Part:BBa_K3717011
Endo-β-Galactosidase with T7 + RBS, N-Terminal 6x His-Tag, and Double Terminator
The composite part utilizes a T7 promoter + RBS (BBa_K525998), Endo-β-galactosidase (BBa_K3717008), and double terminator (BBa_B0015).
Endo-β-galactosidase catalyzes the cleavage of A and B type blood antigen trisaccharides such that the remaining sugar can be classified as a precursor to the 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, the enzyme converts both A and B blood types to universal O type.
Figure 1. Endo-β-Galactosidase with T7 Promoter, strong RBS, N-Terminal 6x Histidine tag and Double Terminator Construct
Construct Design
We obtained the amino acid sequence of the Endo-β-gal protein, derived from Streptococcus pneumoniae, that Kwan et. al performed iterative steps of directed evolution on to increase the activity of the enzyme by around 170-fold [2]. This sequence served as our Open Reading Frame (ORF; BBa_K3717008). We attached a T7 promoter, derived from the T7 phage, strong ribosome binding site (RBS; BBa_K525998) upstream of the open reading frame (ORF), a 6x Histidine tag through a flexible Glycine-Serine linker in the open reading frame, upstream of the Endo-β-gal sequence for protein purification purposes, and a double terminator (BBa_B0015) downstream of the ORF. The composite gene BBa_K3717011 was synthesized through IDT.
Characterization
Protein Expression and Purification
We tested protein expression of the composite parts by transforming our plasmids into BL21(DE3) E. coli cells. We grew cultures at 37°C overnight, diluted those cultures, and then grew to OD600 0.5~0.6 at 37°C. We then induced expression with 0.5 mM IPTG and allowed cultures to grow overnight at room temperature.
We harvested the cells after the overnight induction and lysed them either through sonication or with xTractor Lysis Buffer spiked with 500mM Imidazole stock (to a final concentration of 20mM in the lysate solution) [3]. We purified the Histidine tagged proteins using Ni sepharose affinity chromatography. We then utilized SDS-PAGE to confirm the sizes of purified proteins.
Our results indicate no protein bands, showing that our protein purification of BBa_K3717011 was unsuccessful.
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. Kwan, David H., et al. “Toward Efficient Enzymes for the Generation of Universal Blood through Structure-Guided Directed Evolution.” Journal of the American Chemical Society, vol. 137, no. 17, American Chemical Society, May 2015, pp. 5695–705. ACS Publications, https://doi.org/10.1021/ja5116088.
3. XTractorTM Buffer & xTractor Buffer Kit User Manual. (n.d.). 10.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 2073
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 698
Illegal SapI.rc site found at 2886
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