Core Design
We primarily use SnapGene for plasmid design and construction, and the online tool NovoPro for reverse translation from protein to DNA and codon optimization of the DNA sequence. Based on the protein information generated by the model's optimization, we generate the corresponding gene sequence according to the amino acid sequence and ligate the gene sequence onto a plasmid suitable for protein function characterization to construct an engineered plasmid.
After establishing the plasmid model in SnapGene, we conduct experiments in the laboratory. We order synthesized genes and the primers required for plasmid construction from a partner laboratory company. We use PCR technology to synthesize the target gene, ligate the target gene with the plasmid backbone to construct the plasmid vector, and then introduce the vector into E. coli for functional characterization. Alternatively, we enrich the culture with E. coli, then extract the plasmid from the E. coli, and introduce it into Bacillus subtilis for functional characterization. According to the optimization direction of the model, we design corresponding functional evaluation methods and standards to determine whether the model optimization is successful.
Icons of various tools
Example of Experimental Design
Experimental design is the starting point of wet experiment work and also the most challenging and interesting process. Here we design functional validation experiments for protein GFP together. The design of the GFP protein function optimization validation experiment can well represent the design process of our validation experiments for other proteins.
First, we got the optimized amino acid sequence of the protein provided by the PROTEUS model. We got a preliminary DNA gene sequence based on the reverse translation of the amino acid sequence. We want to use the PHT01 plasmid of Bacillus subtilis for gene expression in Bacillus subtilis, so we need to codon-optimize the initial DNA sequence to improve its expression efficiency in Bacillus subtilis. All of this work can be done using online tools.
Figure 22: GFP sequence optimization process
We thus got the gene that expresses the GFP protein. From now on, SnapGene's software will be our main battleground. Next, we'll insert the gene into the original plasmid. Since the dead grass itself does not express the GFP protein and there is no similar gene on the plasmid, we will add the promoter and terminator sequences to the GFP gene. Among them, the promoter sequence is crucial, as a strong promoter can enhance the expression efficiency of the gene. This is also one of the reasons why we chose Bacillus subtilis to express the gene.
Figure 23: Plasmid design with promoter and terminator sequences
With the addition of promoters and terminators, we got a complete plasmid expression vector in software. But a lot of primers are needed to work together to make it a reality. First, we design primers to synthesize genes. The primers we use here are around 70bp long, very long. We want the primers to be templates to each other, with the primers connected end to end.
Figure 24: Primer design for gene synthesis
In the PCR reaction system, we add 12 synthetic primers and the related enzymes. During the reaturation process, the primers bind in pairs and end to end; During the extension process, the polymerase fills the gap between the primers. After 30 rounds of PCR, we got a certain amount of synthetic genes. Then we used this round's product as a substrate template for another round of PCR, where we used upstream primers and downstream primers as primer combinations to amplify the target gene. After two rounds of PCR, we conducted an electrophoresis test on the product, and the size was correct, indicating that we had synthesized the target gene.
Figure 25: PCR amplification results and electrophoresis verification
The insertion of the gene into the plasmid was also achieved using special primers. On the map displayed by the software, locate the two ends of the gene and set two primers (upstream GFP-L-1 and downstream GFP-L-2) at the junction of the gene and the plasmid. The two primers are used to amplify the skeleton fragment from the plasmid. Use ligases to link genes to the skeleton.
Figure 26: Primer locations for gene insertion into plasmid
After that, we added the promoter and terminator sequences using primers. Since both the promoter sequence and the terminator sequence are short, after inserting the sequence into the appropriate position on the map, add the corresponding sequence directly to the 5 'end of the primer, and then add the sequence to the plasmid through the primer.
Figure 27: Final plasmid construction with promoter and terminator addition
We synthesized the desired plasmid through five rounds of PCR (two rounds of gene synthesis, one round of plasmid skeleton amplification, one round of promoter addition, and one round of terminator addition) and ligation. The next step is the process of introducing the plasmid into the recipient cell for enrichment and expression. It will not be elaborated here.