（1）Activation of bacteria: prepare 5 mL LB(no antibiotics, 1:100 inoculation) the night before, shock culture at 37 degrees overnight (less than 16 hours), at the same time, add a tube of antibiotics to see whether bacteria grow (let bacteria resuscitate, enter the early and middle logarithmic phase. It's easier to make competent cells （2) The next morning, the medium was transferred to 100mL with a density of 1:200-500 and cultured for 2h (slight turbidity is enough according to the naked eye, or dim palmlines are enough if the palm is placed on the bottom of the bottle, rather than too much cultivation). （3) Prepare the ice box and insert multiple 50mL centrifuge tubes vertically for precooling. In the ultra-clean platform, the bacteria liquid was transferred to a 50m pre-cooled centrifugal tube for ice bath for 20min. Then, centrifugation was performed at 4000 RPM at 4℃ for 10 min. （4) After centrifugation, the supernatant was poured out of the ultra-clean platform, and 5mL of pre-cooled 0.1-m cacl2-MgCl2 solution was added to each tube with repeated blowing and suction with the head of a gun. The mud was suspended again, and placed in ice for 10min. Then, centrifugation was performed at 4000 RPM at 4℃ for 10min. （5) After centrifugation, the supernatant was poured out of the ultra-clean platform, and 2mL of pre-cooled 0.1m cacl2-glycerin solution was added to each tube and repeatedly blown and sucked with the head of a gun. Then the mud was suspended again and placed in ice for 10min （6) After the ice bath, the competent cells were divided into 1.5mL EP tubes, 100uL for each tube, and immediately placed on ice.（7) End of packing. The competent cells were placed in a ziplocked bag, labeled and stored in a refrigerator at -80℃.
1.The preserved competent cells were taken out and thawed on ice for 10min 2.Add 3U plasmids into the competent cells respectively 3.Adhesion: Set on ice for 20min 4.Heat shock: metal bath 42℃ 60s 5.Recovery: Ice bath for 2min 6.Add 300U LB culture solution under ultra-clean stage 7.Culture on shaking table at 37℃ for 1h 8.Centrifugation: 8000rpm for 1min 9.The supernatants were respectively adsorbed with 300μ suspension mud 10.Apply the above reserved LB plates, and place them upside down at 37℃ for overnight culture in a biochemical incubator
Purchased SBF powder, when added water each pack can make 500mlSBF
The following system is configured to operate on ice
Fig.1Enzyme digestion system
Blend with vortex then place in a metal bath at 37℃ for 20min
Discard the supernatant from the petri dish Rinse 3 times with 3mL ddH2O Add CV solution 10mL and stain for 20min Pour off the dye, add ddH2O and rinse several times until the rinse water is clear, and observe that the bottom layer of the medium cannot be rinsed
1.Prepare 1~4ml of bacteria solution centrifuge at 12000rpm for 1 min, discard the supernatant (Repeat the step two times) 2.Add 150µL of Buffer P1 and vortex to blend until no bulk precipitation visible 3.Add 150µL of Buffer P2 and gently invert 6~8 times to mix. 4.Immediately add 350µL of Buffer P5. Mix quickly upside-down 12 to 20 times, thoroughly. Flocculent precipitation should be seen at this point.Centrifuge at 12000rpm for 2min to sperate plasmid and impurity. 5.Apply the supernatants to a CP3 spin column and centrifuge 12000rpm for 30s, discard the liquid in the collection tube. 6.Wash the CP3 spin column by adding 300µL of Buffer PWT. Centrifuge at 12000 rpm for 30s, discard the liquid in the collection tube. 7.Put back CP3 spin column and repeat step 7 without adding Buffer PWT. Discard the ethanol impurities. 9.CP3 was adsorbed into the collection tube, 50-100 µ l elution buffer TB was dropped to the middle part of the adsorption film, centrifuged for 30s, and plasmid solution was collected into the centrifugal tube
Fig.2The Gibson system
Incubate with PCR instrument at 50 ℃ for 30min
1.The linking reaction system was prepared in 1.5mL PCR tube on ice, and each component was added according to the table on the next page (20 uL as an example). Note: Add enzymes last!
Fig.3T4 DNA Ligase system
2.Pipette suction or vortex oscillator briefly shock, mix the system, and then centrifuge with a desktop centrifuge for 1-2 seconds to concentrate the reaction liquid at the bottom of the PCR tube. 3.Incubate at room temperature (25 °C) or 16°C for 1h, or overnight at 4 °C. 4.Chemical transformation of 10uL products into competent cells.
1. When OD=0.6-0.8, add IPTG solution. (concentration of IPTG solution* sample volume=0.3Mm*flask volume) 2. Put into the 25℃ shaker
The strain of Composite parts was in a Petri dish containing M63 medium during culture. We assessed the ability of the engineered extracellular matrix to act as a template for HA mineralization. After a 7-d incubation in 1.5× SBF (Change it every other day) at 37 °C, Detailed morphological characterization by scanning electron microscopy (SEM) strongly indicated that the surfaces of the biofilms were covered with lath-like mineralized HA structures
Place the strain in a dark environment, exposed for 3-7d to blue light emitted from a portable LED projector.
Fig.4Blue light exposure diagram
1.The strain was cultured overnight in liquid LB medium. 2.Add the bacteria solution 1:200 into fresh LB medium (possibly containing IPTG) and culture at 220 RPM at 37 ℃ for 8 hours. 3.Take 200 microl cultures into 96-well plates and measure 485/510 fluorescence and OD600 using a microplate reader. The ratio of fluorescence value to OD600 was used to indicate the expression level of sfGFP (vertical axis below).
Fig.5 PhlO and PhlF assay results
The expression level of MFP-CSGA protein could be determined by bioflim staining
Fig.6;Staining results of protein expressed by plasmid PT7-B0034-CSGA_linker_mFP3SPEP, suggests that There's something sticking to the petri dish
Fig.7The results of staining after three days of induction culture after co-transformation of two plasmids, still material that cannot be washed and can be stained
After 9 days of SBF culture, THE MFP-CSGA was scanned by electron microscopy (compared with the sample on day 0).
Fig.8 SEM image showing the surface morphology of the biofilm (i.e., unmineralized). Scale bars:3μm
Fig.9 SEM image showing the surface morphology of the mineralized composite (mineralization 7d). Scale bars: 3μm
