Team:Nanjing-China/Notebook

The engineered bacteria containing the target plasmid (produced by the company according to the plasmid sequence and stored in a glycerol tube) were streaked on the Kana plate and cultured overnight at 37°C.

(2) LB culture

1) Prepared a 250ml Erlenmeyer flask, added 50ml LB, 50μl (1000x) Kana

2) Used a pipette tip to pick the monoclonal strain on the plate into an Erlenmeyer flask and shook it well. Cultured on shaker overnight at 37°C (12h).

3) Measured the absorbance of the bacterial solution at OD600 with LB zero adjustment, determined the amount of bacterial solution added to the synthetic wastewater culture medium, and ensured that the initial OD600 of the synthetic wastewater culture medium containing engineered bacteria was 0.2.

(3) Cultivation of synthetic wastewater

1) Centrifuged the bacteria-containing LB medium at 5000r for 1min, discarded the supernatant, resuspended it with a small amount of synthetic wastewater medium, pipette and mix well, centrifuged at 5000r for 1min, discarded the supernatant, and resuspended with a small amount of synthetic wastewater.

2) Prepared a 500ml Erlenmeyer flask, added 200ml synthetic wastewater culture medium, 200μl (1000X) Kana to each bottle, calculated the amount of synthetic wastewater culture solution after resuspension. Incubated on a shaker at 37°C for 12-14h.

(4) Determination of polyP content

1) Centrifuged at 12000r for 1min to separate the bacteria and the solution.

2) Took the supernatant and used the molybdate method to determine the phosphorus content in the solution. The reduced phosphorus was considered to be synthesized as polyP and stored in the bacteria.

2.Extraction of polyP

(1) Bacteria pretreatment

The recombinant engineered bacteria (containing polyP) cultured in synthetic wastewater for 14 hours were freeze-dried to obtain freeze-dried powder, and placed in a muffle furnace to heat at 600 degrees for 1 hour to obtain a carbonized black solid.

(2) Ground the black solid, added deionized water to dissolve it, the supernatant solution was the product solution containing polyP, placed it in a freeze dryer and freeze-dry to obtain the solid.

3. Purification of polyP (separation of polyP with different chain lengths)

(1) Used NaOH or KOH (2.5M) to adjust the pH of the polyP aqueous solution to 7, meanwhile, the total volume of the solution was V1

(2) In order to precipitate polyP with medium chain length, 0.156V1 volume of ethanol (96%v/v) was added to the solution and mixed well. The solution was stood for 1 hour to allow the polyP to fully aggregate together.

(3) Centrifuged the solution at 10000g for 5min after standing to separate the insoluble matter from the supernatant. The insoluble matter contained medium chain polyP (polymerization degree 30-40).

(4) Desiccation.opened the cap of the above centrifuged tube and placed it on a desiccator (a device filled with silica) for one week.

(5) Separated short-chain polyP, and collected the supernatant in step (3) into a new tube. At this time, the supernatant contained short-chain polyP (polymerization degree is about 11).

(6) Added 0.885V1 volume of absolute ethanol and mixed well. The solution was allowed to stand for 1 hour to allow the polyP to fully aggregate together.

(7) Centrifuged the solution at 10000g for 10min after standing, the insoluble matter contained short-chain polyP

(8) Desiccation, same as step (4).

Attachments:

