1.#
Design 1#
In this project, we first synthesized the expression sequence based on the gene sequence of PDXA and PDXJ, optimized the codons, digested it, and inserted it into the expression vector pRSFDuet, and then introduced the expression sequence of GFP to enable visual expression, facilitate the detection of the expression process and the identification and analysis of the product. According to our design, when green fluorescent protein is presented, it indicates that the two key genes PDXA and PDXJ can be successfully expressed, and the initial yield can be judged based on the fluorescence.
Build 1#
We selected pCDFDuet-1 as the biosensor vector to construct the recombinant plasmid (Figure 1).
After synthesizing the relevant target gene sequence, the plasmid is introduced by enzyme digestion to construct a recombinant plasmid. The constructed plasmid is verified, and after confirming that the construction is complete, the recombinant plasmid is transferred into BL21 (DE3) competent cells.
Test 1#
We found that the obtained recombinant strain BL21(DE)3/pRSFDuet-1-PdxA-PdxJ-GFP showed yellow-green color when grown on LB solid medium in the E. coli expression system, indicating the expression of green fluorescent protein (Figure 3). At the same time, the PAGE electrophoresis results also showed a clear expression of the target protein.
Learn 1#
Through comprehensive result characterization such as strain growth experiment, agarose gel electrophoresis identification, fluorescent protein characterization and PAGE (polyacrylamide gel electrophoresis) experiment, we found that both PDXA and PDXJ target proteins were well expressed and their sizes were consistent with expectations, confirming the successful construction of the recombinant expression plasmid, normal expression of the reporter gene, and good results in the expression optimization of the two gene sequences (Figures 3, 4), laying a good foundation for the next step of construction and verification of the cell-free expression system.
2.#
Design 1#
In this project, we first synthesized the expression sequence based on the gene sequence of PDXA and PDXJ, optimized the codons, and constructed a linear expression vector after enzyme digestion. The T7 promoter sequence and related expression elements were introduced by PCR, and efficient expression was performed in the cell lysate. Detection and analysis were performed using conventional means. According to our design, when green fluorescent protein is expressed, the solution in the test tube will appear yellow-green after simple treatment, indicating that the two key genes PDXA and PDXJ can also be successfully expressed in the cell-free system, and a preliminary yield judgment can be made based on the fluorescence.
Build 1#
We used PCR amplification to construct a linear expression vector (Figure 5).
The gel electrophoresis pattern of the two-step PCR amplification results is shown below.
Test 1#
Through gel electrophoresis and fluorescence detection, we can well judge that the target gene was successfully amplified and the linear expression vector was successfully constructed. From the size of the fragment, we can clearly determine that the linear vector was successfully constructed. The cell lysate of the E. coli expression strain can express PdxA-PdxJ-GFP in a test tube. By treating and detecting the reaction solution, it was found that yellow-green fluorescence appeared, indicating that the cell-free system was successfully constructed and the target gene was successfully expressed (Figure 7).
Learn 1#
Based on the results of this part of the experiment, the agarose gel electrophoresis pattern shows that our linear expression vector was successfully constructed, and the green fluorescent protein also achieved a high expression level. The yellow-green fluorescence can be clearly observed in the test tube, and the concentration of the expression product is high, which can be used for the subsequent VB6 synthesis verification. This experiment also well confirmed that the key gene sequence in the circular vector can be smoothly expressed in the linear vector, and the product yield is good. The cell lysate of the expression strain contains enough components for protein expression, which can well perform extracellular protein synthesis. The above experimental results show that the protein expression products of the two key genes PDXA and PDXJ in this stage of the experiment can be used in the next step of VB6 biosynthesis verification experiment (Figure 8).
Design 2#
In this project, we first synthesized the expression sequence based on the gene sequence of PDXA and PDXJ, optimized the codons, and constructed a linear expression vector after enzyme digestion. The T7 promoter sequence and related expression elements were introduced by PCR, and efficient expression was performed in the cell lysate. Detection and analysis were performed using conventional means. According to our design, when green fluorescent protein is presented, the solution in the test tube will appear yellow-green after simple treatment, indicating that the two key genes PDXA and PDXJ can also be successfully expressed in the cell-free system, and a preliminary yield judgment can be made based on the fluorescence.
Build 2#
Combining the previous research experience, we constructed the biosynthetic pathway of VB6, as shown in the figure below.
According to the above experimental results, the in vitro synthesis of VB6 was directly verified by referring to the cell-free expression system of PDXA and PDXJ. In order to obtain higher yield and better effect, the experiment optimized the conditions of different time and temperature. The comparison results are shown in the figure below:
The results showed that the cell-free in vitro expression system can achieve better synthesis results at 37°C in 3 hours. After obtaining the best reaction conditions, we further explored the synthesis of VB6. Combining the experience of predecessors, we focused on the comparative study of synthesis time and yield to determine the feasibility of VB6 in vitro biosynthesis, laying the foundation for subsequent green large-scale production. The results in the above figure show that the peak of the reaction can be reached in 6 hours in vitro, and the expression level can reach 30mg/L in the case of small-scale expression. Compared with the intracellular (E. coli expression system), the yield is significantly increased (p=0.0109, figure below), which is expected to develop a faster and better VB6 green production system.
Test 2#
Through the detection and analysis of the VB6 biosynthesis system, we were able to obtain effective concentrations of VB6. Further high-pressure liquid phase detection was also able to confirm the existence of VB6, indicating that our cell-free expression system can not only obtain key synthases, but also perform efficient synthesis of VB6 (Figures 10, 11).
Learn 2#
In order to further verify the feasibility of the synthesis system, we also used high performance liquid phase to verify the reaction products. As shown in Figure 12, the characteristic peak positions and peak shapes of the experimental samples and standard samples are similar, which can well judge the successful expression of VB6. During the experiment, we obtained the latest reaction time and reaction problems of the current cell-free expression system and VB6 synthesis system. Of course, in large-scale synthesis experiments, the relevant experimental parameters need to be further refined and explored. Due to time constraints, we did not optimize all the key parameters in the reaction system. In this project, we mainly confirmed the feasibility of the VB6 synthesis method we proposed.
In summary, the experimental results of this project show that we have successfully constructed circular and linear expression vectors of PDXA and PDXJ, which have been verified in Escherichia coli and cell-free expression systems respectively; this project has also obtained a set of cell-free expression reaction systems that can achieve rapid expression of target genes. In this project, on the premise of obtaining high concentrations of PDXA and PDXJ in the first step, we explored the new biosynthesis of VB6 using glutamine, 5-phosphoglyceraldehyde and 5-phosphoribose as raw materials. The experimental results can well confirm the success of this project (Figures 10, 11, 12).