Plasmid

Part:BBa_K5375004

Designed by: BOHAN REN   Group: iGEM24_Keystone   (2024-09-23)


pA7-GFP-Profilin 3



Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 4417
    Illegal XbaI site found at 4093
    Illegal SpeI site found at 3367
    Illegal PstI site found at 2413
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 4417
    Illegal SpeI site found at 3367
    Illegal PstI site found at 2413
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 4417
    Illegal BamHI site found at 4127
    Illegal BamHI site found at 4822
    Illegal XhoI site found at 3302
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 4417
    Illegal XbaI site found at 4093
    Illegal SpeI site found at 3367
    Illegal PstI site found at 2413
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 4417
    Illegal XbaI site found at 4093
    Illegal SpeI site found at 3367
    Illegal PstI site found at 2413
  • 1000
    COMPATIBLE WITH RFC[1000]


BBa_K5375004: pA7-GFP-Profilin 3

BBa_K5375004: pA7-GFP-Profilin 3

Profile

Name: pA7-GFP-Profilin 3

Origin: Synthesized by company and constructed by the team

Properties: Fusion expression of protein Profilin3-GFP

Usage and Biology

The pA7 plasmid vector serves as a carrier for the expression of fusion proteins, particularly well-suited for the production of GFP fusion proteins in prokaryotic cells such as E. coli. This vector features a multi-cloning site (MCS), which enables researchers to insert target genes, thereby facilitating the fusion of the target protein with GFP for subsequent expression. Such design allows for visualization and tracking of the target protein through GFP, aiding in investigations into its localization, expression levels, and dynamic behavior within cellular environments. Typically, the pA7 plasmid incorporates a robust promoter—such as lac or tac—to enhance expression efficiency and may include an antibiotic resistance gene serving as a selection marker to identify transformed bacterial strains. Furthermore, this vector may also possess a cleavable tag sequence that permits removal of GFP by specific proteases (e.g., TEV protease) at later stages, thus yielding purified target proteins. The strategic design of this vector not only streamlines protein purification processes but also facilitates functional analysis of proteins.

Cultivation and Purification

Figure 1. Plasmid map of pA7-GFP-PFN3
Figure 1. Plasmid map of pA7-GFP-PFN3.

The vector PA7 originates from a non-respiratory clinical isolate of Pseudomonas aeruginosa from Argentina, later linked with GFP. It is used for protein expression in plants, a plant expression vector including a 35S promoter and ampicillin resistance, and is usually cultivated in a DH5a E. coli strain at 37°C. It was chosen to measure the protein expression of PFN3.

Figure 2. PCR amplification of the fragment used for plasmid construction
Figure 2. PCR amplification of the fragment used for plasmid construction. The pA7-GFP-PFN3 sequence was amplified by PCR, with a length of 396 bp.

Characterization/Measurement

We constructed pA7-GFP-PFN3 using homologous recombination. The pA7-GFP-PFN3 sequence was amplified by PCR, with a length of 396 bp. Then the target gene sequence, including Profilin 3, was inserted. It was reconstructed through homologous recombination. To incubate and culture the reassembled plasmid overnight, it is diluted and spread out onto an LB agar plate. In the figure shown above, the growth of pA7-GFP-Profilin 3 was significant.

Figure 3. Growth of plasmid pA7-GFP-PFN3 transformed bacterial on LB agar plates
Figure 3. Growth of plasmid pA7-GFP-PFN3 transformed bacterial on LB agar plates.

Single colonies from each of the LB agar plates were taken and amplified through PCR. Multiple samples were taken from each of the plates to ensure that even if an error does occur, other samples can cover it.

Figure 4. Colony PCR verification of PA7-PFN3
Figure 4. Colony PCR verification of PA7-PFN3.

The sequencing results of the reconstructed plasmid PA7-GFP-PFN3 were within the expected parameters, indicating no significant deviations. The sequence matched the designed structure, confirming the successful integration of the target gene into the plasmid.

Figure 5. Sanger sequencing map of PA7-GFP-PFN3
Figure 5. Sanger sequencing map of PA7-GFP-PFN3.

By transforming the reconstructed plasmid into the protoplasm of Arabidopsis thaliana, and observing the changes of GFP fluorescence intensity and PFN3 gene expression in the protoplasm after the addition of siRNA that inhibited the expression of this protein, the effectiveness of siRNA was verified.

Figure 6. Enzymatic hydrolysis solution for protoplasts
Figure 6. Enzymatic hydrolysis solution for protoplasts.

Extracting protoplasts from Arabidopsis thaliana is the first step. Protoplasts are isolated from healthy, pest-free Arabidopsis leaves by cutting young leaves into small pieces under sterile conditions and suspending them in a digestive solution containing cellulase and pectinase. This enzyme hydrolytically degrades the cell wall, releasing protoplasts, which are then cultured with gentle agitation for thorough digestion. After this, the larger tissue fragments are filtered out and the protoplasts are purified by centrifugation and washing to remove remaining enzymes and contaminants. The isolated protoplasts are then re-suspended in a medium suitable for molecular biology applications, such as gene transformation research. Successful enzymatic hydrolysis is essential for efficient extraction and subsequent manipulation of protoplasts, as shown in the diagram of enzymatic hydrolysis of Arabidopsis leaves.

Figure 7. Observation under fluorescence microscope
Figure 7. Observation under fluorescence microscope.

After isolating protoplasts from Arabidopsis, we transformed three RNAi targets specifically for the PFN gene: PA7-PFN3+RNAi-A, PA7-PFN3+RNAi-B, and PA7-PFN3+RNAi-C. Microscopic observation showed that GFP fluorescence was weak after transformation, which may indicate that the construct failed to express successfully or was suppressed by RNAi. The empty vector group did not show fluorescence, suggesting that GFP was not successfully converted into protein. Bright-field microscopy revealed that most protoplasts were round, indicating their healthy state. Although microscopic analysis provided qualitative evidence regarding the transformation and expression of the GFP-tagged construct, quantitative assessment of RNAi efficiency is still needed.

Figure 8. The Profilin 3 mRNA transcription level in treated and untreated protoplasts
Figure 8. The Profilin 3 mRNA transcription level in treated and untreated protoplasts.

The siRNA sequence Profilin 3-A successfully reduced the mRNA levels of profilin expression in injected tobacco leaf cells to approximately 0.5 times that of unregulated cells, demonstrating the evident improvements created by our therapy. However, the other two siRNA sequences were not similarly successful; in contrast, the mRNA levels remained largely unchanged compared to the baseline.

References

[1] Kwon, Y. J., Kim, S. H., Lee, S. G., Lee, S.Y., & Kim, T. H. (2001). Construction of a novel expression vector system for enhanced production of recombinant proteins in Escherichia coli. Journal of Industrial Microbiology & Biotechnology, 27(5), 291-296. https://doi.org/10.1038/sj.jimb.7000919

[2] Buchholz, F., & Prehn, S. (2002). The Gateway System: Applications for protein expression and tagging. Current Opinion in Biotechnology, 13(6), 553-558. https://doi.org/10.1016/S0958-1669(02)00362-9

[3] He, X., & Wang, X. (2005). Expression vectors and systems for recombinant protein expression. In Methods in Molecular Biology, Vol. 297, Protein Expression Systems. Humana Press.