Notebook
Day 1
1. Configurate (solid / liquid) LB medium
Component Mass/volume
Tryptone 10g
Yeast Extract 5g
Sodium Chloride 10g
Deionized Water 1L
Solid LB medium: Besides the component above, add 20g agarose (2%), mix well and sterilize under high pressure for later use.
2. Inoculate glycerol bacteria pQE-30
The glycerol bacteria pQE-30 is inoculated into 15ml liquid LB culture medium containing ampicillin/chloramphenicol, and cultured overnight in a constant temperature shaker (37℃, 220 rpm).
Day 2
1. Amplify the hGK2 gene using PCR
2. Extract Plasmid pQE-30
3. Double-digestion of BamHI and SalI
4. Gel electrophoresis identification and Gel extraction
5. Use T4 Ligase to ligate
6. Transform into M15 E. coli competent cells & Coating plate
Day 3
1. Positive transformant amplification
2. Extract plasmid from positive bacteria
3. Double enzyme digestion and Gel electrophoresis, send for Sanger sequencing
Day 4
1. Inoculate the sequenced correct M15 transformants in 20 LB (ampicillin/chloramphenicol) and culture at 37℃ 220rpm overnight.
Day 5
1. Transfer all of the transformant cultured medium into 1L LB (ampicillin/chloramphenicol) culture medium and incubate at 37℃ 220rpm until the OD is arranged 0.6-0.8.
2. Induce protein expression with IPTG after OD values of bacterial fluid is 0.6-0.8, and incubate at 25℃ 220rpm for 20h.
3. Ins-832 / 13 cell culture
1) Cryopreserved cells should be thawed and resuscitated immediately in a 37 ° C water bath after removal. Pipette the cell fluid into a 15 ml centrifuge tube and add 3 ml complete medium.The whole medium was resuspended and moved into a Petri dish. Centrifugate at 100 rpm for 5 min, and discard the supernatant. After resuspension with complete medium, move it into a Petri dish.
2) Culture Ins-832 / 13 cells in RPMI 1640 medium containing 10% FBS, 10mM HEPES, 2mm L-glutamine, 50μM β-M mercaptoethanol, 1 mM propylene, M sodium ketone, 100 U / mL penicillin with 100 μ G / ml streptomycin (penicillin-streptomycin a cell culture incubator containing 5% CO2 at 37 ° C. When the cells grow to 80% - 90%, use 0.25% trypsin for digestion and generation. Generate it every 2 days approximately.
4. Configuration of GK enzyme activity detection reagent
Glucose: 42 mM (7.56 g)
G6PDH: 8.5 mM (7.6 g)
NAD：42U/mL
Buffer: 210 mM HEPES （50g），210 mM KCl （15.645g），17 mM MgCl2（1.62g），8.5 mM DTT （1.31g）
BSA:1.6%
Day 6
1. Bacterial fluid collection
Centrifugation: 500 mL × 4, 4000 rpm for 15 min.
Collect the bacterial fluid.
2. Bacteria Lysate and protein purification
3. Protein identification by SDS-PAGE.
4. Protein dialysis and concentration.
Day 7
1. Screen Glucokinase agonist
stablish a screening evaluation system for glucokinase agonists. The total volume of the reaction system is 120μL.
Component of the reaction system
Component Volume (μL) Final Concentration
Ultrapure Water 51.6
Glucose 12 5mM
G6PDH 12 1mM
NAD 12 5U/mL
10×Buffer 12 25mM HEPES, 25mM KCl,
2mM MgCl2,1mM DTT
BSA 6 0.10%
LGK2 1.2 18.7μg/mL
Compound 1.2
Finally, a flexstation 3 multi-purpose microplate reader workstation was used to add 12 μL ATP to initiate the reaction and immediately detect the change in absorbance at 340 nm. The Maximum reaction rate is reflected by the slope of the curve.
2. Cell plating
After digesting the cells, inoculate them into 24 hole plate at the density of 2×105 each well, or into 96 hole plate at the density of 5×105 each well.
Day 8-11
1. Detect the effect of active compounds on insulin secretion induced by glucose
1) Incubate 2×105 of Ins-832/13 cells in a 24-well cell culture plate with 500 μL medium which is placed in an incubator with 5% CO2, 37°C overnight.
2) The next day, aspirate the medium. Add 500μLof KRB buffer (115 mM NaCl, 5 mM KCl, 24 mM NaHCO3, 10 mM HEPES, 2.5 mM CaCl2, 1 mM MgCl2, 0.1% BSA, pH = 7.3) with 2.8mM glucose. Starve the cells for 2h. And then replace the material with KRB buffer containing 5.5 mM glucose and 20 μM compound 13926. Incubate the cells for 2h.
3) Supernatant of the absorbing medium was transferred to a 1.5 mL centrifuge tube at 600 rpm and centrifuged at 4℃ for 5 min. Take out the supernatant, and dilute it until the volume grew to 10 times the original. Test it with an insulin assay kit. After the cells were lysed at 100μL RIPA, the protein concentration was determined as an internal reference using the BCA protein concentration determination kit.
2. Detect the protective effect of active compounds on pancreatic islet cell
1) Ins-832 / 13 cells were incubated with a density of 5 × 105 for each well. Incubate the cells in 96-well plates. Culture them overnight in a 5% CO2, 37°C cell incubator.
2) On the second day, add STZ with a final concentration of 0.4 mM and 13926 with a final concentration of 20μM. Incubate them for 24 h.
3) On the third day, suck out the medium, add the medium containing 0.5 mg / mL MTT, and continue to incubate for 4 h. After that, aspirate the medium. Add 100 μL DMSO to each hole, and shake the plate at 400 rpm for 10 min. Detect the absorbance at 490 nm with a spectrum max M5 multi-functional micro-plate reader.