Coding

Part:BBa_K3717007

Designed by: Enoch Toh   Group: iGEM21_TAS_Taipei   (2021-09-18)
Revision as of 14:36, 18 October 2021 by Ykuo (Talk | contribs)


α-N-Acetylgalactosaminidase with N-Terminal 6x Histidine tag

α-N-Acetylgalactosaminidase catalyzes the cleavage of the terminal N-acetylgalactosamine of A type blood antigens such that the resultant antigen can be classified as an H antigen, which the anti-A and anti-B antibodies are unable to recognize and hence does not elicit an immune response in the human body [1]. Thus, α-N-Acetylgalactosaminidase can convert A blood types to universal O type.

T--TAS_Taipei--hisnaga.jpg

Figure 1: α-N-Acetylgalactosaminidase with 6x N-Terminal His-Tag and GS linker


Construct Design

We optimized the DNA sequence for expression in E. coli, then attached a 6x histidine tag (6x His-Tag) upstream of the α-N-Acetylgalactosaminidase sequence followed by a glycine-serine linker (GS linker) to form our open reading frame (ORF) (BBa_K3717007) for purification purposes. We flanked our open reading frame with a T7 promoter + RBS (BBa_K525998) upstream of the open reading frame and a double terminator (BBa_B0015) downstream of the sequence. This composite part (BBa_K3717010) was assembled through DNA synthesis by IDT.


Characterization

Protein Expression and Purification

We tested protein expression of the composite parts by transforming our plasmids into BL21(DE3) 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.

We harvested the cells after the overnight induction and lysed them either through sonication or with xTractor Lysis Buffer spiked with 500mM Imidazole stock (to a final concentration of 20mM in the lysate solution) [2]. We purified the Histidine tagged proteins using Ni sepharose affinity chromatography [2]. We then utilized SDS-PAGE to confirm the sizes of purified proteins.

Our results indicated no protein bands, showing that our protein purification of BBa_K3717010 was unsuccessful.

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.

2. XTractorTM Buffer & xTractor Buffer Kit User Manual. (n.d.). 10.



Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 208
    Illegal AgeI site found at 511
  • 1000
    COMPATIBLE WITH RFC[1000]


[edit]
Categories
Parameters
None