Difference between revisions of "Part:BBa K3717011"

Revision as of 09:37, 20 October 2021

Endo-β-Galactosidase with T7 + RBS, N-Terminal 6x His-Tag, and Double Terminator

The composite part utilizes a T7 promoter + RBS (BBa_K525998), Endo-β-galactosidase (BBa_K3717008), and double terminator (BBa_B0015).

Figure 1. Endo-β-galactosidase with T7 promoter, RBS and double terminator construct

Construct Design

We derived the sequence of Endo-β-galactosidase from Streptococcus Pneumoniae [1] and optimized the sequence for E. coli protein expression. We then attached a 6x histidine tag (6x His-Tag) upstream of the Endo-β-galactosidase sequence followed by a glycine-serine linker (GS linker) to form our open reading frame (ORF) (BBa_K3717008) 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_K3717011) 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. We then utilized SDS-PAGE to confirm the sizes of purified proteins.

Our results indicate no protein bands, showing that our protein purification of BBa_K3717011 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