Difference between revisions of "Part:BBa K3717012"
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<partinfo>BBa_K3717012 short</partinfo> | <partinfo>BBa_K3717012 short</partinfo> | ||
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+ | The composite part utilizes a T7 promoter + RBS (BBa_K525998), α-Galactosidase (BBa_K3717015), and double terminator (BBa_B0015). | ||
https://static.igem.org/mediawiki/parts/5/5f/T--TAS_Taipei--t7agalhis.jpg | https://static.igem.org/mediawiki/parts/5/5f/T--TAS_Taipei--t7agalhis.jpg | ||
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+ | <b> Figure 1. α-Galactosidase 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-Galactosidase from <i>Bacteroides fragilis</i> [1] and optimized the sequence for <i>E. coli</i> protein expression. We then attached a 6x histidine tag (6x His-Tag) downstream of the α-Galactosidase sequence preceded by a glycine-serine linker (GS linker) to form our open reading frame (ORF) (BBa_K3717015) 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_K3717012) was assembled through DNA synthesis by IDT. | ||
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+ | However, cells transformed with the plasmids had problems growing on culture plates and therefore, we were unable to commence protein purification. | ||
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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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Revision as of 09:42, 20 October 2021
α-Galactosidase with T7 + RBS, C-Terminal 6x His-Tag, and Double Terminator
The composite part utilizes a T7 promoter + RBS (BBa_K525998), α-Galactosidase (BBa_K3717015), and double terminator (BBa_B0015).
Figure 1. α-Galactosidase with T7 promoter, RBS and double terminator construct
Construct Design
We derived the sequence of α-Galactosidase from Bacteroides fragilis [1] and optimized the sequence for E. coli protein expression. We then attached a 6x histidine tag (6x His-Tag) downstream of the α-Galactosidase sequence preceded by a glycine-serine linker (GS linker) to form our open reading frame (ORF) (BBa_K3717015) 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_K3717012) was assembled through DNA synthesis by IDT.
However, cells transformed with the plasmids had problems growing on culture plates and therefore, we were unable to commence protein purification.
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.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]