Inspiration

Why are we doing mutation library generation?

Mutation is a natural component of life, its participation is crucial for the development of any species, or on a much smaller level, any genetic materials.

Mutation libraries are widely used in various fields, allowing for downstream high throughput scanning of desired product. It’s generation not only gives new insights to the nature of existing products, but also helps to bring about novel outcomes, promoting synthetic biology in its way. Through the development of mutagenesis methods, we hope to make up the missing puzzle by mutating only the target sequence in vivo. We believe that this offers researchers an easy and care-free choice among the existing ones.

Existing mutagenesis methods

Current mutagenesis methods can be divided into two large categories, in vitro and in vivo. In vitro methods like error-prone PCR is most-used among researchers, but it bears the intrinsic problem of separate mutagenesis and selection period, which brings about laborious and costly design-build-test cycles, as well as the limitation of host transformation efficiency.

The most renowned in vivo methods these days would be PACE (Phage Assisted Continuous Evolution). However, apart from its difficulty to set up in common laboratories, it encounters the limitation of linking selection process to phage survival, which greatly limits its range of applicable substrates. Also, the mutagenesis plasmid it used would mutate the whole genome and plasmid alike.

We hope to establish a system to address the above problems and allow for easy manipulation of genetic sequences. We then consulted our idea through mail with Prof. Alper, who had published a paper of a similar system in yeast, and received his supportive response.

A brief description of R-Evolution

R-Evolution is a dual plasmid system, with one plasmid containing the target sequence and necessary flanking sequences; while the other contains the remaining components for the target’s random mutation.

In the system, the target sequence will go through three process—transcription into mRNA, reverse transcription into cDNA, and recombination to allow the mutated sequence replace the original target.

The target sequence is placed under T7 promoter and allowed to express stably. The target sequence is flanked by the sequences supporting reverse transcription, and in the outward loxP sites are placed.

The reverse transcription module, consists of the gag-pol polyprotein from Moloney murine leukemia virus (MMLV), and its matching initiation tRNA, tRNAPro. This is the crucial step where the target sequence will be randomly mutated by the error-prone nature of reverse transcriptase.

The recombination process utilizes Cre recombinase and incompatible loxP sites on either end to allow for the re-insertion of mutated sequence and minimize the possibility of self-splicing. To gain better control of this process, degradation tag is added to enable its fast disappearance in the system.

As transcription serves as a great amplifier and could happen continuously, the cell could obtain various versions of mutated sequence, which are then randomly inserted back into the plasmid. Also, the three processes can happen for multiple times in one round of the system, allowing for mutation accumulation and bringing an increase to mutation rate.