Difference between revisions of "Part:BBa K1483000"

Revision as of 09:35, 19 October 2021

α-N-Acetylgalactosamindase

This part is [http://appft1.uspto.gov/netacgi/nph-Parser?Sect1=PTO1&Sect2=HITOFF&d=PG01&p=1&u=/netahtml/PTO/srchnum.html&r=1&f=G&l=50&s1=20140220553.PGNR. patented] and should therfore only be used for research purposes.

Encodes for an enzyme, that is capable of cleaving off N-acetlygalactosamine from A-group blood antigens, thereby turning them into H-antigens. Can be used to convert erythrocytes from blood type A to O. Part in RFC25 standard.

Following reaction is catalysed by this part

Improvement by TAS_Taipei

We improved a part from the 2014 Tuebingen team: α-N-Acetylgalactosaminidase (NAGA) from Bacteroides fragilis (Part:BBa_K1483000).

Expression of Part From 2014 Tuebingen

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, as well as 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 1 - 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.

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.

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.

Successful Expression of Part

Sequence and Features