1. LB Broth: 10g/L Tryptone, 5g/L Yeast, 10g/L NaCl, sterilized after dissolving above content in water.
2. Stock Ampicillin
3. 0.1M MgCl₂
4. 0.1M CaCl₂
5. Glycerol Stock Bacterial Cells (SHuffle strain in our case)

1. Take out one tube of frozen glycerol stock bacterial cells from -80ºC storage. (SHuffle strain in our case)
2. Thaw the cell and spread onto an antibiotic free LB plate.
3. Grow the plate overnight at 37°C.

1. Pick up a single colony of SHuffle strain from fresh LB plate
2. Inoculate a 5 mL starter culture of LB without antibiotics.
3. Grow culture at 37°C in shaker at 220 rpm overnight.

1. Inoculate 150 mL of LB media with 1.5 mL starter culture (1:100) and grow in 37°C shaker.
2. Measure the OD₆₀₀ till it reaches 0.4-0.6(no exceeding 0.6), collect the culture in 50 ml centrifugation tubes. Then immediately put the cells on ice. Chill the culture on ice for 20-30 minutes, swirling occasionally to ensure that the cultrue is evenly cooled.
3. Harvest the cells by centrifugation at 3000 rcf for 15 minutes at 4°C.
4. Decant the supernatant and gently resuspend the pellet in 50 mL of pre-chilled 0.1M MgCl₂. Then keep it on ice for 20 minutes.
5. Harvest the cells by centrifugation at 3000 rcf for 15 minutes at 4°C. Decant the supernatant and resuspend the pellet in 50 mL of pre-chilled 0.1M CaCl₂. Keep this suspension on ice for 20 minutes.
6. Harvest the cells by centrifugation at 3000 rcf for 15 minutes at 4°C. Decant the supernatant and resuspend the pellet in 5 mL of pre-chilled ice cold.0.1M CaCl₂,15% glycerol solution.
7. Aliquot 100 μL compentent cell into sterile 1.5 mL microfuge tubes and snap freeze with liquid nitrogen. Store frozen cells in the -80°C freezer.

1. LB Broth: 10g/L Tryptone, 5g/L Yeast, 10g/L NaCl, sterilized after dissolving above content in water.
2. Stock Ampicillin
3. Glycerol Stock Bacterial Cells (SHuffle strain in our case)
4. 10 ng vectors for transformation (pGLO and genetically modified pGLO in our case)
5. LB plate with anphicilin (0.1 g/L in our case)

1. Take out one tube of frozen glycerol stock competent bacterial cells from -80ºC storage. (SHuffle strain in our case)
2. Thaw the cell on ice untill little crystalloid could be observed in the tube.
3. Add 10 ng vectors for transformation into the tube.
4. Heat shock at 42ºC water bath for 90 seconds.
5. Keep on ice for two minutes.
6. Add 500 μL antibiotic free LB broth into the 1.5 mL EP tube. Incubate the tubes for 30 minutes at 37ºC shaker at 220 rpm to allow antibiotic resistance gene expressed.
7. Concentrate the cell by conducting centrifugation at 4000 rcf for one minute.
8. Discard the supernatant and leave around 100 μL cell culture, spread the cell onto LB plate with corresponding antibiotics. (Ampicillin in our case)
9. Incubate the plate at 37ºC.

1. Pick up a single colony of SHuffle strain from LB plate into 5mL antibiotics containing liquid LB medium. Incubate in 37ºC shaker at 220 rpm for 8 hours.
2. Send the culture for sequencing.

1. LB Broth: 10g/L Tryptone, 5g/L Yeast, 10g/L NaCl, sterilized after dissolving above content in water.
2. Stock Ampicillin
3. 20% Arabinose solution (mass fraction)
4. Stock bacteria culture (SHuffle strain in our case)

1. Taking 50 μL culture from 5 mL antibiotics containing liquid LB medium into new 5 mL antibiotics containing liquid LB medium. Incubate at 37ºC in the shaker at 220rpm overnight. (If the original culture has been stored in the 4ºC for some days, otherwise if directly following the competent cell transformation step, start from Day 2)

