Difference between revisions of "Part:BBa K3257042"

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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. Naturally, when Moloney Murine Leukemia Virus (MMLV) infects its host, gag-pol polyprotein encoded in the MMLV genome is expressed. This polyprotein will be further cleaved into the capsid protein and the reverse transcriptase by a protease also encoded in the polyprotein mRNA (Figure 1).

Figure 1. Exhibition of gene encoding gag-pol polyprotein

Moloney Murine Leukemia Virus Reverse Transcriptase (MMLV RT) is responsible for the reverse transcription process of the target gene, 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 reverse transcription cycle (please refer to https://parts.igem.org/Part:BBa_K3257043).

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, we have verified its expression in E.coli (Figure 2). 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