Difference between revisions of "Part:BBa K2302008"

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PelB+CALB+His

This plasmid cords for lipase out the membrane,which can hydrolyze fat to fatty acids, it consists four basic parts: promoter T7, RBS, pelB and CALB. pelB is sequence cords for a functional signal peptide, which transport the fused protein CALB out the membrane efficiently, and CALB is a kind of lipase which can hydrolyze fat. By transforming this plasmid, a bacteria can secret lipase out the membrane,hydrolyzing fat in the environment to fatty acids. Sequence and Features

Results

We used Western blot to confirm the expression of the pHisx6-PelB-CALB vector. As shown in Fig. 12A, the expression of CALB protein was very clearly detected in bacteria harboring pHisx6-PelB-CALB vector in the presence of IPTG, as compared to control, indicating that the pHisx6-PelB-CALB vector could express our designed protein successfully. To further examine whether the CALB could be secreted into the medium, the culture medium supernatant of bacteria was directly analyzed by western blot. Fig. 12B indicated that CALB protein expression can be very well detected in the culture medium of bacteria transformed by pHisx6-PelB-CALB.

Figure. 12. Western blot analyses of CALB protein expression

A. CALB protein expression in total bacterial lysates; B. CALB protein expression in culture supernatant. Con, empty pHisx6 vector; CALB, pHisx6-PelB-CALB overexpression plasmid; M, protein molecular weight markers

To further determine whether the recombinant CALB is functional, we made soft agar containing glyceryl tributyrate on culture plates containing growing bacteria (Fig.1). The result showed that Follower D could secrete degrade glyceryl tributyrate as indicated by transparent ring shaped as “NEFU”, suggesting its production of functional lipase.

Fig.1 The forming transparent area shaped as “NEFU”

Next, we used the culture supernatant of the bacteria to determine whether the lipase can be successfully secreted into culture medium. As shown in Fig.2, the culture supernatant could also cause degradation of glyceryl tributyrate and form transparent arear shaped as “IGEM”. The medium was stained by Sudan III. This result indicate that FD could indeed secret functional lipase into the culture medium, as we expected.

Fig.2 The forming transparent area shaped as “IGEM”