1. Prepare the autoclaved LB medium, for 500 mL Erlenmeyer flask, filled with 150 mL LB medium, for 1 L Erlenmeyer flask, filled with 250 mL LB medium.
2. Add ampicillin to the medium to make the concentration to be 0.1g/L.
3. Add bacteria culture using 1:100 dilution by volume. (For 150 mL LB medium add 1.5 mL bacteria strain prepared in Day 1)
4. Incubate at 37ºC in the shaker at 220 rpm untill the OD₆₀₀ of the culture reach 0.4-0.6.
5. Immediately add 20% Arabinose solution (mass fraction) using 1:100 dilution by volume. (For 150 mL LB medium add 1.5 mL 20% Arabinose solution prepared as stock solution, filtrate through 0.22 μL filter membrane)
6. Incubate at 37ºC in the shaker at 220 rpm for 8 hours or at 18 ºC in the shaker at 220 rpm for 16-18 hours.

1. Distribute the culture into 50 mL centrifugation tube and collect the cell by centrifuge at 4000 rcf for 20 minutes.
2. If not broken immediately after this step, discard the supernatant and store the cell pellet at -80 ºC refrigerator.

1. Buffer A: 150 mM NaCl, 20 mM Tris-HCl, pH =8.0
2. High-pressure homogenizer (Union-Biotech(shanghai)Co., Ltd.)
3. Filtration membrane (0.45 μm, Millipore, Catalog number: SLHPR33RB)
4. AKTA purification system (Union-Biotech(shanghai)Co., Ltd.)
5. HisTrapTM HP His tag protein purification column (Cytiva, Catalog number:17524701)
6. Buffer B: 150 mM NaCl, 20 mM Tris-HCl, 1 M imidazole, pH adjusted to 8.0 by HCl
7. PMSF (Phenylmethylsulfonyl fluoride)

1. Prepare the buffer system using Buffer A as substrate, and add extra PMSF untill a final concentration of 0.1 mM. (Buffer A components: 150 mM NaCl, 20 mM Tris-HCl pH= 8.0)
2. Resuspend the cell pellet with around 10 mL buffer system prepared above for high pressure homogenization. (For 150 mL culture system, homogenizing at 750 bar for 3 minutes, using homogenizer from Union-Biotech(shanghai)Co., Ltd.)
3. Centrifugation at 11000 rcf for 30 minutes.
4. Collect the supernatant and filtrate through 0.45 μm filtration membrane for the preparation of running AKTA purification system.
5. HisTrapTM HP His tag protein purification column (Catalog number:17524701) are applied onto the AKTA system (Union-Biotech(shanghai)Co., Ltd.). The program is set to pre-charge the flow path with 10 column volume with Buffer A, then 12 column volume of the supernatant containing sample protein is applied onto the column. After that apply 10 column volume buffer A to wash out the unbonded protein. Finally, apply the linear gradient of the mixture of buffer A and buffer B (starting ratio is Buffer A: Buffer B = 70%:30%, final ratio is Buffer A: Buffer B = 0%:100%) to wash out the his-tagged protein and collected. (Buffer B components: 150 mM NaCl, 20 mM Tris-HCl, 1 M iminazole, pH adjusted to 8.0 by HCl) Then collect eluted samples by fixed volume collection with 1 mL per EP tube.
6. After that, conduct electrophoresis and Coomassie blue staining to see the purity and relative concentration of the protein collected. Further, collect ones with high purity and concentration through ultrafiltration to substitute the buffer B to buffer A with 0.1 mM PMSF added. And after the solvent replacement, perform BCA test to tell the protein concentration. (For detailed information of this step, see the protocol for electrophoresis, ultrafiltration and BCA test)

1. 5x SDS-PAGE Sample Buffer
2. Protein samples
3. Running Buffer (SDS-PAGE 10× SDS Running Buffer: Tris base 30.3 g, Glycine 144.4 g, Sodium Dodecyl Sulfate 10 g Dissolve in 1 L of ddH_O, composition could be different under different cases, dilluted before use)
4. Protein ladder
5. Electrophoresis system
6. Power supply
7. Coomassie blue dye

Note: Amount of protein sample used for electrophoresis could be adjusted according to the loading capacity of the gel used under specific circumstance.

