Difference between revisions of "Part:BBa K5375004"
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<partinfo>BBa_K5375004 SequenceAndFeatures</partinfo> | <partinfo>BBa_K5375004 SequenceAndFeatures</partinfo> | ||
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+ | <meta charset="UTF-8"> | ||
+ | <meta name="viewport" content="width=device-width, initial-scale=1.0"> | ||
+ | <title>BBa_K5375004: pA7-GFP-Profilin 3</title> | ||
+ | <style> | ||
+ | img { | ||
+ | max-width: 80%; | ||
+ | height: auto; | ||
+ | } | ||
+ | .caption { | ||
+ | text-align: center; | ||
+ | font-size: 0.9em; | ||
+ | margin-top: 5px; | ||
+ | margin-bottom: 20px; | ||
+ | } | ||
+ | </style> | ||
+ | </head> | ||
+ | <body> | ||
+ | <h2>BBa_K5375004: pA7-GFP-Profilin 3</h2> | ||
− | + | <h3>Profile</h3> | |
+ | <p><strong>Name:</strong> pA7-GFP-Profilin 3</p> | ||
+ | <p><strong>Origin:</strong> Synthesized by company and constructed by the team</p> | ||
+ | <p><strong>Properties:</strong> Fusion expression of protein Profilin3-GFP</p> | ||
− | + | <h3>Usage and Biology</h3> | |
− | + | <p> | |
+ | 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. | ||
+ | </p> | ||
− | + | <h3>Cultivation and Purification</h3> | |
+ | <div style="text-align:center;"> | ||
+ | <img src="https://static.igem.wiki/teams/5375/bba-k5375004/1.png" alt="Figure 1. Plasmid map of pA7-GFP-PFN3"> | ||
+ | <div class="caption">Figure 1. Plasmid map of pA7-GFP-PFN3.</div> | ||
+ | </div> | ||
+ | <p> | ||
+ | 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. | ||
+ | </p> | ||
− | = | + | <div style="text-align:center;"> |
− | + | <img src="https://static.igem.wiki/teams/5375/bba-k5375004/2.png" alt="Figure 2. PCR amplification of fragment for plasmid construction"> | |
− | + | <div class="caption">Figure 2. PCR amplification of fragment for plasmid construction (396 bp).</div> | |
+ | </div> | ||
− | + | <h3>Characterization/Measurement</h3> | |
+ | <p> | ||
+ | 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). | ||
+ | </p> | ||
− | + | <div style="text-align:center;"> | |
− | + | <img src="https://static.igem.wiki/teams/5375/bba-k5375004/3.png" alt="Figure 3. Growth of plasmid pA7-GFP-PFN3 transformed bacterial on LB agar plates"> | |
+ | <div class="caption">Figure 3. Growth of plasmid pA7-GFP-PFN3 transformed bacterial on LB agar plates.</div> | ||
+ | </div> | ||
− | + | <p> | |
+ | 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). | ||
+ | </p> | ||
− | + | <div style="text-align:center;"> | |
− | + | <img src="https://static.igem.wiki/teams/5375/bba-k5375004/4.png" alt="Figure 4. Colony PCR verification of PA7-PFN3"> | |
+ | <div class="caption">Figure 4. Colony PCR verification of PA7-PFN3.</div> | ||
+ | </div> | ||
− | + | <div style="text-align:center;"> | |
+ | <img src="https://static.igem.wiki/teams/5375/bba-k5375004/5.png" alt="Figure 5. Sanger sequencing map of PA7-GFP-PFN3"> | ||
+ | <div class="caption">Figure 5. Sanger sequencing map of PA7-GFP-PFN3.</div> | ||
+ | </div> | ||
− | + | <p> | |
− | + | 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). | |
+ | </p> | ||
− | + | <div style="text-align:center;"> | |
+ | <img src="https://static.igem.wiki/teams/5375/bba-k5375004/6.png" alt="Figure 6. Enzymatic hydrolysis solution for protoplasts"> | ||
+ | <div class="caption">Figure 6. Enzymatic hydrolysis solution for protoplasts.</div> | ||
+ | </div> | ||
− | + | <div style="text-align:center;"> | |
− | + | <img src="https://static.igem.wiki/teams/5375/bba-k5375004/7.png" alt="Figure 7. Observation under fluorescence microscope"> | |
+ | <div class="caption">Figure 7. Observation under fluorescence microscope.</div> | ||
+ | </div> | ||
− | + | <div style="text-align:center;"> | |
+ | <img src="https://static.igem.wiki/teams/5375/bba-k5375004/8.png" alt="Figure 8. Profilin 3 mRNA transcription levels in treated and untreated protoplasts"> | ||
+ | <div class="caption">Figure 8. Profilin 3 mRNA transcription levels in treated and untreated protoplasts.</div> | ||
+ | </div> | ||
− | == | + | <h3>References</h3> |
+ | <p>[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. <a href="https://doi.org/10.1038/sj.jimb.7000919">https://doi.org/10.1038/sj.jimb.7000919</a></p> | ||
+ | <p>[2] Buchholz, F., & Prehn, S. (2002). The Gateway System: Applications for protein expression and tagging. Current Opinion in Biotechnology, 13(6), 553-558. <a href="https://doi.org/10.1016/S0958-1669(02)00362-9">https://doi.org/10.1016/S0958-1669(02)00362-9</a></p> | ||
+ | <p>[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.</p> | ||
− | + | </body> | |
− | + | </html> | |
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Revision as of 06:12, 1 October 2024
pA7-GFP-Profilin 3
Sequence and Features
- 10INCOMPATIBLE 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 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 4417
Illegal SpeI site found at 3367
Illegal PstI site found at 2413 - 21INCOMPATIBLE 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 - 23INCOMPATIBLE 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 - 25INCOMPATIBLE 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 - 1000COMPATIBLE WITH RFC[1000]
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
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.
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).
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).
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).
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.