Difference between revisions of "Part:BBa K3257043"
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This part is a mutant of MMLV Reverse Transcriptase and previous research shows that the specific amino acid mutation Y586F further improves its error-prone nature of MMLV reverse transcriptase by about 5-fold in a single replication cycle.<sup>[1]</sup> | This part is a mutant of MMLV Reverse Transcriptase and previous research shows that the specific amino acid mutation Y586F further improves its error-prone nature of MMLV reverse transcriptase by about 5-fold in a single replication cycle.<sup>[1]</sup> | ||
| + | Using SDS-PAGE, we have verified its expression and self-cleavage in ''E.coli'' (Figure 2). | ||
| + | |||
| + | [[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 19:13, 21 October 2019
Lower Fidelity MMLV gag-pol Polyprotein
This part is a mutant of MMLV Reverse Transcriptase and previous research shows that the specific amino acid mutation Y586F further improves its error-prone nature of MMLV reverse transcriptase by about 5-fold in a single replication cycle.[1]
Using SDS-PAGE, we have verified its expression and self-cleavage in E.coli (Figure 2).
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 174
Illegal BglII site found at 1286
Illegal BglII site found at 2592
Illegal BglII site found at 3824
Illegal BamHI site found at 2609
Illegal BamHI site found at 2915
Illegal XhoI site found at 940 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 1825
Illegal AgeI site found at 3396 - 1000COMPATIBLE WITH RFC[1000]
References
[1] Wen-Hui Zhang, Evguenia S. Svarovskaia, Rebekah Barr, and Vinay K. Pathak. Y586F mutation in murine leukemia virus reverse transcriptase decreases fidelity of DNA synthesis in regions associated with adenine–thymine tracts[J]. Proc Natl Acad Sci U S A. 2002 Jul 23; 99(15): 10090–10095