Team:Nankai/Basic Part
Basic Parts
Promoters
According to our promoter strength assay (showed in Figure 1), BBa_K1628001 (Pbca), BBa_K1628003 (BJ27UP), BBa_K1628004 (C2up), BBa_K1628005 (A2up), BBa_K1628006 (P43), and BBa_K1628007 (PamyA) show different properties. Promoter Pbca is the original promoter of pgsBCA operon.Promoter BJ27UP C2up and A2up are an artificially synthesized promoters. Promoter P43 is a strong promoter in Bacillus subtilis 168. Promoter PamyA is a strong promoter in Bacillus amyloliquefaciens LL3.
Judging from the promoter strength assay, BBa_K1628004 (C2up) is the strongest promoters we used in our project and BBa_K1628007 (PamyA) is the second strongest promoter in our project. The strength of BBa_K1628001 (Pbca) is very weak. While the strength of BBa_K1628003 (BJ27UP) is stronger than Pbca, it is still too weak compared with other promoters. What's more, BBa_K1628005 (A2up) is a very strong promoter and BBa_K1628006 (P43) is a weak promoter.
Figure 1. Promoter strength assay in Bacillus amyloliquefaciens NK-1.
Coding genes
BBa_K1628101 (pgsB) and BBa_K1628102 (pgsAC) are genes in pgsBCA operon. pgsB is a gene responsible for γ-PGA synthesis.Protein PgsBCA is a membrane protein and subunit PgsB’s main function is gathering substrate glutamic acid for γ-PGA synthesis (showed in Figure 2). Subunit PgsC is responsible for glutamic acid’s polymerization and subunit PgsA is responsible for the secretion of γ-PGA.We transformed the plasmids pHT01-xylR and pCB-Pxyl into the NK-1 strain, to verify the activity of metabolic toggle switch (see it on our wiki). Fresh colonies of Bacillus 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 2, 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 2. The synthetic pathway of γ-PGA
Funtion of part pgsB and pgsCA were validated through fermentation experiment (showed in Figure 3). Wild type Bacillus amyloliquefaciens NK-1 strain could produce 3-4g/L γ-PGA after 48 hours of fermentation. In previous work in our laboratory, we also tested heterologous expression of γ-PGA in E. coli. We constructed different expression vectors containing pgsBCA genes and transformed them separately into E.coli JM109. The average production of γ-PGA in E. coli JM109 is around 0.5g/L
Figure 3. γ-PGA produced by B. amyloliquefaciens NK-1