1. LB liquid medium formula
Dissolved 0.5g of yeast powder, 1g of peptone, and 1g of NaCl in water and diluted to 100ml.
2. Synthetic wastewater culture medium formula (100* mother liquor, used sterile water to diluted and mix well
(1) N source (high pressure steam sterilization)
Dissolved NaCl0.5g, MgSO4·7H2O2.36g, and NH4Cl1.8g in water, and diluted to 100ml.
(2) P source (high pressure steam sterilization)
Dissolved 1.48 g of dipotassium hydrogen phosphate trihydrate in water and diluted the volume to 100 ml.
(3) Y source (high pressure steam sterilization)
Dissolved 1g of tryptone and 0.1g of yeast extract powder in water and diluted to 100ml.
(4) Source C (filter sterilization)
Dissolved 3g glucose and 1.5g anhydrous sodium acetate in water and diluted to 100ml.
2. Determination of phosphorus content in solution by molybdate method
(1) Configuration of vitamin C and molybdate solution
1) Prepared 10g/100ml vitamin C solution and stored it in a brown bottle.
2) Dissolved 13g of ammonium molybdate tetrahydrate in 100ml of water and slowly added it to 300ml of 49% sulfuric acid. Dissolved 0.35g potassium antimony tartrate into 100ml water, slowly added and mixed well, stirring while adding. Stored in a brown bottle.
(2) Used 10ml molecular water+200μl vitamin C solution+400μl molybdate solution to zero, set 9.8ml molecular water+200μl synthetic wastewater+200μl vitamin C solution+400μl as the total amount, centrifuged 9.8ml molecular water+200μl Clear liquid + 200μl vitamin C solution + 400μl as the experimental group, the value of OD700 was measured under an ultraviolet spectrophotometer. According to the formula of the remaining amount of phosphorus in the experimental group (unit mg/l) = (total OD700-experimental group OD700) * 100, calculated the polyphosphate content in the bacteria.

+36GFP preparation

1. Monoclonal streaking inoculation

The engineered bacteria containing the target plasmid (produced by the company according to the plasmid sequence and stored in a glycerol tube) were streaked on the Kana plate and cultured overnight at 37°C.

2. Monoclonal culture

(1) Prepared a 250ml conical flask, added 50ml LB, 50μl (1000x) Kana

(2) Used a pipette tip to pick the monoclonal strain on the plate into an Erlenmeyer flask and shook it well. Cultured on shaker overnight at 37°C (12h).

3. LB culture (purpose: protein expression)

(1) Prepared a 500ml Erlenmeyer flask, added 200ml LB, 200μl (1000X) Kana, 5ml monoclonal culture medium to each flask, and cultured at 37°C on a shaker.

(2) After transferring the last bottle, measured the OD600 of the first bottle, recorded the time and OD value

(3) Measured the OD every 2h. When the OD reached 0.1, bacterial would enter the logarithmic growth phase. After that, the OD measurement interval should be shortened appropriately. Estimated to reach the ideal OD value in 4h

(4) When the OD reached 0.6-0.8 (1.0 is fine), stopped shaking and added 1 mM IPTG to each bottle. (When the plasmid vector is PET29a (+), the amount of IPTG added was 1mM, and the configured IPTG concentration is 100mM, that was, 1000μL of 100mM IPTG was added to 100mL LB medium)

(5) Put it in a 37°C shaker and shake for 8-10 hours, and the medium turned green.

4. Collection and fragmentation of E. coli

(1) Collectd 1L of fermentation broth and centrifuged at 4℃, 6000rpm, 5min.

(2) Discarded the supernatant, resuspended the pellet, and transferred to a beaker.

Resuspended directly with high salt binding buffer: 1.5 M NaCl, 20 mM NaH2PO4-Na2HPO4 (pH 7.4),30 mM imidazole, 5 mM mercaptoethanol and protease inhibitor cocktail (Sigma-Aldrich).

(3) Bacteria breaking: ultrasonic breaking: placed the mixed bacteria in an ice box (without the liquid surface of the bacteria) and conducted ultrasonic breaking for 2s, The interval was 3s, the power was 100%, and the alarm temperature was 20°C. Observe whether there were lumps in the solution until the bacteria become translucent.

5.Ni column purification

(1) Centrifuged the crushing liquid: 13000rpm, 4℃, 30min.

(2) Passed the supernatant through a 5mL HisTrap HP column (used a syringe or pump to push the pressure).

(3) Eluent elution (Eluent: 1.5 M NaCl, 20 mM NaH2PO4-Na2HPO4 (pH 7.4), 500 mM imidazole, 5 mM mercaptoethanol and protease inhibitor mixture).

