Composite

Part:BBa_K2967028

Designed by: Shuang Wu   Group: iGEM19_NEU_CHINA   (2019-10-06)
Revision as of 06:04, 7 October 2019 by Hatsuse (Talk | contribs)


Expressing vector for YebF-Myrosinase

Expressing vector has been constructed using pcold-1 backbone. We demonstrate western blotting to show the expression of myrosinase. Expressing vector harboring myrosinase 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 myrosinase is 57.46 kDa.

800px-T--NEU_China--part--Myro_vector.png

Figure 1. Diagram for myrosinase expressing plasmid in pcold-1. Promoter cspA, a super strong promoter when incubating at low temperature. RBS, ribosome binding site, downstream is gene myrosinase.

Figure 2. Expressing vector harboring myrosinase gene is transformed into BL21 strain using chemical method. After induction of IPTG, the culture is incubated at 37℃ overnight. The concentration of protein loaded on this two lanes are different.

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
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 689
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 918
    Illegal BsaI site found at 1947


[edit]
Categories
Parameters
None