Difference between revisions of "Part:BBa K2817003"

 
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IL10 is a natural immunosuppressive protein. We add a flag tag for detection and a secretion tag to increase the extracellular concentration.
 
IL10 is a natural immunosuppressive protein. We add a flag tag for detection and a secretion tag to increase the extracellular concentration.
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== 2019 NEU-CHINA ==
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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 humanIL-10. Expressing vector was constructed.
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**********
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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.
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**********
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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.
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=== Protocol ===
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1. Incubating the inoculum at 37℃ overnight.
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<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Revision as of 06:30, 5 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

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 humanIL-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. Incubating the inoculum at 37℃ overnight.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]