Period 2 sequence snapgene8/17/2023 In the meantime, due to the different experimental condition, it is impossible to ensure that each group of test samples will have the same bacterial concentration, Here, the content of phosphoric acid, which is absorbed by the engineering bacteria, was expressed by the reduction of phosphate concentration (ΔCpi). In the supernatant after centrifugation, which was measured by ammonium molybdate spectrophotometry and expressed by the absorbance at the wavelength of 710nm We have determined the cell concentration (CE), which was represented by the absorbance at the wavelength of 600nm (A(600nm)), and phosphate concentration (Cpi) To confirm the function of engineering bacteria, we designed a series of experiments and finally we concluded that the engineering bacteria which was transferred anĪdditional Pst gene could absorb the phosphate more efficiently. (Just replace the solution in step 2 with deionized water)ġ2.Use the reference solution and sample solution to measure the light absorption at a wavelength of 710 nm Then add 2mL 26g/L molybdate solution and set the volume to the scale with water.ġ0.Shake volumetric flask and set aside for 15 minutes.ġ1.Prepare a reference solution in the same way. When the pressure reaches 1.1kg/cm2 and the temperature reaches 120℃, keep it for half an hour and then stop heating.ĩ.Take 2mL solution obtained in step 8 to into a 50mL volumetric flask, then add 3mL 20g/L ascorbic acid solution and wait for half a minute. (The methods to prepare them are as follows)Ģ.Take 5mL bacterial solution then centrifuge and take the sediment.ģ.Add about 5mL deionized water to the sediment and resuspend it.Ĥ.Repeat step 2&3 two or three times to wash the sediment.ĥ.Add a little bit of water (maybe 1 to 2mL) and resuspend it.Ħ.Move the solution to 15mL test tube with stopper and add deionized water to 10mL.ħ.Add 1.6mL 5% potassium peroxydisulfate solution and tie the stopper tightly with gauze and thread (or other ways) and put all the test tubes into a beaker.Ĩ.Heat the large beaker in a high-pressure steam sterilizer. Method for detecting phosphorus concentration in bacteria.ġ.Prepare 5% potassium peroxydisulfate solution, 20g/L ascorbic acid solution and 26g/L molybdate solution. (Detect the phosphate concentration every half hour, the detection method is as Then add 1mL 1mM IPTG solution and 5μL kanamycin.ħ.Grow the engineering bacterium in the condition of 37℃, 220rmp for 4 hours to 6 hours. Verify the function of engineering bacterium.ġ.First prepare the MOPS medium and LB medium.Ģ.Inoculate the engineering bacterium on LB medium till OD600=0.6~0.7.ģ.Take 5 mL suspended solution then centrifuge the bacterial solution and take the sediment.Ĥ.Add about 5mL MOPS medium to the sediment and resuspend it.ĥ.Repeat step 3&4 three times to wash the sediment to remove the residual LB medium.Ħ.Add MOPS medium to sediment up to 5mL and resuspend it. After 6 h induction, significant differenceĬan be seen, and the experimental groups with the inducer IPTG concentration greater than 1 mM all have significant expression. Significant difference in expression between the experimental group and the control group after 2h and 4h induction. His Tag is attached to the C segment of PstB protein, which is 257 AA and weighs 28 kD. Immunoblots were then visualized with chemiluminescence reagent kit. Were incubated with the appropriate secondary antibody (1:10000) at room temperature for 1 h. On the following day, wash the membrane in three washes of TBST, 10 min each. Then electrophoresed and transferred to nitrocellulose membranes (0.2 µm), which were blocked with 5% non-fat blocking grade milk and incubated with theįollowing primary antibodies overnight at 4 ℃: anti-His (1:2000). Pour out the supernatant, suspend each tube with 5mL cold PBS, and crush it with ultrasonic crusher for 5-10min.Īfter crushing, take 1mL of crushed bacterial solution from each tube, add 50ul nickel medium, and incubate it for 2h at 4 degrees with rotation.Īdd 7.5uL SDS-PAGE Loading in each tube,boil at 100 ℃ for 10min.ħ.5uL sample was loaded onto SDS-PAGE gel.Run the 5% stacking gel for 30min at 80V and run the separating gel for 1 h at 120 V. Set two variables:the concentration of IPTG and the induced time.Īfter induction, 4000 rpm, centrifugate for 15 min.
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