Difference between revisions of "Part:BBa K2967031"

Revision as of 08:17, 10 October 2019

Expressing vector for YebF-mouse IL10

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

Figure 1. 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 2. Expressing vector harboring IL-10 gene is transformed into BL21 strain using chemical method. After induction of IPTG, final concentration at 1mM, the culture is incubated at 37℃ for 12h followed by western blotting. Samples in two control lanes and samples in two mIL-10 lanes have no difference.

Sequence and Features