Team:WHU-China/Protocol




Protocol




BC reinforcements



1. Aging the silk


  1. Cut the single colored silk into several pieces at the size of 2cm*5cm and the patterned silk into 4cm*4cm. Add 10% NaOH to age the silk

  2. Remove the NaOH, wash the silk with ddH2O, add hydrochloric acid to control the pH to 6.5. Wash the material again with ddH2O.

  3. Divide the silk into three equal pieces and put them into sterilizer to sterilize.

  4. Dry the silk at 60°C.

2. BC pre-treatment


  1. Use a blender to make the BC into homogenate

  2. Add 10% NaOH into BC while boiling, then keep boiling for about 1 hour. (keep the amount of water)

  3. Change the pH to 6.5 after cooling down.

3. A.xylinum bacteria fluid produce


  1. Inoculate a single colony to 5ml seed culture medium, culture at 30°C, 150rpm for 3-4 days until they form BC balls with diameters around 1cm

  2. inoculate the bacteria fluid into the fermentation culture medium at a ratio of 1:10, culture at 30°C for 4 hours.

4. restoration


  1. in-situ: pour the fluid containing A.xylinum into a spraying can and spray it on the silk
    ex-situ: spray the BC homogenate on the silk
    aging contrast and unaged contrast: spray water on silk.

  2. culture at 30°C for 6 days, keep the silk wet during culture.

  3. use ddH2O to wash 3-4 times after restoration ends

  4. put into the bake out oven for 1h to dry the silk, then dry at room temperature, choose a part to dry in the oven for 3 hours (samples for electronic microscope)

5. mechanical strength


  1. put the silk into universal mechanical tester, set the parameters: use 2kN probe, at the stretching speed of 10mm

  2. measure the stretching strength and the fracture extension rate. Measure 6 samples in each group

6. color difference analysis


  1. Take photos of the silk, observe and use software to analyze

7. observe under electronic microscope:


  1. sample production: put dried silk sample on the carrier of the electronic microscope, spray gold

  2. observe


BC production



1. Inoculate BL21 containing two kinds of plasmids into 5-10mL culture medium (1/1000 chloromycetin and kanamycin) and culture overnight at 37°C.


2. Add the bacteria fluid into 2ⅹYT culture medium (1/1000 chloromycetin and kanamycin), culture at 37°C until OD reaches 0.5-0.7 (no higher than 0.8).


3. Add IPTG to the fluid (concentration to 0.4mM), add L-arabinose (concentration to 0.2%), culture overnight at 28°C.


Examine the cellulose in the material:


Experimental reagents:


67% H2SO4


Acetic acid/nitric reagent: mix 150ml 80% acetic acid and 15ml concentrated nitric acid


Anthrone reagent: dissolve 0.2g anthrone with 100mL concentrated sulfuric acid, prepared fresh daily. Chill for 2 hours prior to use.


Prepare 100°C water bath for use.


Experiment procedure:


1. Homogenize sample of microbial culture, get 10-100ml suspension into a centrifuge tube, centrifuge at 3000rpm for 10-15 minutes, decant and discard the supernatant.


2. add 3ml acetic acid/nitric reagent to make a mixture


3. put the centrifuge tube into 100°C water bath for 30 minutes (keep the water level high enough)


4. centrifuge at highest speed and remove the supernatant.


5. resuspend the sediment with 10ml ddH2O, centrifuge again and remove the supernatant.


6. add 10ml 67% sulfuric acid, let stand for 1 hour.


7. put 1ml fluid into 50ml EP tube, add 4ml ddH2O and put into ice (the amount of sample fluid and ddH2O is changeable, dependent on the cellulose concentration in the sample).


8. add 10ml anthrone reagent into the tube, mix up in ice.


9. 100°C water bath to heat up for 16min


10. put into icy-cold for 2-3 minutes and then wait 5-10 minutes at room temperature.


11.measure the OD value at 620nm.


