Bacterial strains:E. coli
Function:Follower D secretes lipase to degrade sewage grease into fatty acids.
Vector Construction:pHisx6 is an IPTG-induced expression vector with a 6xHis tag at the N-terminal of its fused protein and a PelB-CALB exocrine lipase gene inserted at the multiple cloning site. Since a signal peptide of an expressed protein can be cleaved in the periplasmic space of bacteria, the His tag at the N-terminal is generally excised together with the signal peptide. To trace the synthesis and secretion of recombinant lipase, we also added another Hisx6 tag at the C-terminalof the Pelb-CALB lipase coding sequence. Meanwhile , to enhance the flexibility of Hisx6 tag, seven glycine serine were used to link the C-terminal Hisx6 tag with the exocrine lipase. As showed in Fig. 11A, the predicted cDNA size of Pelb-CALB is 1024 bp, which matched our experimental results. The diagram of the expression cassette was shown in Fig. 11B.
Figure. 11. Electrophoresis of PelB-CALB coding fragment (A) and expression cassette of pHisx6-Pelb-CALB (B). M, the DL2000 DNA ladder.
Protein Expression
Western Blot
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
Functional Verification:
Qualitative verification:
To further determine whether the recombinant CALB is functional, we made soft agar containing glyceryl tributyrate on culture plates containing growing bacteria. 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. 13. 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. 14, 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.
Figure. 14. The forming transparent area shaped as “IGEM”.
Quantitative verification:
According to the theory that copper can combine with the fatty acids and then can be extracted by organic solvent, we measured the copper to detect the quantity of fatty acids degraded from Follower D by testing the liquid OD value.
After diluting standard sample into different concentrations,we got the standard curve about fatty acid concentrations and the OD value.
Figure. 15. The fatty acids concentration
Based on the results above, we could confirm that CALB were successfully expressed in the rossatta cell. Particularly, according to Fig. 1B, Fig. 3 and Fig. 5, the target proteins can be tested out in the medium, indicating that they are secreted out when expressed in rossatta strain.
