Difference between revisions of "Part:BBa K3717012"

Revision as of 09:42, 20 October 2021

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).

Figure 1. α-Galactosidase with T7 promoter, RBS and double terminator construct

Construct Design

We derived the sequence of α-Galactosidase from Bacteroides fragilis [1] 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.

Sequence and Features