(4) Diluted the collected eluate with cation exchange buffer 10 times (50 mM MES (pH 6.0), 1 mM DTT, 1 mM EDTA) to reduce the NaCl concentration to 150 mM.

(5) The diluted sample was passed through 5 mL HiTrapTM SP FF column (GE Healthcare), and the eluent was eluted (eluent: 50 mM MES (pH 6.0), 1 mM DTT, 1 mM EDTA, 1M NaCl, pH Adjust to 8).

(6) The eluate containing the target protein was concentrated and desalted by ultrafiltration (Using an ultrafiltration tube).

(7) Concentration and desalination using ultrafiltration centrifugation.

6.SDS analysis

The green fluorescent protein could be observed with naked eyes, which was used as a rough judgment. We used SDS-PAGE gel electrophoresis to perform accurate analysis based on the gel image.

Attachments

1.Purchased a Millipore ultrafiltration tube (15 ml, 10KD) for centrifugation to change the fluid and remove salt.
(1) Added ultra-pure water (MilliQ) water before use, the water volume was completely over the membrane, and pre-cooled in an ice bath or refrigerator for a few minutes.
(2) Poured out the water and added ≤15 ml of protein solution (the operation should be slight. Before adding the protein solution, the ultrafiltration tube needed to be inserted on ice to pre-cool; the pipette tip should not touch the filter membrane to prevent its damage)
(3) Equilibrated the sample and centrifuged for 15-30 minutes. note:
1) Balanced between quality and center of gravity.
2) When using a fixed-angle rotor, the direction of the device should be such that the membrane panel faces upwards, and centrifuged at a maximum of 5000×g for 15-30 minutes. The specific time needed to be determined according to specific experiments.
3) The centrifugal rotation speed and acceleration should not be too fast, otherwise the filter membrane would be damaged, and the actual rotation speed should be lower than the rotation speed in the manual to extend the life of the centrifuged tube.
4) After the rpm of different centrifuges was converted into g, please refer to the centrifuged manual for the difference.
(4) The final volume of the specific concentrated protein solution should be determined according to the required protein concentration, generally not more than 500μl (in this experiment, it can be determined according to the concentration of your own protein eluate, generally higher than 1mg/ml)
(5) If intended to change the Buffer, when the total protein solution was concentrated to about 1ml, gently added the new Buffer (in this experiment, PBS buffer ultrafiltration was used to change the fluid) (Ultrafiltration with 0.22um ultrafiltration membrane), and then Concentrate to about 1ml for three consecutive times. The final volume of the final concentration depends on the required protein concentration, generally not more than 500μl, and may be concentrated to within 200μl. According to the volume concentration of at least 10 times each time, it can reach more than 1000 times three times, which can basically achieve the purpose of changing the buffer.
(6) The operation of taking out the final protein concentrate was done on ice. Take it with a yellow pipette tip (200μl), gently insert the pipette tip along the edge, gently pipette and mix the protein solution, taking care not to touch the ultrafiltration membrane. Then suck up the concentrated solution, close to 200µl each time, until it was exhausted. There was no need to absorb the last bit of concentrate remaining at the bottom of the tube, otherwise it would be too difficult and the ultrafiltration membrane may be damaged. Finally, added MilliQ water to the ultrafiltration tube to prevent the membrane from losing water and drying out.
2. Desalination
(1) diluted the collected eluate with a cation exchange buffer containing 50 mM MES (pH 6.0), 1 mM DTT and 1 mM EDTA by 10 times to reduce the NaCl concentration to 150 mM.
(2) After that, added the diluent to a 5 mL HiTrap TM SP FF chromatographic column (GE Healthcare), and then eluted the target protein with pH 8.0, 1 M NaCl cation exchange buffer.
(3) Concentrated by ultrafiltration, and then desalted by 5 mL HiTrap TM desalting column (GE Healthcare), eluent: 10 mM HEPES, 150 mM NaCl and 1 mM EDTA.
3. Maintenance of Millipore ultrafiltration tube:
(1) Repeated use
Poured out the solution in the ultrafiltration tube, rinse rinsed gently with sterile water (if there is was visible protein precipitation at the bottom of the tube, add , added ultrapure water and blow with a pipette until the precipitate is was suspended, and then pour it out), then add then added 0.1 M Soak in NaOH for 1 hour, pour out the remaining liquid, soak in a beaker filled with sterile water for 30 minutes (change the water once), and then wash 3 times with sterile water on the centrifuge centrifuged (that is, add , added sterile water and centrifuge centrifuged 3 times). ) for reuse.
(2) Normal storage
Took out the tube core and filled it with MilliQ water. The 50ml tube was also filled with MilliQ water. Put the tube core into the 50ml tube and drain some of the water, then closed the lid and stored at 4 degrees.
(3) Long-term storage
Stored with 20% ethanol, and made sure to keep the filter membrane moist to prevent bacterial growth.
4. Storage of purified protein:
(1) Measured the concentration of purified protein (protein content measured by BCA method or direct instrument measurement), and replaced the solution with a prepared PBS buffer (pH=7.4).
(2) After changing the liquid, added 20% glycerol with a final volume of 20%, aliquot one tube per 100 µl, and store at -80°C
(3) PBS buffer (pH=7.4) configuration
Weighed 8g NaCl, 0.2g KCl, 1.42g Na2HPO4, 0.27g KH2PO4 and dissolve in ddH2O, adjusted the pH to 7.4 with phosphoric acid, and diluted to 1L,disinfected with autoclave and stored at room temperature.

