Difference between revisions of "Part:BBa K5375005"
 
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         <img src="https://static.igem.wiki/teams/5375/bba-k5375005/2.png" alt="Figure 2. PCR amplification of the fragment use for plasmid construction">
 
         <img src="https://static.igem.wiki/teams/5375/bba-k5375005/2.png" alt="Figure 2. PCR amplification of the fragment use for plasmid construction">
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         <div class="caption">Figure 2. PCR amplification of the fragment use for plasmid construction (2123 bp).</div>
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         <div class="caption">Figure 2. PCR amplification of the fragment use for plasmid construction. The pA7-GFP-PFN3 sequence was amplified by PCR, with a length of 2123 bp.</div>
 
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     <h3>Characterization/Measurement</h3>
 
     <h3>Characterization/Measurement</h3>
 
     <p>
 
     <p>
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         We constructed pA7-GFP-HSP70 using homologous recombination. The pA7-GFP-PFN3 sequence was amplified by PCR, with a length of 2123 bp. The target gene sequence including HSP70 was inserted and reconstructed via homologous recombination. After overnight incubation, significant bacterial growth was observed on LB agar plates (Figure 3).
+
         We constructed pA7-GFP-HSP70 using homologous recombination. The pA7-GFP-PFN3 sequence was amplified by PCR, with a length of 2123 bp. The target gene sequence including HSP70 was inserted and reconstructed through homologous recombination. To incubate and culture the reassembled plasmid overnight, it is diluted and spread out onto an LB agar plate. The growth of pA7-GFP-HSP70 was significant.
 
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         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). The sequencing results of the reconstructed plasmid PA7-GFP-HSP70 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).
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         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.
 
     </p>
 
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         <div class="caption">Figure 4. Colony PCR verification of PA7-HSP70.</div>
 
         <div class="caption">Figure 4. Colony PCR verification of PA7-HSP70.</div>
 
     </div>
 
     </div>
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    <p>
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        The sequencing results of the reconstructed plasmid PA7-GFP-HSP70 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.
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    </p>
  
 
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         By transforming the reconstructed plasmid into the protoplasm of <i>Arabidopsis thaliana</i>, and observing the changes of GFP fluorescence intensity and HSP70 gene expression in the protoplasm after the addition of siRNA that inhibited the expression of this protein, the effectiveness of siRNA was verified.
 
         By transforming the reconstructed plasmid into the protoplasm of <i>Arabidopsis thaliana</i>, and observing the changes of GFP fluorescence intensity and HSP70 gene expression in the protoplasm after the addition of siRNA that inhibited the expression of this protein, the effectiveness of siRNA was verified.
 
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         <div class="caption">Figure 6. Enzymatic hydrolysis solution for protoplasts.</div>
 
         <div class="caption">Figure 6. Enzymatic hydrolysis solution for protoplasts.</div>
 
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<p>
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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.</div>
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  </p>
  
 
     <div style="text-align:center;">
 
     <div style="text-align:center;">
 
         <img src="https://static.igem.wiki/teams/5375/bba-k5375005/7.png" alt="Figure 7. Observation under fluorescence microscope">
 
         <img src="https://static.igem.wiki/teams/5375/bba-k5375005/7.png" alt="Figure 7. Observation under fluorescence microscope">
 
         <div class="caption">Figure 7. Observation under fluorescence microscope.</div>
 
         <div class="caption">Figure 7. Observation under fluorescence microscope.</div>
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    After isolating protoplasts from Arabidopsis, we transformed three RNAi targets specifically designed for the HSP70 gene: PA7-HSP70+RNAi-1, PA7-HSP70+RNAi-2, and PA7-HSP70+RNAi-3. Microscopic observation revealed weak GFP fluorescence, possibly indicating unsuccessful expression of the GFP-fused proteins or suppression by RNAi. The empty vector group did not show fluorescence, suggesting that GFP was not successfully converted into protein. This analysis provides qualitative evidence for the transformation and expression of the HSP70-related constructs, but quantitative assessment of RNAi efficiency is still needed.
 +
    </p>
  
 
     <p>
 
     <p>
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         After isolating protoplasts from Arabidopsis, we transformed three RNAi targets specifically designed for the HSP70 gene: PA7-HSP70+RNAi-1, PA7+RNAi-2, and PA7-HSP70+RNAi-3. Microscopic observation revealed weak GFP fluorescence, possibly indicating unsuccessful expression of the GFP-fused proteins or suppression by RNAi. The empty vector group did not show fluorescence, suggesting that GFP was not successfully converted into protein. This analysis provides qualitative evidence for the transformation and expression of the HSP70-related constructs, but quantitative assessment of RNAi efficiency is still needed.
+
         The siRNA sequences HSP70-2 and HSP70-3 both successfully reduced the mRNA levels of profilin expression in injected tobacco leaf cells; however, the impact of HSP70-2 was more pronounced, as the mRNA expression rate declined to approximately one-fifth of the normal level, demonstrating greater efficiency compared to siRNA HSP70-3, which exhibited a reduction rate of around 70%. Although there is an outlier, specifically HSP70-1, the deviations were minimal; nonetheless, the overall trend indicates a positive result, supporting the hypothesis that siRNA can effectively silence HSP70 expression in protoplasts.
 
