Here are the measurements we used along the way. The page includes their detailed steps, the purpose of the experiment and the results.
Our plasmid expresses an adhesive compound protein (CsgA-Mfp3s-pep). We need to determine whether the protein is expressed and whether it sticks to the surface. To achieve our goal, we chose to dump the induced expressed strain and stain the bottom of the petri dish
Steps: Discard the supernatant from the petri dish Rinse 3 times with 3 mL ddH2O Add CV solution 10 mL and stain for 20 min 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
Fig.1Staining results of protein expressed by plasmid PT7-B0034-CSGA_linker_mFP3SPEP, suggests that There's something sticking to the petri dish
Fig.2The 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 7 days of mineralization in SBF for E. Coli, we need to confirm its mineralization. We chose SEM for observation as a direct and effective method. We need to confirm the protein mineralization of E. coli induced in SBF, and using SEM is the most direct and effective way.
Fig.3SEM image showing the surface morphology of the biofilm (i.e., unmineralized). Scale bars:3μm
Fig.4 SEM image showing the surface morphology of the mineralized composite (mineralization 7d). Scale bars: 3μm
To characterize the strength of the promoter, BBa_K3254024 element was connected downstream from the original T7 promoter and the modified T7 promoter with PhlO binding site, and the 485/510 fluorescence and OD600 values of the bacterial fluid were measured using a microplate meter to compare the expression levels of sfGFP
Steps: 1. We inserted the strain into liquid LB medium and incubated it overnight. 2. Add Bacterial broth 1:200 was inserted into fresh LB medium (containing IPTG) and incubated at 37℃, 220 rpm for 8 hours. 3. 200 µl culture was taken into 96-well plates and 485/510 fluorescence and OD600 were measured using an enzyme marker. sfGFP expression was expressed as the ratio of fluorescence value to OD600.
Fig.5 PhlO and PhlF assay results
