Part:BBa_K3717002
α-N-Acetylgalactosaminidase with T7 promoter and strong RBS
Figure 1: α-N-Acetylgalactosaminidase with T7 promoter, RBS, and a terminator
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. Thus, the enzyme can convert A blood types to universal O type. While the enzyme has been previously shown to successfully convert A type erythrocytes to O type, it was inefficient in doing so, and lacked a cost-effective method of production.
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 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 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 T7 promoter and strong ribosome binding site (RBS; BBa_K525998) upstream of the protein amino acid sequence to create a part BBa_K3717002.
Figure 2 - Colony PCR check for T7 promoter α-N-Acetylgalactosaminidase (NAGA) (Part: BBa_K3717002) using VF2 and VR primers. Uncut plasmid (T7 only control) has a band at the expected part size of 332bp, indicated by white triangle. Confirms successful ligation as a band is produced at the expected size of 1661bp, indicated by the red triangle.
We tested protein expression of these two composite parts by transforming our plasmids into BL21 E. Coli cells. We grew an overnight culture of the BL21 cells with our plasmids then diluted our cells to a standardized OD600 of ~0.1 and let it grow until an OD600 of 0.5~0.6. The diluted cultures of OD600 0.5~0.6 were then induced for expression with 0.5 M IPTG stock (to a final concentration of 0.5mM in the culture) and allowed to grow and induce overnight at room temperature.
Figure 3 - SDS-PAGE of cell lysate for each strain: T7 promoter α-N-Acetylgalactosaminidase (NAGA) and 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 both T7 promoter + NAGA and 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 #), which explained the non-expression of the proteins.
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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.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 445
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