Results

Experimental Results

1. Validation the Feasibility of TRACE system-mediated directed evolution

Figure 1. Feasibility validation of TRACE system.

(1) Construction of pTarget-EGFP

We synthesized the coding sequence of EGFP and obtained an EGFP fragment with homologous arms through PCR amplification. The pTarget plasmid (pCDH-CMV-T7) was subjected to double digestion with the restriction enzymes EcoR I and BamH I. Gel electrophoresis was conducted for both the digested fragments and the PCR products, followed by excision and purification of DNA bands corresponding to the desired sizes.
The purified DNA fragments were then ligated using homologous recombination (Gibson Assembly). The Gibson Assembly we used is a method of homologous recombination that involves the use of homologous arms. Homologous arms are short sequences added to the ends of the target DNA fragment, which have sequence overlaps with the recipient DNA. The Gibson Assembly method involves mixing the target DNA fragments, linearized vector DNA, and a recombinase enzyme system, and promoting the overlapping pairing between the homologous arms at iso-thermal conditions, resulting in the formation of ligated products. Specifically, the enzyme system includes an exonuclease, a polymerase, and a ligase. The exonuclease removes a small segment of bases from the 3' end of the target DNA fragment, generating a single-stranded overhang. Then, the polymerase uses this overhang as a primer to initiate the synthesis of a new DNA chain, connecting the target DNA fragment with the recipient DNA. Finally, the ligase joins the adjacent DNA chains of the ligation product to form a complete DNA construct.

Figure 2. Construction of pTarget-EGFP plasmid.

Subsequently, the ligation product was transformed and plated onto agar plates containing ampicillin, followed by overnight incubation at 37°C. The following day, single colonies were selected and inoculated into an ampicillin-resistant LB medium for overnight cultivation. Plasmid extraction and sequencing were performed on the overnight cultures, resulting in the successful verification of the correct sequencing of the pTarget-EGFP plasmid (sequencing results specifically showed the bases near the site of the site-directed mutation, with the remaining sequences confirmed to be accurate).

(2) Construction of pTarget-BFP

Site-directed mutagenesis is a molecular biology technique used to introduce single nucleotide changes into DNA or RNA sequences selectively. This technique allows for the precise alteration of one or more nucleotides in a specific gene, resulting in changes to the target protein sequence. Site-directed mutagenesis is typically achieved by using a pair of primers that contain the desired changes in the nucleotides of interest for PCR amplification. During PCR, these primers anneal to complementary regions in the template DNA sequence and introduce the desired changes to the target nucleotide(s) during amplification. Through selective amplification and screening of individual colonies, DNA sequences with the desired mutations can be isolated from a mixed pool of DNA.
To obtain the pTarget-BFP plasmid, we used the pTarget-EGFP plasmid as a template and performed site-directed mutagenesis. Specifically, by using a pair of site-directed mutagenesis primers, we introduced a point mutation through PCR to change the tyrosine residue at position 67 of EGFP (green fluorescent protein) to histidine, resulting in the generation of BFP (blue fluorescent protein).

Figure 3. Construction of pTarget-BFP plasmid.

(3) Transition from Blue to Green Fluorescence Protein: Validating the TRACE System

To validate the feasibility of the TRACE system, we introduced the pTarget-EBFP plasmid into HEK293FT cells that were pre-transfected with the pEditor plasmid to enable expression of EBFP. If the TRACE system was functional, upon activation of the TRACE system, we expected to observe a transition from blue fluorescence protein(BFP) to green fluorescence protein(GFP) due to the C-to-T mutation at position 199. The TRACE system was initiated by adding 1µM doxycycline, which induced the expression of AID-T7 RNAP-UGI to edit the EBFP gene downstream of the T7 promoter. We monitored the cells daily for the presence of green fluorescence. After 8 days of TRACE system activation, we successfully observed green fluorescence and recorded the images, indicating that the TRACE system was functioning properly.

Figure 4. Conversion of BFP to GFP by the TRACE system.

2. Directed evolution of NAMPT gene by the TRACE systemevolution

Figure 5. Search for highly active NAMPT mutants.

After verifying the feasibility of the TRACE system, we can utilize this system to evolve the key rate-limiting enzyme NAMPT involved in NAD+ biosynthesis. Before that, we require a reporting system for real-time monitoring of intracellular NAD+ concentration. Through literature search, we discovered that the team of Yuzheng Zhao from East China University of Science and Technology in Shanghai has developed the FiNad reporting system, which is specifically designed to monitor changes in NAD+ concentration both inside cells and in vivo. We obtained the ORF of FiNad from their published article, synthesized the fragment through DNA synthesis, and constructed it into the pCDH plasmid for the implementation of NAMPT-directed evolution experiments (With the help from team advisors).