+36GFP combined with polyP

1. The search for phase separation

Mixed the solution according to the following concentration gradient, and searched for the phase separation boundary of the combination of polyP and +36GFP.

Determined the concentration of polyP and GFP for the binding experiment according to the phase separation boundary found, and selected the concentration in the phase separation boundary (main), boundary and outside of the boundary to set the gradient for the experiment.

2. The effect of the combination of polyP and +36GFP on the stability of GFP

(1) Mixed 5.56μM +36GFP and 10μM +36GFP with different concentrations of polyP in equal volumes, respectively incubated for 0h, 3h, 24h, 48h and then detected the fluorescence intensity.

(2) Mixed polyP (6400μM) and +36GFP (5.56μM) in equal volumes, in the phase boundary of liquid-liquid separation, respectively incubated at room temperature for 30min, then 25, 30, 40, 50, 60, 70, 80 Incubated for 10 min at 90, and 100℃ respectively. After cooling, detected the fluorescence intensity with a microplate reader.

(3) The final concentration of polyP (0,100,400,800,1600,3200μM) and the final concentration of +36GFP (2.78μM) protein were mixed in equal volume, respectively incubated at room temperature for 30min, and then incubated at 25,30,40,50,60,70,80,90 and 100℃for 10 minutes. After cooling, the fluorescence intensity was measured by a microplate reader.

(4) The final concentration of polyP (3200μM) and the final concentration of +36GFP (2.78μM) protein were mixed in equal volumes (50μl +50μl); the final concentration of +36GFP (2.78μM) protein was mixed in equal volumes with 20mM HEPES buffer (pH7), incubated at room temperature for 30 minutes, then respectively incubated with buffers of different pH (100 μl) for 30 minutes, and detected the fluorescence intensity with a microplate reader.

(5) +36GFP+polyP: polyP (0,200,800,1600,3200,6400) and +36GFP (5.56μm) mixed in equal volume (50μl +50μl), incubated at room temperature for 30min. After incubation, they were mixed with denaturing solution (100μl) and incubated for 30min. Microplate reader detected the fluorescence intensity.

Attachments:

Denaturing solution
1X denaturing solution; 4M urea, 0.5% SDS, 3M guanidine hydrochloride;
2X: 8M urea, 1% SDS, 6M guanidine hydrochloride.

About Spidroin & +36GFP-sp

The preparation method of spidroin and +36GFP-sp fusion protein was the same as +36GFP.

NOTEBOOK #1

PROTOCOL #2