1. Prepare 16ul protein samples and add 4ul 5x SDS-PAGE Sample Buffer to each protein sample.
2. Heat up the protein sample to 95ºC and keep for 10 minutes.
3. Prepare the electrophoresis chamber and running buffer. Take out required number of 12-well precast gels and set up the system. Fill the chamber with running buffer.
4. Load all the protein samples onto the gel. And add a protein ladder to one well to serve as a molecular weight marker for reference.
8. Transfer all the rest of the running buffer into the chamber and making sure that the gel is completely submerged.
9. Connect the power supply to the electrophoresis system and run the system by 80 V for 20 minutes to check if the circuit is well developed. Then switch the voltage to 140 V and run for another 1 hour. The specific running time will depend on the size of the gel and the separation requirements of specific experiment. Generally, stop running when the dye front reaches the bottom of the gel or reaches a desired distance.
10. Take down the chamber and wash the gel with deionized water by snap-boiling the system in micro-wave oven twice.(Turn down the micro-wave immediately when the deionized water boil)
11. Stain the gel using Coomassie blue dye for 30 minutes.
12. Decolor the gel with deionized water by snap-boiling the system in micro-wave oven multiple times when background coloring is insignificant.
13. Observe the bands to see the purity of the purified protein.

Our BCA test are conducted by using Enhanced BCA Protein Assay Kit from Beyotime (Catalog number: P0009).
Standard Instruction protocol could be found in the link
And we strictly follow the instruction given by Beyotime.

1. SHuffle-pGLO bacteria single colony
2. LB Broth: 10g/L Tryptone, 5g/L Yeast, 10g/L NaCl, sterilized after dissolving above content in water.
3. Stock Ampicillin
4. 0.5 M FeCl₃ solution
5. 0.22 μL filter membrane

Pick up a single colony of SHuffle-pGLO bacteria from LB plate (with 0.1 g/L ampicillin) into 5 mL LB Broth (0.1 g/L ampicillin). Incubate at 37ºC in the shaker at 220rpm overnight.

1. Prepare the autoclaved LB medium. For 500 mL Erlenmeyer flask, fill with 150 mL LB medium. For 1 L Erlenmeyer flask, fill with 250 mL LB medium.
2. Add ampicillin to the medium to make the concentration to be 0.1g/L.
3. Add bacteria culture using 1:100 dilution by volume. (For 150 mL LB medium, add 1.5 mL bacteria strain prepared in Day 1)
4. Incubate them at 37ºC in the shaker at 220 rpm untill the OD₆₀₀ of the culture reaches 0.1.
5. Immediately add FeCl₃ solution to a final concentration of 5 mM Fe³⁺. (In our case, 0.5 M FeCl₃ solution are prepared as stock solution which was filtrated through 0.22 μL filter membrane, and was added with a 1:100 dilution)
6. Incubate at 37ºC in the shaker at 220 rpm for one day in 37ºC.

1. Distribute the culture into 50 mL centrifugation tubes and collect the cell by centrifuge at 4000 rcf for 20 minutes.
2. If they don't break immediately after this step, discard the supernatant and store the cell pellet in the -80 ºC refrigerator.

1. NHS-PEG-Maleimide (MW 3400)
2. DMSO
3. 3kDa Ultrafiltration tube (Catalog number: UFC9003, Millipore)
4. 10kDa Ultrafiltration tube (Catalog number: UFC5010BK, Millipore)
5. Stock Nanoparticle solution

1. Dissolve 0.05/0.10g NHS-PEG-Maleimide in DMSO with a concentration of 100µM and store

1. Mix maleimide solution and nanoparticle solution with a volume ratio 2:1, reacting for 1 h at room temperature
2. Dilute the solution to a proper volume for ultrafiltration. Ultrafiltrate (3kDa), centrifuging at 5000g for at least 20 mins. Finish until the liquid volume above the filtrate is less than 1.5mL.
3. Collect the liquid above the filtrate and combine the solution with the liquid before, add 30 µg/mL antibody solution the same volume as maleimide solution consumed before, reacting for 1.5 h at room temperature
4. Ultrafiltrate (10 kDa), centrifuging at 14000g for at least 20 mins. Finish until the liquid above the filtrate less than 100 µL.
5. Collect the liquid above the filtrate by converting the ultrafiltration tube, centrifuging at 1000g for 1 min.

