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Applications of BBa_K1891011

β-tubulin-YCE 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: β-tubulin-YCE is 1629bp

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 before(Fig.2) and after(Fig.3) breaking the bacteria, and Western blot(Fig.4) was also applied for the further confirmation.

Fig.2 SDS-PAGE of centrifuged cells before ultrasonic breaking.
A: cluc-α-tubulin(74 kDa), α-tubulin-nluc, YCE-β-tubulin(66 kDa), β-tubulin-YCE(66 kDa), YCE-α-tubulin(66 kDa), α-tubulin-YCE(66 kDa), expressed empty vector.
B: left to right: expressed empty vector, α-tubulin(55 kDa), β-tubulin(55 kDa), α-tubulin-YNE(75kDa), YNE-α-tubulin(75kDa).
Arrows show the correct bands.

Fig.3 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)

Fig.4 western blot of rossatta cells expression.
B: left to right: negative control in pellet, β-tubulin in pellet(55 kDa), β-tubulin-YCE in pellet (66 kDa), protein marker, negative control in supernatant, β-tubulin in supernatant (55 kDa), β-tubulin-YCE in supernatant (66 kDa), extracted β-tubulin(55 kDa).

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

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