Part:BBa_K3257042
MMLV gag-pol Polyprotein
When the organization has once begun to vary, it generally continues varying for many generations. – Charles Darwin The Origin of Life
We would like to present this part as our best basic part. Moloney Murine Leukemia Virus Reverse Transcriptase (MMLV RT) is responsible for the reverse transcription process of the target Gene of Interest (GOI), which is rather one of the most important processes in our system.
MMLV RT acts as a reverse transcription tool with a relatively low fidelity so that it is qualified for a random mutation generator. Also, a previous study showed that point mutation (nucleotide from A to T) of at the 586th amino acid residual (from Y to F) of the polymerase itself (encoded merely by pol gene) (BBa_K3257043 https://parts.igem.org/Part:BBa_K3257043) further improves its error-prone nature by ≈5-fold in a single replication cycle.[1]
MMLV RT was first designed to be transcribed into a single mRNA with a stop codon between the gag gene (encoding capsid protein) and pol gene (encoding the reverse transcriptase), which could not be properly expressed in vivo (of E.coli) when we fused an EGFP after the gag-pol CDS.
Frustratedly, we set out to solve the problem by cloning it into various kinds of vectors, after different promoters and mutating the stop codon between gag and pol into Glutamine (from T to C), an intermediate amino acid residue as a read-through. (In wild type Moloney Murine Leukemia virus, there is a stop codon between mRNA transcribed from the gag gene and pol gene while it can be read through as a Glutamine when translated by ribosome to form gag and gag-pol polyprotein at a ratio of 20:1. We have changed the stop codon into a Glutamine codon to assure its readability in E.coli.)
Using SDS-PAGE and qRT-PCR, we have verified its expression in E.coli (Figure 1a) and its function as a reverse transcriptase (Figure 1b). Furthermore, its mutation rate is able to be measured by high throughput sequencing. In the future, this part can be used to express gag-pol polyprotein inside the bacteria to initiate the reverse transcription process in vivo.
Sequence and Features
- 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]
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