Part:BBa_K1891013:Experience

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Applications of BBa_K1891013

We construct β-tubulin-YCE fusion protein vectors by Gateway Large-scale Cloning technology. We used Invitrogen pENTR/D TOPO to clone β-tubulin into entry vector. Primers were designed based on β-tubulin sequence and PCR was done for verification. Electrophoresis result (Fig.1) showed that β-tubulin was successfully cloned into the entry vector.

img width="65%" src="https://static.igem.org/mediawiki/2016/6/63/T--BNU-China--Results14.jpg">

Fig.1 PCR verification result of the constructed entry vectors

In order to do LR reaction, we used the restriction endonuclease Not I to digest the entry vector. Electrophoresis result (Fig.2) showed that single digestion was efficient.

Fig.2 Single endonuclease digestion result of entry vectors

Using Invitrogen Gateway LR Clonase II Enzyme Mix, the entry vectors were ligated with pCambia1300-cluc. Thus the destination vectors were complete. After transformation and running PCR with β-tubulins primers, electrophoresis result (Fig.3) showed high positive rates, indicating β-tubulins was successfully cloned into the vectors.

Fig.3 PCR verification result of the objective vectors

Also, signaling fragments were also needed to be tested. By using the reverse primer of β-tubulin and the forward primer of cluc for PCR verification, we found that cluc-β-tubulin fusion protein vector is successfully constructed. Electrophoresis result is shown in Fig.4.

Fig.4 PCR verification result of pCambia-nluc, pCambia-cluc
Arrows show the correct bands

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