PS: if the OD is too high, dilute the fluid after treating with 67% sulfuric acid.



Verification of light system



1.Activation: Choose a single colony from the plate and culture it in LB liquid (1:1000 adding chloromycetin). Use silver paper to wrap up the test tube to avoid light. Culture overnight at the condition of 37°C, 220rpm.


2.Activation (2): Add the cultured bacteria fluid into 150ml culture medium (with chloromycetin), continuing culturing without light for about 4 hours.


3.Diaphragming: When the OD of the fluid reaches 0.6, make 8 samples in test tubes, each contains 5ml bacteria fluid. 4 of the samples are wrapped up with silver paper to make sure no light can affect them, the other 4 are to be induced by red light.


4.Light induction: Put the 8 samples into the illumination incubator (30°C, 180rpm), turn on the light to begin induction. (arrange the test tubes properly to make sure every tube can accept light)


5.Sample collection: End the induction after about 16 hours, gather 1ml bacteria fluid into EP tube from each test tubes. Centrifuge to collect the bacteria and use 1ml lysis buffer to resuspend the samples.


6.Sample examine: Add 200ul sample from each EP tube to the florescence 96-well plate add measure the florescence intensity. Get the data and do the anaylsis.




Verification of the CBD binding ability



Qualitative test:


1.Mix 0.2g BC with 200μl PBS, wait for 5 minutes at room temperature. Then centrifuge at 13000rpm for 10 min, pour the supernatant.


2.Uses 500μl lysis buffer to resuspend the BC sediment, wait for 30 minutes and then centrifuge at 13000rpm for 10min, pour the supernatant.


3.Add 1ml H2O (0.01% Tween) to resuspend the sediment, wait 5 minutes. Then centrifuge at 13000rpm for 10 minutes, pour the supernatant.


4.repeat the operation above for 3 times, then observe the florescence intensity under UV.


Quantitative test:


1.Add 200ul BC solution into florescence 96-well plate, then dry the liquid at 37°C (need about 20h). The BC film in the wells are considered formed after that.


2.Add 200μl bacteria lysis into each well, wait 12h at 4°C. Use pipette to remove the supernatant (no visible liquid exist) and measure florescence intensity. Then add 200μl H2O (0.01% Tween，mixing against the wall 3 times), vibrate the plate for 10 times and remove the supernatant. Measure the florescence intensity after repeating the operation for 3 times.




Streptavidin Detect



Qualitative:


1.Collect 100μl biotinylated beads in EP tube, add 1ml PBS buffer to resuspend, then wait for 5min at room temperature.


2.Put the EP tube on the , wait for 3min and then remove the supernatant and resuspend the with 100ul PBS.


3.Mix with 100μl dCBM3a lysis and oscillate overnight.


4.Put the EP tube on the …, wait for 3min and preserve a sample.


5.Add 1000μl H2O(Tween) to resuspend the beads, put it on the vortex for 1 minute.


6.Put the EP tube on the …, wait for 3min and preserve a sample.


7.Repeat for 3 times


8.Add 30μl 1ⅹSDS loading buffer, 100°C dry bath for 15 minutes.


9.Put the EP tube on the …, wait for 3min and preserve a sample.


10. SDS-PAGE




Biotinylation detect



Experimental reagents:


Lysis buffer: 50 mM Na2HPO4, 0.3 M NaCl, pH=8.0


Washing Buffer: 50 mM Na2HPO4, 0.3 M NaCl, 10 mM imidazole pH =8.0


Elution Buffer: 50 mM Na2HPO4, 0.3 M NaCl, 250 mM imidazole pH =8.0


High Affinity Ni-NTA Resin: From Genscript No. L00250


Modified Bradford Protein Assay Kit: From Sangon Order NO. C503041


HABA/Avidin Reagent: From Sigma Product No. H 2153


Protocols of Comfirmation of BirA Enzyme:


1. Construct three plasmids, pet28a-BirA (pB), pet28a-sfGFP+Avitag (pGa) and pet28a-sfGFP+Avitag-BirA (pBGa) and transfer the plasmids into BL21 respectively.


