Team:XMU-China/protocols

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Protocols

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 MgCl₂ solutions 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).

- 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.

Table 1. The digestion system

- 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.

- 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.

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

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

- 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

- 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 %.

- 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℃.

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.

- 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.

- 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.