be approximately 60 min for its solidification.
- After solidification, pour out the alcohol and make stacking gel following the formula below. Then mix them up, add the solution onto the running gel in the glass panes until it being filled up with the gel. Insert a clean comb into stacking gel. Wait for about 40 min for stacking gel solidification.

2.2.3 Electrophoresis
- Take out the glass pane with finished gel and then fasten it in an electrophoresis tank. Add some 1× Tank Buffer to detect whether liquid leak or not.     
- Take out the comb slowly and use pipette to add approximately 10 to 20 μL processed samples into the wells in stacking gel.
- Add 1× Tank Buffer until the liquid level is above the platinum line in the electrophoresis tank.
- Cover up the electrophoresis tank and connect it with the electrophoresis device. Set the program 120 V- 60 min and start it up.
- When the green marker band run to the bottom of running gel, stop the device.

2.2.4 Dyeing (Colloidal Coomassie Brilliant Blue)
- Take out the gel and put it into a clean petri dish. And add appropriate Commassie Blue Staining Solution. Please make sure that the solution can cover all the gel.
- Put the petri dish onto the orbital shaker and dye for approximate 30 min.
- Pour out the staining solution then add enough destaining solution. Destain about 30 min.
- Renew the destaining solution for about 2 or 3 times until the blue background of gel being taken off.
- Pour out the destaining solution and add appropriate water to clear it.

2.2.5 Scanning
Scan the processed gel and save the picture for analysis.

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3 Immobilization
3.1 Prepare Sodiumcellu-losesulfate (NaCS)
- Deep freeze H₂SO₄ and absolute ethanol at -20 ℃ for at least 2 hours;
- Prepare a sulphuric acid and ethanol solution at the proportion of 1.51:1(120 mL H₂SO₄ and 80 mL alcohol), maintaining it at -18 ℃ for at least 2 hours;
- Put the sulphuric acid and ethanol solution and 500mL industrial alcohol in an ice box, maintaining them at 0 ℃ for at least 1 hour;
- Immerse 4 g dry absorbent cotton in the solution in ice-bath for 66 min. Then squeezed out the solution and rinsed the reacted linters with 0℃ industrial alcohol in draught cupboard;
- Squeeze out the alcohol, then put the linters in 400 mL deionized water and regulated pH to about 3. Stir and dissolve it for 10 min, then filtrate it;
- Collected the filtrate and regulated pH to 9.3 accurately;
- Add industrial alcohol gradually to the solution until there appears the largest volume of white retiary floccule on the top of the solution;
- Centrifuge the floccule for 5 min at 5,000 rpm and collect it;
- 65 ℃ drying for at least 24 hours until it is completely dry, then collect the final production.

3.2 Prepare microcapsules

- Centrifuge 10 mL bacteria sample for 3 min at 6000 rpm and collect the deposit;
- Add 10 mL NaCS solution and mix it completely with the cells;
- Put a 6% PDMDAAC solution on a magnetic stirrer and stir it at a certain speed, maintaining a small eddy in the center of liquid surface;
- Drop the mixture into the fringe of the eddy by a 1 mL injector until it form a spheroidic membrane. It takes 10 min to react completely and form microcapsules;
- Tip all microcapsules to a strainer and rinse it with sterile water. Then transfer all microcapsules into LB medium with Ampicillin, 37 ℃ shaker incubate at 100 rpm.

3.3 Immobilize cells into calcium alginate beads:

Solutions	Preparation
3% w/v sodium alginate solution	3 g sodium alginate+100 mL deionized water
0.05 mol/L CaCl2 solution	5.55 g CaCl2+1000 mL deionized water（121 ℃ autoclaving for 20 min）

- Centrifuge 10 mL sample at a time for 1 min at 6,000 rpm, drain off supernatant after each spin. Repeat the procedure until 0.2~1.0 g deposits are collected.
- Resuspend the deposits with sterile water and centrifuge for 1min at 6000 rpm，drain off supernatant.
- Mix the deposits with sterile water in a mass ratio of 1:5.
- Add equivalent volume of sodium alginate solution, mixing thoroughly.
- Draw the mixture in a 1 mL injector and then drip it one by one through the pinhead into 100 mL stirring CaCl₂ solution. Set it aside and let the beads fully harden for 2 hours.
- Drain off the CaCl₂ solution and rinsed the calcium alginate beads with sterile water for 1~2 times. After that, add 100mL CaCl₂ solution to the beads and set it overnight.
- Separate the calcium alginate beads and CaCl₂ solution by a colander. The immobilization of cell sample is finished.

3.4 Immobilize cells into intra-hollow Ca-alginate capsules:

Solutions	Preparation
1% w/v sodium alginate solution	1.5 g sodium alginate powder+ 100 mL sterile distilled water（121 ℃ autoclaving for 15 min）
Cationic Mixture	1.2 g CMC powder（121 ℃ autoclaving for 15 min） 50 mL 4% w/v CaCl2 solution（121 ℃ autoclaving for 15 min） Under sterile condition, add 50mL of sterile distilled water into the CMC powder. Dissolve the CMC and mix it with the 50 mL 4% w/v CaCl2 solution.
3% CaCl2 Solution	12 g CaCl2 powder+ 400 mL distilled water. （121 ℃ autoclaving for 20 min）

- Place 3 mL cationic mixture with 0.6mL cell suspension into a 4 mL centrifuge tube, mixing thoroughly.
- Put a magnetic stirrer at the bottom of a beaker, agitate a 100 mL sodium alginate solution at a constant rate, which maintains a small eddy in the center of liquid surface.
- Draw the mixture (CaCl₂/CMC/E. coli) with a 1 mL syringe, and then drip it one by one from a height of 10 cm into the sodium alginate solution. Keep stirring for 5 min.
- Before sifting out the capsules, dilute the gelation reaction system by adding 100 mL aseptic distilled water. Then rinse the capsules with aseptic distilled water on a colander.
- Transfer the capsules to a 3% w/v CaCl₂ solution and stir for 20 min.
- Finally, rinse the capsules with distilled water to remove excess CaCl₂.
- The immobilization of cell sample is finished.

3.5 Immobilize cells into NaCS-PDMDAAC microcapsules
Solutions:
200 mL sulfuric acid and ethanol solution(1.51:1) maintained at 0 ℃ ;
1000 mL industrial alcohol maintained at 0 ℃;
Deionized water;
NaOH solution.

All of the above procedures are carried out at room temperature and all steps are performed under aseptic condition.