Part:BBa_K5375005

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
  

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. This strategic design streamlines protein purification processes and facilitates functional analysis of proteins.

Cultivation, Purification and SDS-PAGE

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. The plant expression vector includes a 35S promoter and ampicillin resistance, and it is usually cultivated in a DH5α *E. coli* strain at 37°C. It was chosen to measure the protein expression of HSP70.

Figure 2. PCR amplification of the fragment used for plasmid construction.

We constructed pA7-GFP-HSP70 using homologous recombination. The pA7-GFP-HSP70 sequence was amplified by PCR with a length of 2123 bp.

Figure 3. Growth of plasmid pA7-GFP-HSP70 transformed bacteria on LB agar plates.

Then, the target gene sequence including HSP70 was inserted. It was reconstructed through homologous recombination. To incubate and culture the reassembled plasmid overnight, it was diluted and spread out onto an LB agar plate. The growth of pA7-GFP-HSP70 was significant.

Measurement and Characterization

Figure 4. Colony PCR verification of PA7-HSP70.

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 occurs, other samples could cover it.

Reference

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. [1](https://doi.org/10.1038/sj.jimb.7000919)

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

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.