Team:Nankai/Results
1. Promoter strength assay in Bacillus amyloliquefaciens NK-1
Six candidate promoters were digested by reletive endonucleases and ligated to the verification vector pCB with a reporter gene bgaB which encodes β-galatosidase (Figure 1).
Figure 1. Plasmids verification. Marker III (M), control plasmids(1), digested plasmids(2).
As shown in Figure 2, promoter C2up is the strongest promoter, about 60 times stronger than Pbca. Promoter PamyA and A2up are both strong promoters, about 45 and 42 times stronger than Pbca. Promoter P43 and BJ27up, though, are weak promoters, about 5 times and 3 times stronger than Pbca.
Figure 2. Promoter strength assay in B.amyloliquefaciens NK-1.
2. Metabolic toggle switch function assay
To verify the function of the metabolic toggle switch, the xylR gene was ligated into the pHT01 plasmid to construct a XylR expression plasmid pHT01-xylR. Pxyl was ligated into the pCB plasmid to construct the reporter plasmid pCB-Pxyl. The electrophoretograms of both plasmids are shown in Figure 3.
Figure 3. Plasmids digested by double endonucleases. Marker III (M), control plasmids (1), digested plasmids(2).
We transformed the plasmids pHT01-xylR and pCB-Pxyl into the NK-1 strain, to verify the activity of metabolic toggle switch. Fresh colonies of B. amyloliquefaciens strains (NK-1 strain containing plasmids pHT01-xylR and pCB-Pxyl and the control NK-1 strain containing plasmids pHT01 and pCB-Pxyl) were first cultured overnight in test tubes containing 5 mL LB liquid and then inoculated into 100 mL fresh fermentation medium. We added 1mM IPTG into the medium after 12h of cultivation. The β-galactosidase activity were measured at 12h, 18h, 24h, 30h, 36h, 42h to test the effect of metabolic toggle switch on the expression of bgaB.
As shown in Figure 4, β-galactosidase enzyme activity dropped considerably after 30 hours of fermentation. The inhibited expression of bgsB in experiment group (NPP+IPTG) indicated that the metabolic toggle switch we constructed is functional in B. amyloliquefaciens NK-1 strain.
Figure 4. Verification of the metabolic toggle switch’s function. IPTG was added to the medium after 12 h cultivation.
3. Metabolic toggle switch’s affect on γ-PGA production
We first constructed a recombinant plasmid pKSU-Pxyl (Figure 5) by intergrating Pxyl promoter into plasmid pKSU. Then we inserted Pxyl into the B. amyloliquefaciens NK-1’s chromosome by markerless gene replacement method (see it in experiment and protocols), to replace Pbca, the orgianal promoter of pgsBCA genes (Figure 6). The genetrated strain was designated as B. amyloliquefaciens NK-TP.
To find out the proper IPTG adding time, we added 1mM IPTG into the medium at different time points (0h, 3h, 6h, 9h, 12h, 15h, 18h, 21h and 24h). According to the γ-PGA fermentation results, we found that the proper IPTG adding time is 15 hours. The highest γ-PGA titer was 4.15 g/L which is about 62.5% higer than that of wild type NK-1 strain (2.55g/L) and 87.5% higher than that of control group (2.21g/L).
4. Replacement of promoters
In order to replace promoter Pbca (the original promoter of pgsBCA) with the candidate promoters, we constructed five promoter recombinant plasmids. As shown in Figure 7, the five promoter recombinant plasmids have been successfully constructed.
Figure 7. Plasmids verification. Marker III (M), control plasmids (1), digested plasmids(2).
Among those candidate promoters, the promoter BJ27up has been successfully inserted into the chromosome of B. amyloliquefaciens NK-TP (showed in Figure 8). The BJ27up promoter insertion strain was designated as NK-BJ27up.
Figure 5. (A)pKSU-Pxyl plasmid verification. (M)Marker III, (1)complete plasmids, (2) digested plasmids.(B) Confirmation of the B. amyloliquefaciens NK-TP. (M) Maker III, (1, 2) NK-TP strain , (3) NK-1 control strain.
Figure 6. The effects of IPTG adding time on γ-PGA production. 1mM IPTG was added into the medium at different time points 0h, 3h, 6h, 9h, 12h, 15h, 18h, 21h and 24h.
Figure 8. Confirmation of the B. amyloliquefaciens NK-BJ27up. Marker III (M), NK-BJ27up (1,2,3), NK-1(4).