Difference between revisions of "Part:BBa K2817003"

Revision as of 09:56, 8 October 2019

YebF-IL10-flag

IL10 is a natural immunosuppressive protein. We add a flag tag for detection and a secretion tag to increase the extracellular concentration.

2019 NEU-CHINA

Characterization result 1

Secretory tag YebF was introduced to secret the protein, however we did not have time to show this tag really work last year. This time, western blotting is demonstrated to show the secretion of human IL-10. Expressing vector was constructed.

Figure 1. Diagram for  human IL-10 expressing and secreting in pCDFDuet-1 plasmid. T7 promoter, the gene downstream of this promoter will be transcribed when there is T7 RNA polymerase. LacO, the sequence represses the nearby promoter when there is no inducer (e.g. IPTG). RBS, ribosome biding site. Secretory tag YebF is introduced to secret protein downstream.

Figure 2. Expressing vector harboring YebF and hIL-10 gene is transformed into BL21 strain. After induction of IPTG, we span down the cells, get them eliminated using filter. Secreted protein is collected from the medium by salting out with ammonium sulfate. The molecular weight of protein YebF-hIL-10 is around 35 kDa.

Protocol

1. Incubate the inoculum at 37℃ overnight.

2. Dilute the culture to OD600=0.2, followed by incubation at 37℃ for around 2 hours until the OD600=0.6.

3. Add IPTG at final concentration 1mM.

4. Incubate the culture overnight.

5. Remove cells by centrifugation at 5000rpm for 10min.

6. Remove cells by using filter.

7. Add saturated ammonia sulfate to the supernatant, and then agitate softly for 2 hours while sitting on ice.

8. Span the protein down by centrifugation at 13000rpm for 30min.

9. Resuspend the protein by cold water and remove any faults by filter.

10. Add Loading Buffer and then incubate the mixture at 98℃ for at least 10min.

11. Western blot.

Characterization result 2

To make sure that amplifier is able to strengthen the expression of gene downstream to promoter hrpL. A coomassie brilliant blue is demonstrated to show that more protein at 35kDA, which should be IL-10, is witnessed when introducing IL-10 gene downstream of promoter hrpL.

Characterization result 3

In order to see whether the nitric oxide sensor will work when it comes to expressing a certain protein, we add the IL-10 downstream and demonstrated the western blotting to show the result. It has been proved that the expression level can be induced when adding nitric oxide.

Sequence and Features