Difference between revisions of "Part:BBa K2967031"

Latest revision as of 09:22, 16 October 2019

Expressing vector for YebF-mouse IL10

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 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. 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
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
INCOMPATIBLE WITH RFC[21]
Illegal BglII site found at 1128
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI.rc site found at 338
Illegal SapI.rc site found at 971

@@ Line 3: / Line 3: @@
 <partinfo>BBa_K2967031 short</partinfo>
-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&#8451; 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.
+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&#8451; for 12h, CFU was inoculated to LB medium followed by 12h incubation. The culture was then diluted to OD<sub>600</sub>=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.
 https://static.igem.org/mediawiki/parts/thumb/4/46/T--NEU_China--part--YebFmil10_gene_circuit.png/800px-T--NEU_China--part--YebFmil10_gene_circuit.png
@@ Line 11: / Line 11: @@
 https://static.igem.org/mediawiki/parts/thumb/d/d5/T--NEU_China--result--il10_wb.png/600px-T--NEU_China--result--il10_wb.png
-'''Figure 2. Protein expression of mouse IL-10 gene which transformed in BL21 strain.''' 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.
+'''Figure 2. 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.