Difference between revisions of "Part:BBa K1628202"

Revision as of 18:08, 21 October 2021

Pgrac

Promoter Pgrac is a promoter of lactose operon regulated by repressor LacI. Pgrac together with promoter PlacI, repressor LacI (BBa_K1628201), promoter Pxyl (BBa_K1628002) and repressor XylR (BBa_K1628203) formed a metabolic toggle switch. We used this device to regulate the expression of odhAB genes in Bacillus amyloliquefaciens NK-1 (showed in Figure 1).

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.

NPU-CHINA 2021:Through the information searched by our team members[1], according to figure 3(A) pNDH33 and the DNA sequence of the Pgrac promoter (in capital letters) including the upstream AT-rich UP element, the lac operator (lacO; in capital letters) and the ribo-some-binding sequence (underlined) (showed in Figure 3)

NPU-CHINA 2021:According to the literature[2], we found Pgrac100 not only tightly controls the background expression level in E.coli, but also allowed high protein production levels at low IPTG concentrations(showed in Table 1).

Reference

[1]Phan TT, Nguyen HD, Schumann W. Novel plasmid-based expression vectors for intra- and extracellular production of recombinant proteins in Bacillus subtilis. Protein Expr Purif. 2006 Apr;46(2):189-95. doi: 10.1016/j.pep.2005.07.005. Epub 2005 Aug 9. PMID: 16125412.

[2]Phan, Trang & Linh Thuoc, Tran & Schumann, Wolfgang & Nguyen, Hoang. (2015). Development of Pgrac100-based expression vectors allowing high protein production levels in Bacillus subtilis and relatively low basal expression in Escherichia coli. Microbial cell factories. 14. 72. 10.1186/s12934-015-0255-z.

Sequence and Features