Figure 6. Construction of pCDH-CMV-mCherry-FiNad plasmid.

Due to the time constraints of the iGEM competition, we were unable to complete the entire process of NAMPT evolution experiments. However, through patent search, we discovered that some sites on the NAMPT protein have been confirmed through experiments such as prokaryotic expression, protein purification, and enzyme activity assays to enhance the enzymatic activity of NAMPT. We went forward and successfully constructed these mutated plasmids by our own, which can be used for subsequent enzyme activity validation.

(1) Construction of the wild-type NAMPT expressing plasmid

Biomolecules are separated by applying an electric field to move the charged molecules through an agarose matrix, and the biomolecules are separated by size in the agarose gel matrix. A ladder-like pattern of DNA fragmentation appeared upon agarose gel electrophoresis. Agarose gel electrophoresis is used to know how many different DNA fragments are present in a sample and how large they are relative to one another.
By comparing the bands in a sample to the DNA ladder using the gel documentation system, we can determine their approximate sizes. As a result, we confirmed that NAMPT fragments were successfully amplified, and pCDH vectors were also successfully linearized with restriction enzymes.

Figure 7. NAMPT fragment and linearized vector preparation for molecular ligation.

We use the NanoDrop to quantify the amount of DNA sample.Nucleic acids absorb light at a wavelength of 260 nm. If a 260 nm light source shines on a sample, the amount of light that passes through the sample can be measured, and the amount of light absorbed by the sample can be inferred. For double-stranded DNA, an Optical Density (OD) of 1 at 260 nm correlates to a DNA concentration of 50 ng/μL so that DNA concentration can be easily calculated from OD measurements.

Figure 8. NAMPT plasmid concentration measurement by Nanodrop.

(2) Site-directed mutagenesis of NAMPT genes

Agarose Gel Electrophoresis

Biomolecules are separated by applying an electric field to move the charged molecules through an agarose matrix, and the biomolecules are separated by size in the agarose gel matrix. A ladder-like pattern of DNA fragmentation appeared upon agarose gel electrophoresis. Agarose gel electrophoresis is used to know how many different DNA fragments are present in a sample and how large they are relative to one another.
Using the gel documentation system, it was confirmed that the NAMPT circular plasmid was successfully amplified.

Figure 9. Site-directed mutagenesis of NAMPT genes.
Pick multiple single clones for sequencing identification

Bacteria grow on solid media as colonies. A colony is defined as a visible mass of microorganisms all originating from a single mother cell, therefore a colony constitutes a clone of bacteria all genetically alike. As shown in the pictures below, the plates of bacteria cultured by different groups of students all grew monoclonal colonies.
Each student picked 3 individual bacterial colonies using sterile toothpicks, cultured them into a medium containing ampicillin antibiotic, and incubated them at 37°C overnight with shaking (300 rpm).

Figure 10. The status of colony formation for each NAMPT mutant.
Plasmid sequencing

The monoclonal bacteria were amplified overnight and sent to the corresponding sequencing company for sequencing.The Sanger sequencing technique is the most commonly used method for determining DNA sequences of recombinant plasmids. Sanger sequencing involves the use of a DNA polymerase, a primer, unlabeled deoxynucleotide triphosphates (dNTPs), and fluorescently labeled dideoxynucleotide triphosphates (ddNTPs), where each base is labeled with a unique fluorophore. Incorporation of a ddNTP into the newly synthesized strand prevents the addition of subsequent nucleotides, stopping the further elongation of the DNA molecule and resulting in a DNA product with a fluorescently labeled ddNTP at the end of the strand. The sequence of nucleotides can then be determined by separating the DNA products by size.
By using SnapGene software, we could align DNA sequences with a reference sequence to verify mutagenesis. As shown in the pictures below, we successfully constructed five point mutations including D354E, G384C, L386P, V221A, and V221G.

Figure 11. The sequence results for each NAMPT mutant.

3. [Future plan] Biochemical validation of highly active NAMPT mutants for NAD+ Bio-production

Figure 12. Biochemical validation of highly active NAMPT mutants.

As long as we obtain the active NAMPT mutant(s), we will first validate the screen results through a series of biochemical assays including site-directed mutagenesis, prokaryotic expression, protein purification, and enzyme activity assays of NAMPT. If our mutant(s) passes these assays, we will go further and test its efficacy for NAD+ bio-production.