Team:Fudan-TSI/Basic Part
Basic Parts
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.
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, we have verified its expression in E. coli (Figure 1a).
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.
Project by Team:Fudan-TSI
Mutation library generation is critical for biological and medical research, but current methods cannot mutate a specific sequence continuously without manual intervention. We hereby present a toolbox for in vivo continuous mutation library construction. First, the target DNA is transcribed into RNA. Next, our reverse transcriptase (RT) reverts RNA into cDNA, during which the target is randomly mutated by our RT's enhanced error-prone ability. Finally, the mutated version replaces the original sequence through recombination. These steps will be carried out iteratively, generating a random mutation library of the target with high efficiency as mutations accumulate along with bacterial growth. Our toolbox is orthogonal and provides a wide range of applications among various species. R-Evolution could mutate coding sequences and regulatory sequences, which enables the evolution of individual proteins or multiple targets at a time, promotes high-throughput research, and serves as a foundational advance to synthetic biology.