Biosensor sensing naringenin

a) Inductor BL21 was added to two liquid media (containing 3um naringenin), after cultured overnight at 16 ° C, centrifuged, it is observed that the fluorescence intensity of the bacterial mass (in the morning).

b) Add 0.5mm IPTG to the two liquid culture media, induce culture at 16 ° overnight, centrifuge, and observe the fluorescence intensity of the bacterial mass (in the afternoon).

c) Prepare two pieces of protein glue, centrifuge the preserved solution, ultrasonic wave, take the supernatant, add it into the protein loading buffer, boil it, and electrophoresis (electrophoresis staining, afternoon).

Delphinidin Synthesis Operon (Naringenin Added)

Prepare 50 ml LB culture solution containing 3um naringenin and 50ug / ml Kan, and prepare 50 ml LB culture solution containing no naringenin and only 50ug / ml.

c) Select the BL21 transformed monoclonal containing plasmid 5, inoculate it in LB containing antibiotics, grow it overnight, dilute it with fresh lb to OD600 of 0.1, and culture it in a shake flask. Incubate at 37 ° C until OD600 is 0.8, add 0.5 mm isopropyl-d-mercaptogalactoside (IPTG) for induction, and grow at 250rpm-300rpm at 30 ° C for 3 h to overexpress the protein. The cells were collected by centrifugation and resuspended in M9 liquid medium at pH5, containing necessary antibiotics, 1% glucose, 20mm naringenin, 0.5mm IPTG, 0.1 mm 2-oxoglutarate, 2.5 mM sodium ascorbate. The cells were incubated at 30 ° C for 48h (the supplement at the middle 24h can be selected: 1% glucose (20ul), 2um naringenin). After that, the cells were centrifuged to observe the color of the bacterial mass (adding HCl to make it pink, adding NaOH to make it grass green). d) BL21 solution of carrier 5 was cultured in LB medium of 3um naringenin and 50ug / ml Kan for 16h. Respectively take 1ml of culture, 12000rpm, 1min, observe the color of the bacteria at the bottom of the tube, and take photos.

IN THIS PICTURE

Plasmid 5 contains four fusion proteins, among these proteins, F3H catalyzes the conversion of naringenin to dihydrokaempferol, F3 '5' H catalyzes the conversion of dihydrokaempferol to dihydromyricetin, DFR catalyzes the conversion of Dihydromyricetin to colorless delphinidin, and ans catalyzes the conversion of colorless delphinidin to colorless delphinidin.

The coding genes of the first two proteins are driven by the first T7 promoter of the vector for transcription, and the coding genes of the last two proteins are driven by the second T7 promoter of the vector for transcription, and finally translated into four corresponding proteins.

e) The BL21 bacterial solution of carrier 5 was cultured in LB medium of 0um naringenin and 50ug / ml Kan for 16h, and 1ml of culture was taken respectively at 12000rpm for 1min. The color of the bacterial mass at the bottom of the tube was observed and photographed.

f) BL21 solution of carrier 5 was cultured in LB medium of 3um naringenin, 50ug / ml Kan and 0.5 mm IPTG for 16h. Respectively take 1ml of culture, 12000rpm, 1min, observe the color of the bacteria at the bottom of the tube, and take photos.

g) BL21 solution of carrier 5 was cultured in LB medium containing 0 um naringenin, 50 UG / ml Kan and 0.5 mm IPTG for 16 h. Respectively take 1ml of culture, 12000rpm, 1min, observe the color of the bacteria at the bottom of the tube, and take photos.

Naringenin synthesis operon (add 4-coumaric acid)

h) Prepare a plate containing 25ug / ml ampicillin and 12.5ug/ml chloramphenicol. The competent cells dh5a and BL21 were co transformed with the receptor plasmid and plasmid 4.

i) Prepare 50ml LB liquid medium containing 0.2g/l coumaric acid, add 12.5ug/ml cm and 25ug/ml amp

j) Prepare LB liquid medium without coumaric acid, add 12.5ug/ml cm and 25ug/ml amp

k) Plasmid was extracted and transformed.

l) Prepare a receptive state.

m) Plasmid 5 and plasmid 4 were transformed into dh5a and BL21 competent cells, respectively.

n) The receptor plasmid and plasmid 4 were co transformed into BL21, expanded and shaken, and screened and cultured in LB plates containing 25ug / ml ampicillin and 12.5ug/ml chloramphenicol. After 16 hours, monoclonal antibodies were selected, cultured in LB medium containing coumarin and without coumarin, induced, centrifuged, and observed the changes of green fluorescence of the bacteria. The bacterial fluid of receptor and BL21 were selected for comparison.