1. 0.1M PBS 2. 0.2M EDC·HCl 3. 0.2M NHS 4. 10kDa Ultrafiltration tube (Catalog number: UFC5010BK) 5. Stock Nanoparticle solution 6. Antibody solution

1) Transfer 200 μL of our bio-synthetic nanoparticles and 0.4mg (8μL in volume) chemo-synthetic nanoparticles to two eppendorf tubes.
2) Add 200 μL 0.1M PBS to the second tube to make up for the difference
3)Then add 200 μL 0.1M PBS to each of the tubes to mix it thoroughly.
4)Then, add 60 µL of 0.2 M EDC·HCl and 120 µL 0.2 M NHS to each tube.
5)After 10 min, add 30 μg antibody to the EDC/NHS-activated NPs solutions.
6)After 24 h, transfer the solution to 10 KD ultrafiltration tubes and centrifuge for 20 min at 14000 rpm to remove the unbounded antibody.
7)Finally, collect the obtained antibody-conjugated NPs (IONPs-antibody) in EP tubes.

1. Deionized water
2. High-pressure homogenizer (Union-Biotech(shanghai)Co., Ltd.)
3. 100 kDa Ultracentrifugation tube (Catalog number: UFC9100, Millipore)
4. 3 kDa Ultracentrifugation tube (Catalog number: UFC9003, Millipore)
5. Buffer A: 150 mM NaCl, 20 mM Tris-HCl
6. Ultrasonic Cleaner

1. Resuspend the cell pellet for each 50 mL centrifuge tube with around 10 mL deionized water for high pressure homogenization. (For 150 mL culture system, homogenize at 750 bar for 3 minutes, the broken cell system could be concentrated in one 50 mL centrifugation tube after homogenization, using homogenizer from Union-Biotech(shanghai)Co., Ltd.)
2. Centrifugation at 11000 rcf under 4ºC for 30 minutes.
3. Discard the supernatant, add 10 ml deionized water, vortex the system for 1 minutes. (Notice that now there should still have a lot iron salt precipitate at the bottom of the tube, while the cell debris should already be resuspended, this could prevent jamming in the ultrafiltration step followed).
4. Carefully put the tube into an ice bath and then put the whole system into ultrasonic cleaners and run the cleaners for 15 minutes.
5. Without disrupting the precipitate filtrate, the supernatant through 0.45 μm and 0.22 μm filtration membranes as a prevention for ultrafiltration membrane blockage, then move the filtrated supernatant into activated 100 kDa Ultracentrifugation tube.
6. Centrifuge at 5000 rcf under 4ºC for 25 minutes. Note that if there still have more than 2 mL liquids above the filtration membrane, centrifuge at 5000 rcf under 4ºC for another 20 minutes.
7. Discard the filtration membrane and carefully put the tube into an ice bath and then put the whole system into ultrasonic cleaners and run the cleaners for 15 minutes.
8. Transfer the flow through into an activated 3 kDa Ultracentrifugation tube.
9. Centrifuge at 5000 rcf under 4ºC for 25 minutes. Note that if there still have more than 5 mL liquids above the filtration membrane, centrifuge at 5000 rcf under 4ºC for another 20 minutes untill there only have around 2 mL liquid above the filtration membrane.
10. Collect the liquid above the filtration membrane. Carefully put the tube into an ice bath and then put the whole system into ultrasonic cleaners and run the cleaners for 15 minutes. This sample could be used for TEM characterization. Note: In this experiment design, we use deionized water for the solution system to avoid the interference of mineral salt in TEM analysis. But if the target is to get nanoparticle with all membrane protein on the surface biologically active, please use Buffer A to take place of deionized water.

1. DMEM High Glucose H-21 Media (Catalog number: 11965084)
2. RPMI 1640 basal Media (Catalog number: 12633012)
3. Fetal Bovine Serum (Catalog number:10099141C)
4. Penicillin, Streptomycin
5. Trypsin/EDTA
6. Dulbecco's Phosphate Buffered Saline
7. Cell strains
8. Tissue culture dish
9. 15 ml Falcon tube
10. 37ºC water bath
11. 37ºC 5% CO₂ incubator
12. Insulin (Catalog number: I860440-25mg)

Culture medium: 450 ml DMEM High Glucose H-21 Media, 50 ml (to 10%) Fetal Bovine Serum (Catalog number:10099141C) , 5 ml Pen Strep

