Composite

Part:BBa_K314104

Designed by: Tomas Huber, Justin Siegel   Group: iGEM10_Washington   (2010-10-08)

T7 expression cassette

Lactose (IPTG) inducible T7 protein expression insert includes the f1 origin BBa_K314110, a Lac I generator BBa_K314111, a T7 promoter BBa_K314112 that includes the Elowitz standard RBS BBa_B0034. When ligated with a protein coding sequence at the SpeI site the scar site created will create proper spacing for protein expression.

Usage and Biology

Washington2010 vector assay data.png BBa_E0040 (GFP) was inserted between S and P using standard BioBrick assembly. The resulting constructs were grow in DH5a (constitutive promoters BBa_J23100 or BBa_J23114, as well as inducible promoter BBa_R0011) or BL21(DE3)* (T7 promoter, BBa_K314104) cells overnight at 298K in either the presence or absence of 0.5mM IPTG. The cells were harvested, washed in PBS, and then fluorescence for the whole cells was measured on a fluorometer (ex. 485, em. 525) and normalized to cell density (OD600). Cells that did not contain GFP were used as a blank. As observed, the addition of IPTG significantly increased the observed fluorescence of the cells.


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 NgoMIV site found at 126
  • 1000
    COMPATIBLE WITH RFC[1000]


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