Difference between revisions of "Part:BBa K2817002"

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===Usage and Biology===
 
===Usage and Biology===
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We inserted this part into plasmid pCDFDuet-1. So the expression of this part can be induced by IPTG. We transformed the IL10-flag plasmid into BL21, and incubated at 37 ℃ overnight to dilute to OD = 0.2. After 2 h of growth at 37 ℃, IPTG was added and induced at 30 ℃ for 16 h. Then, the bacterial solution was lysed and the expression of IL10 was detected by western blot (Figure 1). We used the human IL10 sequence optimized by E. coli from the previous iGEM team (the original part is BBa_K554004). So we have reason to believe that we have successfully expressed IL10. As a widely validated and used cytokine, we believe it can play an immunosuppressive role in vivo.
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We inserted this part into plasmid pCDFDuet-1. So the expression of this part can be induced by IPTG. We transformed the human IL10-Flag plasmid into BL21, incubated at 37 ℃ overnight and then diluted to OD600 = 0.2. After 2 h of growth at 37 ℃, IPTG was added to induce IL10 expression at 30 ℃ for 16 h. Then, the bacterial solution was lysed and the expressed IL10 was detected by Western Blot (Figure 1). From this result, IL10 was successfully expressed in our constructed plasmid. As a widely validated and used anti-inflammatory interleukin, IL10 has been identified with critical functions in immunosuppression in human [1].
  
 
https://static.igem.org/mediawiki/2018/a/ac/T--NEU_China_A--results-7.png
 
https://static.igem.org/mediawiki/2018/a/ac/T--NEU_China_A--results-7.png

Revision as of 17:34, 17 October 2018


IL10-flag

Interleukin-10 is a natural immunosuppressive cytokine that is normally released by regulatory T cells and plays an important role in intestinal homeostasis. In our engineered bacteria, we hope to induce the release of interleukin-10 through the inflammatory signal nitric oxide to achieve drug release controllability.

Usage and Biology

We inserted this part into plasmid pCDFDuet-1. So the expression of this part can be induced by IPTG. We transformed the human IL10-Flag plasmid into BL21, incubated at 37 ℃ overnight and then diluted to OD600 = 0.2. After 2 h of growth at 37 ℃, IPTG was added to induce IL10 expression at 30 ℃ for 16 h. Then, the bacterial solution was lysed and the expressed IL10 was detected by Western Blot (Figure 1). From this result, IL10 was successfully expressed in our constructed plasmid. As a widely validated and used anti-inflammatory interleukin, IL10 has been identified with critical functions in immunosuppression in human [1].

T--NEU_China_A--results-7.png

Figure 1. Western blot analyses using a flag antibody on bacterial lysate to detect IL10. Lane 1: Negative control (cell lysate without IPTG induction); Lane 2: 0.5mM IPTG, Lane3: 1mM IPTG induction for 16h at 30℃.

[1] Steidler, L. Treatment of murine colitis by Lactococcus lactis secreting interleukin-10. Science 289, 1352–1355 (2000).

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]