Composite

Part:BBa_K3257106

Designed by: Yi Qiao   Group: iGEM19_Fudan-TSI   (2019-10-13)

gag-Q-pol with T5 Promoter-IPTG Inducible

This part is an expression composite of the Moloney Murine Leukemia Virus Reverse Transcriptase (MMLV RT) and T5 Promoter. Under different regulation by different promoters, the expression level varies accordingly.

Cloned into pET16b vector, transformed into E.coli BL21(DE3) and analyzed induced protein expression by SDS-PAGE, we have shown that gag-pol polyprotein can be expressed and self-cleaved.

Figure 2. SDS-PAGE of gag-pol polyprotein expression This SDS-PAGE analysis proves the expression and self-cleavage of gag-pol polyprotein. As displayed in the result, WT represents the gag-Q-pol polyprotein (BBa_K3257042 https://parts.igem.org/Part:BBa_K3257042) and Mutant represents the Y586F mutant of gag-Q-pol polyprotein (BBa_K3257043 https://parts.igem.org/Part:BBa_K3257043). - represents the uninduced bacteria culture sample and + represents the induced bacteria culture. Comparing + and -, we can see that IPTG induction enables protease (about 13.5 kDa) to be expressed and we can also see an increase of capsid protein (about 60.4 kDa) and reverse transcriptase (about 69.1 kDa) expression.

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 250
    Illegal BglII site found at 1362
    Illegal BglII site found at 2668
    Illegal BglII site found at 3900
    Illegal BamHI site found at 2685
    Illegal BamHI site found at 2991
    Illegal XhoI site found at 1016
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 1901
    Illegal AgeI site found at 3472
  • 1000
    COMPATIBLE WITH RFC[1000]


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