Background

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) into life cycle of modified Enterobacteria phage M13 (Annotation see M13 part), SYSU-China intends to build Integrated Evolution Machine (IgEM), a system enabling us to obtain desired protein in a short time automatically.

In our system, the primitive protein coding sequence would evolve spontaneously as modified M13 phage infects the host and replicates itself. With the assistance of mutagenesis module in the engineered host, mutation could be rapidly introduced into the sequence. Different from the previous design (Liu DR et al., 2011), Bacterial Two-hybrid System provides the selective pressure in system, screening the protein trending with specific protein-protein interaction activity. As the modified gVIII deleted M13 phage is budding-deficient, only the favorable candidate proteins could activate compensating gene transcription and rescue phage budding, thus surviving during evolution. Later, under the control of RNAT module, phage carrying prospective protein coding sequence would generate its progeny and gradually become dominance in population.

IgEM vs. Traditional directed evolution

Traditional directed evolution

Traditionally, directed evolution system consists of three steps, mutagenesis, selection and 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 pressure 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.

What if IgEM is available?

Apparently, all traditional steps need manual intervention, making the process slow and complicated. It usually takes researchers days to accomplish a single round of selection. It has been reported that in vivo directed evolution system, focusing on DNA-protein interaction, could realize single round of screening as soon as in 37 minutes (Liu DR et al., 2011). What if IgEM is available?