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From 2014.igem.org

Background

Protein is one of the most powerful gift created by nature. Artificial protein products have been broadly applied in scientific researches, pharmaceutical industry, new energy fields and etc. Though protein's composition have been well-studied, even till nowadays, acquiring protein with new function is a laborious task for human. The current method to engineer protein, such as ration design and directed evolution, is both inefficient and labor-intensive.

To overcome this dilemma, this year, by integrating the separated steps of traditional directed evolution, SYSU-China intends to build an Integrated Evolution Machine, a system enabling us to obtain desired new protein in a short time automatically.

For the natural evolution process, inherent mutation provides the raw materials, then the natural selection drive the fittest dominating. In artificial evolution system, the selective force is manually chosen, directing the raw materials evolve to gain the expected function. However, for traditional directed evolution system, mutagenesis, selection and domination all need manual intervention, which makes the process slow and complicated.

In our system, therefore, in order to avoid laborious manipulation, the raw protein coding sequence is integrated into the genome of Enterobacteria phage M13, which enables the protein mutating spontaneously with the assistance of mutagenesis module in the engineered host. Different from the previous design(Liu DR et al., 2011), Bacterial Two-hybrid System provides the selective force in system, screening the protein with specific protein-protein interaction activity. Later, under the control of built-in RNAT module in host, phage carrying wanted protein sequence would be able to generate progeny and gradually become dominance.

Artificial protein products have been broadly applied in scientific researches, pharmaceutical industry, new energy fields, etc. Though being well-studied, even till nowadays, acquiring proteins with aimed new function is still a laborious task. The current method of engineering protein, such as ration design and directed evolution, is both inefficient and labor-intensive for involving manual operation all the steps.

To overcome this dilemma, by integrating the separated steps of traditional directed evolution (Annotation see below), SYSU-China intends to build an Integrated Evolution Machine (IgEM), a system enabling us to obtain desired new protein in a short time automatically.

In our system, to avoid laborious manipulation, steps of directed evolution are integrated into life cycle of Enterobacteria phage M13 (Annotation see M13 part). In this way, with the assistance of mutagenesis module in the engineered host, primitive protein coding sequence could mutate spontaneously as the M13 phage infects and replicates. Different from the previous design (Liu DR et al., 2011), Bacterial Two-hybrid System provides the selective force in system, screening the protein with specific protein-protein interaction activity. Later, under the control of built-in RNAT module in host, phage carrying wanted protein sequence would be able to generate progeny and gradually become dominance.

Annotation of Traditional directed evolution Traditionally, directed evolution system consists of three steps, mutagenesis, selection and domination (amplification?). Firstly mutagenesis, usually realized by error-prone PCR, generates a pool of candidate protein coding sequences. Then, using screening techniques, such as phage display and yeast-two-hybrid system, certain candidates with expected property would be selected out. Finally, isolated ideal mutant sequence could be amplified by PCR. As the selective force is manually chosen, after several rounds of screening, the raw materials would be directly evolved to gain the expected function. Apparently, all steps above need manual intervention, which makes the process slow and complicated.