When the organization has once begun to vary, it generally continues varying for many generations. – Charles Darwin, The Origin of Life
Best basic part
As best basic part we’d like to present the Moloney Murine Leukemia Virus Reverse Transcriptase (MMLV-RT) BBa_K3257042. 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.
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 (about 60.4kDa) and the reverse transcriptase (about 69.1kDa) by a protease (about 13.5 kDa) also encoded in the polyprotein mRNA (Figure A).
Figure A. Exhibition of gene encoding gag-pol polyprotein.
MMLV-RT acts as a reverse transcription tool with a relatively low fidelity so that it is qualified for a random mutation generator. Also, Zhang et al. 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), further improves its error-prone nature by ≈5-fold in a single replication cycle.
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 residual 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 A) and its function as a reverse transcriptase (Figure B). 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.
Figure B. gag-pol polyprotein is successfully expressed and underwent excision in the cell.
For more information of other basic parts, please refer to the part collection page and the iGEM Part Registry ranging from BBa_K3257000 to BBa_K3257076.
Reference
- [1] Zhang W H, Svarovskaia E S, Barr R, et al. Y586F mutation in murine leukemia virus reverse transcriptase decreases fidelity of DNA synthesis in regions associated with adenine-thymine tracts. Proceedings of the National Academy of Sciences, 2002, 99(15):10090-10095.