Difference between revisions of "Part:BBa K3717003"

Revision as of 09:08, 19 October 2021

α-Galactosidase with strong promoter and RBS

Figure 1: α-galactosidase from 2014 Tuebingen with strong promoter, RBS, and a terminator

Construct Design

α-Galactosidase is an enzyme that 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 thus does not elicit an immune response in the human body. Thus, the enzyme can convert B blood types to universal O type. The enzyme has been previously shown to successfully convert B type erythrocytes to O type but lacked a cost-effective method of production.

We obtained the amino acid sequence of the α-Galactosidase protein, derived from Bacteroides fragilis, from the iGEM DNA Repository Plate (BBa_K1483002), 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

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_K3717003.

Figure 2 - Colony PCR check for strong promoter (K88) α-Galactosidase (α-Gal) (Part: BBa_K3717003) 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 2107 bp, as 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-Galactosidase (α-Gal) (Part: BBa_K3717001) and strong promoter (K88) α-Galactosidase (α-Gal) (Part: BBa_K3717003). Green triangles indicate expected size for α-Gal (65.7 kDa). We found that the sequences for the target proteins do not contain a start codon, thus have no expression, as shown by the triangles.

Our SDS-page (Fig. 3) 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: 65.7 kDa band for both T7 promoter + α-Gal and K88 promoter + α-Gal 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_K1483002) with full sequences that were offered by other studies online, we discovered that the enzyme sequences were missing a few amino acids, also including the start codon, which explained the non-expression of the proteins (Fig. 4).

Figure 4 - Top sequence: First 24 amino acids of Team Tuebingen's 2014 α-Galactosidase part BBa_K1483002. Bottom sequence: First 60 amino acids of TAS_Taipei'sα-Galactosidase part BBa_K3717006; the first 15 amino acids include a 6x His tag and linker used for purification. Based on the alignment of the two sequences, Tuebingen's part is missing the first 21 amino acids of the α-Galactosidase protein.

Sequence and Features