Part:BBa_K3717014
Endo-β-Galactosidase with T7 promoter and strong RBS
Figure 1: Endo-β-galactosidase from 2014 Tuebingen with T7 promoter, RBS, and a terminator.
Contruct Design
Endo-β-galactosidase an enzyme that catalyzes the cleavage of the A and B type blood antigens (trisaccharides) 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 thus does not elicit an immune response in the human body. Thus, the enzyme can convert both A and B blood types to universal O type. The enzyme has been previously shown to successfully convert A,B, and AB type erythrocytes to O type but lacked a cost-effective method of production.
We obtained the amino acid sequence of the endo-β-galactosidase protein, derived from Clostridium perfringens, from the iGEM DNA Repository Plate (BBa_K1483001), which served as our Open Reading Frame (ORF). We attached a T7 promoter, derived from the T7 phage, and strong ribosome binding site (RBS; BBa_K525998) upstream of the open reading frame (ORF). The composite gene was synthesized through DNA cloning.
Results
We obtained the amino acid sequence of the Endo-β-Galactosidase protein from the iGEM DNA Repository Plate (BBa_K1483001), as entered into the iGEM parts collection database by the Tuebingen iGEM team in 2014
In order to test protein expression of the enzyme, we added a T7 promoter and a strong ribosome binding site (RBS; BBa_K525998) upstream of the protein amino acid sequence to create a part BBa_K3717014.
However, the post-ligation PCR gel run showed no DNA, indicating that we were unsuccessful in adding the promoter and RBS to the DNA. Hence, we could not test protein expression of DNA part BBa_K1483001.
References
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.
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.
UniProtKB - Q6RUF5 (EABC_CLOPF). UniProt, 2 June 2021, www.uniprot.org/uniprot/Q6RUF5. Accessed 19 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]
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