Difference between revisions of "Part:BBa K3257106"
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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. | 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. | ||
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| + | 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. | ||
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| + | [[File:Gag-pol-sds page.png|center|500px|thumb|'''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.]] | ||
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Latest revision as of 22:07, 21 October 2019
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:
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE 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 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 1901
Illegal AgeI site found at 3472 - 1000COMPATIBLE WITH RFC[1000]