Part:BBa_K4909007

pET28a-Fat

Contribution by iGEM Team SubCat-GD 2023

Composite Part: BBa_K4909007

Profile

Name: pET28a-Fat1

Base Pairs: 5644 bp

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 recognize a range of 16-20 carbon substrates for ω-6 PUFAs, which in turn is able to catalyze 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.

Figure 1. 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 are expressed successfully.

Figure 4. Western Blotting for Fat1 protein detection

The other part that makes up BBa_K4909007 are as follows:

Basic Part: BBa_K4909000

Name: Fat-1

Base Pairs: 1223 bp

Origin: Synthetic

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.

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 Caenorhabditis 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, 2001, 98: 4050-4054.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NotI site found at 5637
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 4402
  Illegal BamHI site found at 4422
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 2622
  Illegal NgoMIV site found at 2782
  Illegal NgoMIV site found at 4370
  Illegal NgoMIV site found at 5583
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 4606
  Illegal SapI.rc site found at 4646