Difference between revisions of "Part:BBa K3717015"
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| − | <b> Figure 1: α-Galactosidase with His-Tag and GS linker </b> | + | <b> Figure 1: α-Galactosidase with 6x C-Terminal His-Tag and GS linker </b> |
<b><font size="+1.2"> Construct Design </font></b> | <b><font size="+1.2"> Construct Design </font></b> | ||
| − | We optimized the | + | We derived the sequence of α-Galactosidase from <i>Bacteroides fragilis</i> and optimized the sequence for <i>E. coli</i> 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. | However, cells transformed with the plasmids had problems growing on culture plates and therefore, we were unable to commence protein purification. | ||
Revision as of 14:49, 18 October 2021
α-Galactosidase with C-Terminal 6x Histidine tag
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 6x C-Terminal His-Tag and GS linker
Construct Design
We derived the sequence of α-Galactosidase from Bacteroides fragilis 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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]