Part:BBa_K3521004

pet28a-backbone

This part is used to effectively express the target protein. It contains T7 promoter and terminator, N-terminal and C-terminal His tags, kanamycin resistance gene, and the replication origin site of E. coli

Contribution by Team 2023 TSBC-SZ

Summary

In this project, we used pET28a-Backbone (BBa_K3521004) as a vector to construct recombinant plasmid pET28a-Gox1 (BBa_K4841018) in order to express proteins in E. coli host.

Documentation

a. Usage and Biology

pET28a-Backbone is a commonly used fusion protein type prokaryotic expression vector that contains anti-kanamycin genes and is induced by T7 RNA polymerase provided by host cells. E. coli DH5α and BL21(DE3) are commonly used as the receptor strain. It contains His tags, mainly used for protein purification[1].

Figure 1: Double enzyme gel cleavage diagram of pET-28a

Linear plasmid fragments were obtained through dual enzyme digestion of NheI and XhoI, and then connected to gene fragments through DNA ligases to form recombinant plasmid pET28a-Gox1 (BBa_K4841018).

References

1. Renz, B., Davies, J. K., Carling, D., Watkins, H., & Redwood, C. (2009). Determination of AMP-activated protein kinase phosphorylation sites in recombinant protein expressed using the pET28a vector: a cautionary tale. Protein expression and purification, 66(2), 181–184.

Contribution by iGEM Team SubCat-GD 2023

Added new information to the pET-28a (BBa_K3521004)

The new contribution composite Part: BBa_K4909007

Profile

• Name: pET28a-Fat1
• Base Pairs: 5644bp
• Properties: The Fat-1 gene is able to encode an ω-3 PUFAs desaturase, which dehydrogenates substrate ω-6 PUFAs to produce the corresponding ω-3 PUFAs, leading to alter the intracellular ratio of ω-6/ω-3 PUFAs.

Usage and Biology

The Fat-1 gene is able to encode an ω-3 PUFAs desaturase, which dehydrogenates substrate ω-6 PUFAs to produce the corresponding ω-3 PUFAs, leading to alter the intracellular ratio of ω-6/ω-3 PUFAs[1].

In 1997 Spychalla et al. demonstrated the function of the fat-1 (fatty acid metabolism 1) gene derived from the small showy nematode (C. elegans) in the model organism Arabidopsis thaliana [2]. It is capable of converting ω-6 PUFAs to ω-3 PUFAs. The unsaturated fatty acid deoxygenase encoded by the fat-1 gene is able to recognise a range of 16-20 carbon substrates for ω-6 PUFAs, which in turn is able to catalyse the conversion of the different ω-6 PUFAs to the corresponding ω-3 PUFAs [3].

Construction Design

Construction of the pET-28a-Fat1(BBa_K4909007) plasmid: Primer-assisted codon-optimized fat-1 CDS(BBa_K4909000) amplified by PCR. Then fat-1 CDS and pET-28a(BBa_K3521004) were then digested with 5'BamHI/3'NotI, and After that, T4 DNA ligase is used to ligate and convert the target gene and vector. The following day, recombinant plasmids were extracted, enzyme-digested to identify them, and positive clones were selected, amplified, and removed. The sequencing business received the positive recombinant plasmids for additional sequencing identification.

Figure1. The genetic map of pET28a-Fat1

Experimental Approach

Construction of the pET-28a-Fat1 plasmid: Primer-assisted codon-optimized fat-1 CDS amplified by PCR. Then fat-1 CDS and pET-28a were then digested with 5'BamHI/3'NotI, and After that, T4 DNA ligase is used to ligate and convert the target gene and vector. The following day, recombinant plasmids were extracted, enzyme-digested to identify them, and positive clones were selected, amplified, and removed. The sequencing business received the positive recombinant plasmids for additional sequencing identification.

Figure 2. The plasmid pET28a-Fat1 sequencing results

Figure 3. TAE agarose gel electrophoresis to verify the construction of pET-28a-Fat1

To test Fat1 proteins, we ran a Western blot using Supernatant of cell lysate and Pellet of cell lysate. The Fig4 showed that Fat1 protein is found on Pellet of cell lysate, marker 50KDa, indicating that Fat1 proteins is expressed successfully.

Figure 4. Western Blotting for Fat1 protein detection

References:

1. Guo T. The basic study on the fat-1 transgenic cattle [D]. Inner Mongolia Agricultural University. 2013.
2. Spychalla JP, Kinney AJ, Browse J. Identification of animal omega-3 fatty acid desaturase by heterologous expression in Arabidopsis[J]. Proc Natl Acad Sci USA, 1997, 94: 1142-1147.
3. Rang ZB, Ge YL, Chen ZH, et al. Adenoviral gene transfer of cacnorhabditis elegans n-3 fatty acid desaturase optimizes fatty acid composition in mammalian cells[J]. Proceeding of the national academy of science of the United States of America, 200

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  
• 12
  INCOMPATIBLE WITH RFC[12]

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• 21
  INCOMPATIBLE WITH RFC[21]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal BglII site found at 4402
  
• 23
  INCOMPATIBLE WITH RFC[23]

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• 25
  INCOMPATIBLE WITH RFC[25]

  Plasmid lacks a prefix.
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  Illegal NgoMIV site found at 2622
  Illegal NgoMIV site found at 2782
  Illegal NgoMIV site found at 4370
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.