Team:XMU-China/protocols

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Protocols

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

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℃ until they reach an OD = 0.3-0.4 at 600nm (1cm pathlength cell). This usually takes 1.5-2 hours.
- Ice down the LB with growing cells for 10 min.
- Aliquot into sterile 1.5mL tubes and Spin down at 6000rpm for 10 minutes at 4℃; discard supernatant and.
- Ice down sterile 100mM CaCl2 and 100mM MgC2solutions during centrifugation.
- Gently resuspend each pellet in 8mL 0.1M MgCl2 and 2mL 0.1 M CaCl2 and combine into 2 tubes (this should take 1-2 minutes per tube)
- Centrifuge 6000rpm for 10 minutes and discard supernatant.
- Resuspend each pellet on ice in 100μL 0.1M of ice cold CaCl2 and combine into one tube

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

Purity Plasmid Miniprep
- Centrifuge sample in Eppendorf tube approximately 1.5 mL at a time, draining off supernatant after each spin and adding more cell solution
- Resuspened 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 1-2minutes 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 12,000rpm 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 12,000rpm. 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 1minute at 12,000rpm 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 1minute at 12,000rpm 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 12,000rpm.
- Discard the column and store the purified plasmid DNA at -20°C.

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 4 h at 37 °C, afterwards inactivated by adding 10x loading buffer and standing for 10 min at room temperature.

Standard BioBrick Assembly
- Digestion of insert: 2 μg~5 μg DNA / 100 µL volume, 10x H buffer, EcoRI, SpeI. 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 x M buffer, EcoRI, XbaI. 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.

Suffix Insertion
- Digestion of insert: 2 μg~5 μg DNA / 100 µL volume, 10x M buffer, XbaI, PstI. Digestion and inactivation. Clean up the insert.
- Digestion of vector : 2 μg~5 μg DNA / 100 µL volume, 10x H buffer, SpeI, PstI. Digestion and inactivation. Clean up the vector.

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

Restriction analysis
- Pick one colony with a sterile tip and cultivation in 20mL 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 %.

Gel Extraction
- Weigh a 1.5mL 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 to the DNA as possible 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 100μL of solution per 100mg 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 minute at room temperature.
- Centrifuge the Spin Columns assembly in a microcentrifuge at 12,000 rpm for 1 minute, then discard the flow-through.
-  Wash the columns by adding 500 μL of Wash Buffer PE to the Columns. Centrifuge the columns assembly for 1 minute at 12,000 rpm, then 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 minute 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 minute at 12,000 rpm. Discard the columns and store the microcentrifuge tube containing the eluted DNA at –20℃.

Characterization
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 °C at 200 rpm.
- The culture is checked for OD600 next day and then subculture by the same medium with antibiotics at 37 °C 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 minutes: absorbance (600 nm filter) and fluorescence (485nm and 520nm for GFP).
- The results then transfer to excel sheet and interpret.

Immobilization
Prepare Sodiumcellu-losesulfate (NaCS)
- Deepfreeze H2SO4 and absolute alcohol at -20℃ for at least 2 hours;
- Prepare a sulphuric acid and ethanol solution at the proportion of 1.51:1(120mL H2SO4 and 80mL 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 4g 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 400mL 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 appear the largest volume of white retiary floccule on the top of the solution;
- Centrifuge the floccule for 5 min at 5,000rpm and collect it;
- 65℃ drying for at least 24 hours until it is completely dry, then collect the final production.

Prepare microcapsules

- Centrifuge 10mL bacteria sample for 3 min at 6000rpm and collect the deposit;
- Add 10mL NaCS solution and mix it throughly 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 1mL injector until it form an 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 100rpm.