2. Three kinds of BL21 bacteria, pB, pGa and pBGa, were selected by Kan. Inoculate one colony in a test tube separately. Culture tubes overnight at 37℃, 220r. Add the bacterial suspension to a conical flask containing 150 ml LB liquid medium at a ratio of 1:20. At the same time, add bioitn solution to make the final concentration 200 mM. Besides, add 50mg/ml Kan at 1:1000 ratio. When OD600 in conical bottle is 0.6-0.8, add 0.1 mM IPTG and induce for 22 hours at 13℃, 110r.


3. Collect the bacteria at 8,000r, 4℃, 10 min. Wash them once with Lysis Buffer. Then centrifuge again under the same conditions and finally freeze bacteria at -20℃.


4. Thaw the bacteria on ice. Add 8 ml Lysis Buffer and suspend them fully. Break the bacteria on ice by ultrasound until the liquid is clear.


5. 12, 000 r, 4℃, 15 min, centrifuge twice and collect supernatant.


6. Balance 1ml Ni resin according to Genscript No. L00250 High Affinity Ni-NTA Resin instructions. Then add 1ml Ni resin into supernatant and shake it on ice or 4℃ for 1h.


7. Remove the excess liquid through the column. Wash it with Washing Buffer according to the instructions, and then elute the target protein with Elution Buffer.


8. Run SDS-PAGE gel for further detection of target protein.


9. Purification of the target protein can be further carried out by chromatography.


10. After the protein concentration is measured by Bradford method, the biotinylated protein can be measured by HABA/Avidin reagent under OD500 according to the following table:







DSAOP detect



  • Measurement of DPPH Radical scavenging activity

Experimental reagents:


0.2mM DPPH solution (dissolved with ethanol)


0.4mg/ml Vitamin E solution (dissolved with ethanol)


0.35mg/ml protein sample of DSAOP and DSAOP+avitag


ddH2O


experiment procedure:


1.set experimental group and contrast group as follow:





2. Mix the reagents for 10s.


3. React for 20 minutes in dark


4. Measure OD under the wavelength of 517nm every 10 minutes, 2 hours in total.


5. Use formμla （DPPH radical scavenging activity (100%) = [1 − (Ai − Aj )/ A0] × 100）to deal with the data.


  • Measurement of hydroxyl radical scavenging activity

Experimental reagents:


3mM FeSO4


3mM salicylic acid


3mM H2O2


0.4mg/ml Vitamin E (in ethanol)


0.35mg/ml sample of DSAOP and DSAOP+avitag


ddH2O


Ethanol


Experiment procedure:


1.Set experiment groups and contrast groups as follow:





2. wait 30min at 37°C


3. measure its OD at 510nm


4. use formula（hydroxyl radical scavenging activity (100%) = [1 − (Ai − Aj )/A0] × 100.）to deal with the data.




AMP detect



  • Purification of glutathione S-transferase (GST) tagged protein

Prepare the buffers


1.Lysis buffer: 20 mM Tris–Cl (pH 7.4), 150 mM NaCl, and 1 mM dithiothreitol (DTT), stored in 4℃


2.Wash buffer:


Hypersaline: 20 mM Tris–Cl (pH 7.4), 500 mM NaCl, and 1 mM dithiothreitol (DTT), stored in 4℃


Hyposaline: 20 mM Tris–Cl (pH 7.4), 150 mM NaCl, and 1 mM dithiothreitol (DTT), stored in 4℃


3.Elution buffer: 50mM Tris–Cl (pH 8.0), 150mM NaCl, 10mM GSH (add it when using)


4.Cleavage buffer:


50mM Tris–Cl (pH 7.0), 150mM NaCl, 1mM EDTA, 1mM DTT, stored in 4℃


5.1XPBS


Spin down 500ml expression culture in 6x100 ml spin bottles for 15 minutes at 4℃ and 4500 rpm;


