Difference between revisions of "Part:BBa K3717007"
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<partinfo>BBa_K3717007 short</partinfo>
 
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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. Thus, α-N-Acetylgalactosaminidase can convert A blood types to universal O type.
  
 
https://static.igem.org/mediawiki/parts/e/ef/T--TAS_Taipei--hisnaga.jpg
 
https://static.igem.org/mediawiki/parts/e/ef/T--TAS_Taipei--hisnaga.jpg
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<b> Figure 1: α-N-Acetylgalactosaminidase with His-Tag and GS linker </b>
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<b><font size="+1.2"> Construct Design </font></b>
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We 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_K3717006) 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. The composite part was assembled through DNA synthesis by IDT.
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<b><font size="+1.2"> Characterization </font></b>
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<b><font size="+0.5"> Protein Expression and Purification </font></b>
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We tested protein expression of the 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.
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Cells were then harvested after the overnight induction and lysed them either through sonication or with xTractor Lysis Buffer (XTractorTM Buffer & XTractor Buffer Kit User Manual, n.d.) spiked with 500mM Imidazole stock (to a final concentration of 20mM in the lysate solution). The Histidine tagged proteins were then purified using Ni sepharose affinity chromatography. SDS-PAGE was then utilized to confirm sizes of purified proteins.
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Our results indicate no protein bands, showing that our protein purification was unsuccessful.
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<span class='h3bb'>Sequence and Features</span>
 
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<partinfo>BBa_K3717006 SequenceAndFeatures</partinfo>
  
  
 
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===Functional Parameters===
 
===Functional Parameters===
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<partinfo>BBa_K3717006 parameters</partinfo>
 
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Revision as of 09:43, 18 October 2021


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. Thus, α-N-Acetylgalactosaminidase can convert A blood types to universal O type.

T--TAS_Taipei--hisnaga.jpg

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


Construct Design

We 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_K3717006) 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. The composite part 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 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.

Cells were then harvested after the overnight induction and lysed them either through sonication or with xTractor Lysis Buffer (XTractorTM Buffer & XTractor Buffer Kit User Manual, n.d.) spiked with 500mM Imidazole stock (to a final concentration of 20mM in the lysate solution). The Histidine tagged proteins were then purified using Ni sepharose affinity chromatography. SDS-PAGE was then utilized to confirm sizes of purified proteins.

Our results indicate no protein bands, showing that our protein purification was unsuccessful.




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]