Difference between revisions of "Part:BBa K5375004"
Line 19: Line 19:
 
     <style>
 
     <style>
 
         img {
 
         img {
−
             max-width: 80%;
+
             max-width: 65%;
 
             height: auto;
 
             height: auto;
 
         }
 
         }

Revision as of 06:15, 1 October 2024


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, 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 as a selection marker. The vector may also possess a cleavable tag sequence for removal of GFP by specific proteases (e.g., TEV protease), thus yielding purified target proteins. This design streamlines protein purification and 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 fragment for plasmid construction
Figure 2. PCR amplification of fragment for plasmid construction (396 bp).

Characterization/Measurement

The pA7-GFP-PFN3 sequence was amplified by PCR with a length of 396 bp. The target gene sequence including Profilin 3 was inserted and reconstructed via homologous recombination. After overnight incubation, significant bacterial growth was observed on LB agar plates (Figure 3).

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 plate were taken and amplified via PCR to verify plasmid integration. Multiple samples were analyzed to ensure coverage of any errors (Figure 4). Sequencing confirmed the correct integration of the target gene (Figure 5).

Figure 4. Colony PCR verification of PA7-PFN3
Figure 4. Colony PCR verification of PA7-PFN3.
Figure 5. Sanger sequencing map of PA7-GFP-PFN3
Figure 5. Sanger sequencing map of PA7-GFP-PFN3.

We transformed the reconstructed plasmid into the protoplasm of Arabidopsis thaliana, observing the GFP fluorescence intensity and PFN3 gene expression after the addition of siRNA that inhibited the expression of this protein. siRNA Profilin 3-A successfully reduced PFN3 mRNA levels (Figure 8).

Figure 6. Enzymatic hydrolysis solution for protoplasts
Figure 6. Enzymatic hydrolysis solution for protoplasts.
Figure 7. Observation under fluorescence microscope
Figure 7. Observation under fluorescence microscope.
Figure 8. Profilin 3 mRNA transcription levels in treated and untreated protoplasts
Figure 8. Profilin 3 mRNA transcription levels in treated and untreated protoplasts.

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.