Thawing Cells:
1. Take one vial immediately in 37ºC water bath. Transfer the content to a 15 ml Falcon tube already containing 10 ml of fresh medium.
2. Spin down at 1200 rpm for 3 min at 4ºC. Aspirate supernatant and resuspend cells in fresh medium and transfer to 10cm tissue culture dish and cultured at 37°C in 5% CO₂. Change medium about every 3 days.
Subculture
After cell culture reaches 80-85% confluence, subculture is conducted.
1. Remove the medium from the dish.
2. Wash 1x with 10 ml of Dulbecco's Phosphate Buffered Saline (without Ca²⁺ or Mg²⁺)
3. Add 1 ml of 0.05% Trypsin and trypsinize for 3-5 min at 37°C.
4. Add 8 ml of medium to neutralize the trypsin and use it to rinse the dish by pipetting up and down 4-5 times to detach the cells off.
5. Spin down at 1200rpm for 3 min. Aspirate supernatant.
6. Add 8 ml of medium to the 15ml tube with cell pellet, and pipette up and down to mix. Subculture ratio is about 1:10.
7. Put the cells into 37°C with 5% CO₂.

Reference Linkage

Culture medium: 500 ml RPMI 1640 basal Media, 100 ml (to about 20%) Fetal Bovine Serum, 5 ml Penicillin, Streptomycin, 2μg/ml insulin

Thawing Cells:
1. Take one vial immediately in 37℃ water bath. Transfer the content to a 15 ml Falcon tube already containing 8 ml of fresh medium.
2. Spin down at 1000 rpm for 3 min. Aspirate supernatant and resuspend cells in fresh medium and transfer to 10cm tissue culture dish and culture at 37°C in 5% CO₂. Change medium about every 2 days.

Subculture
After cell culture reaches 70-75% confluence, subculture is conducted.
1. Remove media from dish.
2. Wash 1x with 10 ml of Dulbecco's Phosphate Buffered Saline.
3. Add 1 ml of trypsin/EDTA for about 10 min at 37°C.
4. Add 6 ml of medium to neutralize the trypsin and use it to rinse the dish by pipetting up and down 4-5 times to detach the cells off.
5. Spin down at 1200rpm for 3 min. Aspirate supernatant.
6. Add 6 ml of media to the 15ml tube with cell pellet, and pipette up and down to mix. Subculture ratio is about 1:3/1:4.
7. Put the cells into 37°C with 5% CO₂.

Reference Linkage

1. Cell Counting Kit-8 (CCK-8) (Catalog number: ab228554, Abcam)
2. 96-well plate
3. 37ºC, 5% CO₂ incubator
4. 37ºC water bath
5. 96-well plate reader
6. WST-8 Solution

1. Plate 10,000 cells in 100 µl per well for a 96 well plate
2. Add test compounds into cells and incubate for 24 hours in a 37ºC, 5% CO2 incubator.
3. For blank wells (medium without cells), add the same amount of test compounds.
4. Add 10 µl/well (96 well plate) or 5 µl/well (384-well plate) of WST-8 Solution to each well. Protect from the light and incubate for 1-4 hours at 37ºC.
5. Measure the absorbance increasement at 460 nm.

Reference 1 Reference 2

1. SDS-PAGE gel
2. PVDF membrane
3. BSA blocking solution
4. TBS-T solution
5. Primary antibody
6. Secondary antibody
7. Exposing reagent
8. Electrophoresis system
9. Power supply
10. Protein ladder

1. Prepare the SDS-PAGE gel and electrophoresis system.
2. Prepare the protein samples and add 5x SDS-PAGE Sample Buffer to each protein sample.
3. Heat up the protein sample to 95ºC and keep for 10 minutes.
4. Load all the protein sample onto the gel. And add a protein ladder to one well to serve as a molecular weight marker for reference.
5. Transfer the protein onto PVDF membrane.
6. Block the membrane with 1% BSA in TBS-T solution for 1 hour.
7. Incubate the membrane with primary antibody at 4ºC overnight.
8. Wash the membrane with TBST for 3 times, 10 minutes each time.
9. Incubate the membrane with secondary antibody at room temperature for 1 hour.
10. Wash the membrane with TBST for 3 times, 10 minutes each time.
11. Develop the membrane with exposing reagent and observe the bands.

Author

• Shuoyi HU: [1] [2] [3] [4] [5] [6] [7] [10] [13]
• Songnan REN:[8]
• Shijie HE:[9]
• Yixiao XIAO: [11] [12]