Part:BBa_K3717012
α-Galactosidase with T7 + RBS, C-Terminal 6x His-Tag, and Double Terminator
The composite part utilizes a T7 promoter + RBS (BBa_K525998), α-Galactosidase (BBa_K3717015), and double terminator (BBa_B0015).
α-Galactosidase catalyzes the cleavage of the galactose off of B type blood antigens such that the remaining sugar can be classified as a H antigen, which the anti-A and anti-B antibodies are unable to recognize and hence does not elicit an immune response in the human body [1]. Thus, α-Galactosidase converts B blood types to universal O type.
Figure 1. α-Galactosidase with T7 Promoter, strong RBS, C-Terminal 6x Histidine tag and Double Terminator Construct
Construct Design
We derived the sequence of α-Galactosidase from Bacteroides fragilis [2] and optimized the sequence for E. coli protein expression. We then attached a 6x histidine tag (6x His-Tag) downstream of the α-Galactosidase sequence preceded by a glycine-serine linker (GS linker) to form our open reading frame (ORF) (BBa_K3717015) for purification purposes. We flanked our open reading frame with a T7 promoter + RBS (BBa_K525998) upstream of the open reading frame and a double terminator (BBa_B0015) downstream of the sequence. This composite part (BBa_K3717012) was assembled through DNA synthesis by IDT.
However, cells transformed with the plasmids had problems growing on culture plates and therefore, we were unable to commence protein purification.
References
1. Rahfeld, Peter, and Stephen G. Withers. “Toward Universal Donor Blood: Enzymatic Conversion of A and B to O Type.” Journal of Biological Chemistry, vol. 295, no. 2, Jan. 2020, pp. 325–34. DOI.org (Crossref), https://doi.org/10.1074/jbc.REV119.008164.
2. UniProtKB - A4Q8F7 (GH109_ELIME). UniProt, 2 June 2021, www.uniprot.org/uniprot/A4Q8F7. Accessed 20 Oct. 2021.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
| None |