Part:BBa_K2302008
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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 1023
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 25
Illegal NgoMIV site found at 51
Illegal NgoMIV site found at 744
Illegal NgoMIV site found at 854
Illegal AgeI site found at 546
Illegal AgeI site found at 804 - 1000COMPATIBLE WITH RFC[1000]
Results
We used Western blot to confirm the expression of the pHisx6-PelB-CALB vector. As shown in Fig. 1A, 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. 1B indicated that CALB protein expression can be very well detected in the culture medium of bacteria transformed by pHisx6-PelB-CALB.
Figure 1. 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.
Figure 2. 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 indicated that FD could indeed secret functional lipase into the culture medium, as we expected.
Figure 3. The forming transparent area shaped as “IGEM”
According to the theory that copper can combine with fatty acids and then be extracted by organic solvent, we measured the levels of associated copper to indirectly quantify fatty acids generated by Follower D. As compared to the control, the fatty acid concentrations dramatically increased in Follower D group, and reached to a plateau from 200 μM to 800μM of glyceryl tributyrate (Fig. 3). The result indicated that the CALB secreted by Follower D could efficiently convert glyceryl tributyrate into fatty acids.
Link
【1】http://2017.igem.org/Team:NEFU_China/Results:Followers
【2】http://2017.igem.org/Team:NEFU_China
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