Suspend the bacteria with 30ml 1xPBS;


centrifuge at 4500 rpm for 15 minutes at 4°C, Collect deposits, freeze overnight at -20 ℃;


Suspend the bacteria with 30ml lysis buffer, broke bacteria with high-pressure for 4 times;


centrifuge at 4500 rpm for 15 minutes at 4°C, take 40ul sample of Supernatant and precipitation to put on a SDS-PAGE gel;


Hung the supernatant on GST affinity column (had balanced with lysis buffer) for 30 minutes in 4℃, take 40ul sample to put on a SDS-PAGE gel;


Wash the affinity column with hyposaline wash buffer and hypersaline wash buffer for three times, take 40ul sample to put on a SDS-PAGE gel;


  • Elute the target protein with 1ml elution buffer, take 40ul sample to put on a SDS-PAGE gel;

Add 100ul glycerin and cryopreserve the protein by liquid nitrogen freezing method and put it in -80℃ for later use.


  • Use cleavage buffer to balance the GST affinity column;

Add 5ml prescission protease stored in cleavage buffer, hung for 16h in 4℃;


Collect the protein in the dialysis bag, put PEG-20000 on the bag to reduce the solvent;


Add glycerin and cryopreserve the protein by liquid nitrogen freezing method and put it in -80℃ for later use.


  • purification of PreScission Protease (from TJUSLS_China)

editor:TJUSLS_China


The plasmids of PreScission Protease are obtained from high-throughput molecular drug screening center . The protein expressed by the recombinant plasmids has a GST tag and is mainly purified by GST affinity chromatography. The PreScission Protease purification protocol is as follows :


1. Transforming PreSeission Protease plasmids into BL21(ED3) Competent Cells.


2. The selected clone was inoculated into a 5mLLB small test tube, cultured at 37 degrees celsius until the bacterial solution was turbid.


3. Transfer the turbid bacteria solution into 800 mLLB medium, and cultured at 37℃ until OD600 reached 0.6.Add 0.5mMIPTG and inducing protein expression for 4h.


4. The bacteria were collected by high speed centrifugation at 5500 rpm for 20 min and suspend the bacteria with 1×PBS.


5. After 4 times of high-pressure bacterias broken, use ultrasonic to continue to break the bacteria for 30 min (ultrasonic power 300w, turn it on for 4s and then turn it off for 6s).


6. Centrifuge at 12000rpm for 30 min and hung the supernatant on GST affinity column for 3-4 times.


7. Wash impurities with 1XPBS until G250 detection does not change blue and then wash with 1×PBS containing 500 mM NaCl (500 mM NaCl, 2.7 mM KCl.10 mM Na2HPO4, 1.8 mM KH2PO4 (pH=7.3)) until G250 detection does not change blue . Alternately wash high and low salt solution for alternately 2-3 times.


8. Dilute 10×GSH to 2×GSH with 1×PBS ,and use it to elute the target protein until G250 does not change blue.


9. Collect the eluent and concentrate to less than 1mL with 15ml and 30KD concentration tubes.


10. Add Ppase solution(50mM Tris-HCI pH8.0 ,150 mM NaCI, 10 mM EDTA,20%Glycerol ,1 mMDTT)to change the protein solution into Ppase solution .


11. The protein was packaged at 4℃ with a concentration of 1 mg/ml, 400μl per tube. Cryopreserve protein by liquid nitrogen freezing method and put it in -80℃ for later use.


  • Molds Test

Preparation of mould spore suspension, dip in with inoculation loops take a small amount of suspension, satisfactory to PDA plate central, 30 ℃ to cultivate 1 d to mould colonies radius is 3 cm, tag the mold on the edge of the colony, 0.3 cm on the edge of the colony marks positive control, negative control and test groups three vaccination, and the corresponding place leaching to detect liquid aseptic filter wafer, continue to cultivate 1 d, observe the bacteriostatic ring size.