Part:BBa_K1891012:Experience

This experience page is provided so that any user may enter their experience using this part.
Please enter how you used this part and how it worked out.

Applications of BBa_K1891012

YCE-β-tubulin was cloned respectively via fusion PCR. After ligating these fusion gene fragments to pET30a(+) empty vectors, we transformed the target plasmids to Trans5α. When colony PCR was done for screening, we picked correct colonies shown in electrophoresis gel (Fig.1) for plasmid amplification.

Fig.1 Result of colony PCR
Arrows show the correct size of fusion gene fragments: YCE-β-tubulin is 1629bp.

Sequencing results further confirmed that YCE-β-tubulin was constructed successfully.

Rossatta(DE3) is a kind of E.coli strain that can express rare codons and improve the expression level of eukaryotic protein. Thus we applied this strain to optimize our protein expression.

SDS-PAGE were done to verify the expression results after(Fig.2) breaking the bacteria.

Fig.2 SDS-PAGE of supernatant after ultrasonic breaking the rossatta cells
Left to right: expressed empty vector, α-tubulin(55 kDa), β-tubulin(55 kDa), α-tubulin-YNE(75kDa), YNE-α-tubulin(75kDa), α-tubulin-YCE(66 kDa), YCE-α-tubulin(66 kDa), β-tubulin-YCE(66 kDa), YCE-β-tubulin(66 kDa), α-tubulin-nluc, cluc-α-tubulin(74 kDa)

Based on the results above, we could confirm that YCE-β-tubulin fusion protein was successfully expressed in rossatta cell.

User Reviews

UNIQ05ed6fbe0ad6ecb8-partinfo-00000001-QINU UNIQ05ed6fbe0ad6ecb8-partinfo-00000002-QINU