Engineering Success

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Directed evolution techniques not only facilitate the rapid optimization of proteins, but also enable the optimization of specific cell types or their genetic backgrounds, providing a platform for developing targeted drugs. In 2018, for her outstanding contributions in the field of "directed evolution of enzymes", Professor Frances H. Arnold was awarded the Nobel Prize in Chemistry.

Figure 1. Prof. Frances H. Arnold wins the 2018 Nobel Prize in Chemistry.

The basic process of directed evolution can be divided into three main steps:
(1) Constructing a mutant library.
(2) Expressing the library in a suitable expression system.
(3) Performing fast, efficient, and high-throughput selection or screening.
In sum, directed evolution aims to simulate natural evolution under specific laboratory conditions. By inducing mutations and recombination, a large library of mutants is created. After expressing the mutations in host cells, specific conditions are used for selection or screening to identify proteins with desired characteristics, achieving molecular-level evolution simulation.

Figure 2. The schematic illustration for Directed Evolution.

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To construct a mutation library, a recently developed system called TRACE (T7 polymerase-driven continuous editing) has been introduced in recent years. This system utilizes AID (activation-induced cytidine deaminase) fused with T7 RNA polymerase (T7 RNAP) as the editor, enabling continuous and targeted mutations in mammalian cells. When this system is activated, the editor recognizes the target gene sequence containing the T7 promoter and introduces random mutations on the target gene through cytidine deaminase during transcription. During the preparation of our iGEM competition, Professor Yang Yu from the Institute of Biophysics, Chinese Academy of Sciences introduced this powerful system to us, which has been extensively used in his lab, and told us we are welcome to harness this tool to achieve our goal to improve the activity of NAD+ production enzymes.

Figure 3. The schematic illustration for the TRACE System.

Therefore, we first obtained the cell line that was pre-built with the Editor system from Professor Yang Yu's laboratory. This cell line incorporates the AID-T7 RNAP-UGI Editor system inducibly expressed by doxycycline (Dox). After expression, this system can target and induce mutations in the target gene (Target) driven by the T7 promoter.

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Based on previous research, the coding sequence of the blue fluorescent protein (BFP) gene can be used to validate the feasibility of the TRACE mutation system visually. This BFP gene is derived from the EGFP gene, with a mutation of the 199th base from T-A to C-G, shifting the fluorescence color from green to blue.Therefore, if the TRACE system is feasible, the mutated BFP gene obtained from site-directed mutagenesis can be reversed back to the EGFP gene, resulting in a shift in cell fluorescence from blue to green.
The following experiments were co-performed under the guidance of our advisors. To obtain the pTarget-EBFP plasmid, we first cloned the EGFP gene and linearized the pTarget plasmid using restriction endonucleases. Subsequently, we performed homologous recombination to ligate the linearized pTarget vector with the EGFP gene, resulting in the successful generation of the pTarget-EGFP plasmid with correct sequencing. Next, using a pair of site-directed mutagenesis primers, we introduced a mutation at nucleotide position 199 in the EGFP gene, changing T to C. We ultimately succeeded in constructing the pTarget-EBFP plasmid with correct sequencing (sequencing results specifically showed the bases near the site of the site-directed mutation, with the remaining sequences confirmed to be accurate).

Figure 4. The sequence results for the construction of EGFP and EBFP plasmids.

Then, we introduced this BFP plasmid into the cell line that was pre-built with the Editor system as mentioned above. We proceeded to activate the TRACE system by adding doxycycline at a final concentration of 1μM to the culture medium. This induced the expression of AID-T7 RNAP-UGI, initiating continuous mutagenesis of the target gene. In the BFP validation experiment, we observed significant green fluorescence after 8 days of doxycycline induction, confirming the feasibility of the TRACE system.

Figure 5. The immunofluorescence results confirmed the successful directed evolution of GFP from BFP by the TRACE system.

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This exciting success was not achieved overnight, but rather through many failures and subsequent troubleshooting and retries. For example: (1) Initially, we hoped to directly transform the wild-type BFP protein into GFP protein using the directed evolution system. It became apparent that more mutations would be required, and these mutations needed to occur simultaneously on one mRNA in order for it to be successful. Due to very low probability, it did not succeed. (2) After dismissing the first approach, we started thinking about how to use a simpler system to validate the possibility of our directed evolution. This process was time-consuming and demanding, requiring full support and a literature search from our advisory team (3) During the process of finding and confirming the final feasible approach, we also encountered many problems, such as the expressed BFP protein not fluorescing, introducing new mutations through plasmid construction, and the directed evolution time being too short, resulting in no green fluorescence appearing.
Fortunately, through the relentless efforts of team members and guidance from our advisors, we finally saw the exciting green fluorescence, which proved the feasibility of this system and the engineering success required for our next experimental goal.