Part:BBa_K3717004
α-N-Acetylgalactosaminidase with strong promoter and RBS
Figure 1: α-N-Acetylgalactosaminidase with T7 promoter + RBS
Construct Design
α-N-acetylgalactosaminidase is an enzyme that catalyzes the cleavage of the N-acetylgalactosamine off of A type blood antigens such that the remaining sugar can be classified as an 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 [1]. Thus, the enzyme can convert A blood types to universal O type.
We obtained the amino acid sequence of the α-N-acetylgalactosaminidase protein, derived from Elizabethkingia meningoseptica, from the iGEM DNA Repository Plate (BBa_K1483000), which served as our Open Reading Frame (ORF). We attached a strong promoter and strong ribosome binding site (RBS; BBa_K880005) upstream of the open reading frame (ORF). The composite gene was synthesized through DNA cloning.
Results
We obtained the amino acid sequence of the α-N-Acetylgalactosaminidase protein from the iGEM DNA Repository Plate (BBa_K1483000), 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 strong promoter and strong ribosome binding site (RBS; BBa_K880005) upstream of the protein amino acid sequence to create a part BBa_K3717004.
Figure 2 - Colony PCR check for strong promoter (K88) α-N-Acetylgalactosaminidase (NAGA) (Part: BBa_K3717004) using VF2 and VR primers. Uncut plasmid (K88 only control) has a band at the expected part size of 355 bp, indicated by white triangle. Confirms successful ligation as a band is produced at the expected size of 1684 bp, as indicated by the red triangle.
We tested protein expression of this composite part by transforming our plasmids into BL21 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 took samples pre-induction and post-induction and examined them by SDS-PAGE.
Figure 3 - SDS-PAGE of cell lysate for the strain: strong promoter (K88) α-N-Acetylgalactosaminidase (NAGA). Blue triangles indicate expected size for NAGA (50.1 kDa). Sequences for target proteins do not contain a start codon, thus have no expression, as shown by the triangles.
Our SDS-page did not show any overexpression bands for the enzymes of interest. The results indicate that there were no target proteins at their expected band sizes: 50.1 kDa band for K88 promoter + NAGA in the induced sample. As the SDS page is of cell lysis samples, other bands present are due to innate proteins present in the bacteria cell.
Upon comparison of the amino acid sequence from Tuebingen’s part (BBa_K1483000) with full sequences that were offered by other studies online, we discovered that the enzyme sequences were missing the start codon (Fig 4), which explained the non-expression of the proteins.
Figure 4 - Top sequence: First 37 amino acids of Team Tuebingen's 2014 α-N-Acetylgalactosaminidase part BBa_K1483000. Bottom sequence: First 38 amino acids of TAS_Taipei's α-N-Acetylgalactosaminidase part BBa_K3717016. Based on the alignment of the two sequences, Tuebingen's part is missing the first amino acid of the α-N-Acetylgalactosaminidase protein.
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]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 468
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