Part:BBa_K3717013

α-N-Acetylgalactosaminidase with T7 + RBS, C-Terminal 6x His-Tag, and Double Terminator

The composite part utilizes a T7 promoter + RBS (BBa_K525998), α-N-Acetylgalactosaminidase with C-Terminal 6x Histidine tag

(BBa_K3717016), and a double terminator (BBa_B0015).

Construct Design

α-N-acetylgalactosaminidase is an enzyme that catalyzes the cleavage of the N-acetylgalactosamine off of A type blood antigens such that the remaining sugar can be classified as an H antigen, which the anti-A and anti-B antibodies are unable to recognize and thus does not elicit an immune response in the human body. Thus, the enzyme can convert A blood types to universal O type. While the enzyme has been previously shown to successfully convert A type erythrocytes to O type, it was inefficient in doing so, and lacked a cost-effective method of production.

We obtained the amino acid sequence of the α-N-acetylgalactosaminidase protein, derived from Elizabethkingia meningoseptica, from the iGEM DNA Repository Plate, which served as our Open Reading Frame (ORF). We attached a T7 promoter, derived from the T7 phage, strong ribosome binding site (RBS;BBa_K525998) upstream of the open reading frame (ORF), and a 6x Histidine tag through a flexible Glycine-Serine linker in the open reading frame but downstream of the α-N-acetylgalactosaminidase sequence for protein purification purposes. The composite gene was synthesized through DNA sequencing.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 567
  
• 1000
  COMPATIBLE WITH RFC[1000]