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         <div class="caption">Figure 8. The expression levels of HSP70.</div>
 
         <div class="caption">Figure 8. The expression levels of HSP70.</div>
 
     </div>
 
     </div>
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−
    <p>
 
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        The siRNA sequences HSP70-2 and HSP70-3 both successfully reduced the mRNA levels of profilin expression in injected tobacco leaf cells; however, the impact of HSP70-2 was more pronounced, as the mRNA expression rate declined to approximately one-fifth of the normal level, demonstrating greater efficiency compared to siRNA HSP70-3, which exhibited a reduction rate of around 70%. Although there is an outlier, specifically HSP70-1, the deviations were minimal; nonetheless, the overall trend indicates a positive result, supporting the hypothesis that siRNA can effectively silence HSP70 expression in protoplasts.
 
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    </p>
 
  
 
     <h3>References</h3>
 
     <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 <i>Escherichia coli</i>. 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>[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 <i>Escherichia coli</i>. 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>[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>
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     <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.
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     <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>
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Latest revision as of 06:37, 1 October 2024

pA7-GFP-HSP70



Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 5012
    Illegal BglII site found at 5936
    Illegal BamHI site found at 4127
    Illegal XhoI site found at 3302
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 5097
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 4091
    Illegal BsaI.rc site found at 4987
    Illegal SapI.rc site found at 6306



BBa_K5375005: pA7-GFP-HSP70

BBa_K5375005: pA7-GFP-HSP70

Profile

Name: pA7-GFP-HSP70

Origin: Synthesized by company and constructed by the team

Properties: Fusion expression of protein HSP70-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-HSP70
Figure 1. Plasmid map of pA7-GFP-HSP70.

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 HSP70.

Figure 2. PCR amplification of the fragment use for plasmid construction
Figure 2. PCR amplification of the fragment use for plasmid construction. The pA7-GFP-PFN3 sequence was amplified by PCR, with a length of 2123 bp.

Characterization/Measurement

We constructed pA7-GFP-HSP70 using homologous recombination. The pA7-GFP-PFN3 sequence was amplified by PCR, with a length of 2123 bp. The target gene sequence including HSP70 was inserted and reconstructed through homologous recombination. To incubate and culture the reassembled plasmid overnight, it is diluted and spread out onto an LB agar plate. The growth of pA7-GFP-HSP70 was significant.

Figure 3. Growth of plasmid pA7-GFP-HSP70 transformed bacterial on LB agar plates
Figure 3. Growth of plasmid pA7-GFP-HSP70 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-HSP70
Figure 4. Colony PCR verification of PA7-HSP70.

The sequencing results of the reconstructed plasmid PA7-GFP-HSP70 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-HSP70
Figure 5. Sanger sequencing map of PA7-GFP-HSP70.

By transforming the reconstructed plasmid into the protoplasm of Arabidopsis thaliana, and observing the changes of GFP fluorescence intensity and HSP70 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 designed for the HSP70 gene: PA7-HSP70+RNAi-1, PA7-HSP70+RNAi-2, and PA7-HSP70+RNAi-3. Microscopic observation revealed weak GFP fluorescence, possibly indicating unsuccessful expression of the GFP-fused proteins or suppression by RNAi. The empty vector group did not show fluorescence, suggesting that GFP was not successfully converted into protein. This analysis provides qualitative evidence for the transformation and expression of the HSP70-related constructs, but quantitative assessment of RNAi efficiency is still needed.

The siRNA sequences HSP70-2 and HSP70-3 both successfully reduced the mRNA levels of profilin expression in injected tobacco leaf cells; however, the impact of HSP70-2 was more pronounced, as the mRNA expression rate declined to approximately one-fifth of the normal level, demonstrating greater efficiency compared to siRNA HSP70-3, which exhibited a reduction rate of around 70%. Although there is an outlier, specifically HSP70-1, the deviations were minimal; nonetheless, the overall trend indicates a positive result, supporting the hypothesis that siRNA can effectively silence HSP70 expression in protoplasts.

Figure 8. The expression levels of HSP70
Figure 8. The expression levels of HSP70.

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.