Difference between revisions of "Part:BBa K3717010"
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<partinfo>BBa_K3717010 short</partinfo>
 
<partinfo>BBa_K3717010 short</partinfo>
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The composite part utilizes a T7 promoter + RBS (BBa_K525998), α-N-Acetylgalactosaminidase (BBa_K3717007), and  double terminator (BBa_B0015).
  
 
https://static.igem.org/mediawiki/parts/8/8b/T--TAS_Taipei--t7hisnaga.jpg
 
https://static.igem.org/mediawiki/parts/8/8b/T--TAS_Taipei--t7hisnaga.jpg
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<b> Figure 1. α-α-N-Acetylgalactosaminidase with T7 promoter, RBS and double terminator construct </b>
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<b><font size="+1.2"> Construct Design </font></b>
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We derived the sequence of α-N-Acetylgalactosaminidase from <i>Elizabethkingia meningoseptica</i> [1] and optimized the sequence for <i>E. coli</i> protein expression. We 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.
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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(DE3) <i>E. coli</i> 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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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.
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Our results indicated no protein bands, showing that our protein purification of BBa_K3717010 was unsuccessful.
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<b><font size="+1.2"> References </font></b>
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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.
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2. XTractorTM Buffer & xTractor Buffer Kit User Manual. (n.d.). 10.
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<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here
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===Usage and Biology===
 
===Usage and Biology===
  

Revision as of 09:29, 20 October 2021


α-N-Acetylgalactosaminidase with T7 + RBS, N-Terminal 6x His-Tag, and Double Terminator

The composite part utilizes a T7 promoter + RBS (BBa_K525998), α-N-Acetylgalactosaminidase (BBa_K3717007), and double terminator (BBa_B0015).

T--TAS_Taipei--t7hisnaga.jpg

Figure 1. α-α-N-Acetylgalactosaminidase with T7 promoter, RBS and double terminator construct


Construct Design

We derived the sequence of α-N-Acetylgalactosaminidase from Elizabethkingia meningoseptica [1] and optimized the sequence for E. coli protein expression. We 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 NgoMIV site found at 296
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 1316