Part:BBa_K3257043

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:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE 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
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 1825
  Illegal AgeI site found at 3396
  
• 1000
  COMPATIBLE 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