Part:BBa_K2967032

YebF-mouse IL10-flag

In our project, we used our engineered bacteria strain to express anti-inflammatory proteins to relieve IBD symptoms. As a complex chronic disorder, IBD can be induced by various factors which we have mentioned before. Among these factors, immunity imbalance is acting as a critical inducer and over-activated immune cells produce large amount of cytokines that lead to intestinal layer destruction and inflammation. However, cytokines production is extremely regulated in immunocompetent host; thus, Interleukin-10 (IL-10) plays a central role in downregulating inflammatory cascades and maintain the intestinal layer functioning [1]. Furthermore, IL-10 has been determined in preventing cell accumulation which consequently alleviates IBD. However, the administration of purified IL-10 protein via oral and intravenous routes is not practical. Therefore, we used genetically modified E. coli strain to express IL-10 protein to relieve chronic inflammation [2].

Figure1. The effect of our anti-inflammatory E. coli. Excessive inflammation happens when immune cells such as effector T cells and macrophages keep accumulating. But Interleukin-10 can stop this trend [3].

[1] Steidler, L., et al., Treatment of murine colitis by Lactococcus lactis secreting interleukin-10. Science, 2000. 289(5483): p. 1352-5.

[2] Steidler, L., et al., Biological containment of genetically modified Lactococcus lactis for intestinal delivery of human interleukin 10. Nat Biotechnol, 2003. 21(7): p. 785-9.

[3] Liang YE and Huang Z. IL-10 Signalling in Macrophage during Autoimmunity. Austin Biol. 2016; 1(1): 1004.

Characterization (See BBa_K2967031)

Expressing vector has been constructed using pCDFDuet-1 backbone. Western blotting was used to show the expression. Expressing vector harboring IL-10 gene was transformed into BL21 strain using chemical method. After incubating at 37℃ for 12h, CFU was inoculated to LB medium followed by 12h incubation. The culture was then diluted to OD₆₀₀=0.2 and let grow to around 0.5. 1 mM IPTG was added to the culture to induce protein expression followed by 12h incubation. Cells were washed and collected, ready for western blotting. The molecular weight of protein YebF-mIL-10 is 33.46 kDa.

Figure 2. Diagram for mouse IL-10 expressing and secreting vector 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 binding site. Secretory tag YebF is introduced to secret protein downstream.

Figure 3. Protein expression of mouse IL-10 gene which transformed in BL21 strain. After induction of IPTG, final concentration at 1mM, the culture was incubated at 37℃ for 12h followed by western blotting. Samples in two control lanes and samples in two mIL-10 lanes had no difference.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 681
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 681
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 681
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 681
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 681
  
• 1000
  COMPATIBLE WITH RFC[1000]