pTRC-6xHIS-MntR for the inducible expression of MntR
We have generated a plasmid for the IPTG-induced expression of a 6X-HIS tagged manganese metalloregulatory protein (6X-HIS mntR). This is an improvement of part Part: BBa_K902030 designed for the constitutive mntR expression by the Calgary 2012 iGEM team. Since this protein is known to respond to the level of intracellular manganese, an IPTG-inducible system that allows titration of mntR levels in E. coli would have potential utility in the experiments requiring the regulation of manganese homeostasis and response. Further, no commercial antibodies are available for mntR, so the addition of a 6X-HIS tag allows the quantification of mntR protein levels by Western Blot.
We confirmed that IPTG in the range of 0.1mM to 10mM induces 6X-HIS tagged mntR expression induction by Coomassie stain and immunoblot for HIS-tagged proteins of cell lysates from mutant MG1655 E.coli lacking endogenous mntR (MG1655 ΔmntR) expressing the pTrc-6X-HIS-mntR plasmid (Fig.1). We confirmed induction of 6X-HIS-mntR protein by 2hr of IPTG treatment and sustained induction through overnight incubation.
Fig.1. Immunoblot (top) and Coomassie (bottom) of cell lysates from MG1655 ΔmntR E.coli expressing the pTrc-6X-HIS-mntR plasmid. 5 µl of Low MW protein ladder (Thermo Fisher #26616) was run in the first lane. A positive control of pet29b-6X-HIS-GFP was run as a positive control. 0.01 OD600 equivalent of the soluble fraction of cell lysates were run in each lane. Proteins were separated on a 10% SDS-PAGE gel run at 135V and either stained with Coomassie stain or transferred to a 0.45µm PVDF membrane with 0.35A for 60min for immunoblot for GFP. Immunoblot was performed using mouse anti-GFP (Cell Signaling Technology) at 1:1000 in 5% milk overnight at 4oC and goat anti-mouse IgG at 1:5000 in 5% milk for 1 hour at room temperature. Blots were developed using Lightning Plus ECL bioluminescence substrate and imaged on a Fuji LAS 4000.
Additionally, we demonstrated that uninduced co-expression of the pTrc-mntR plasmid with the pSB3K#-pmntP-rs-sfGFP sensor plasmid functions to increase production of sfGFP from the sensor plasmid (Fig.2). Interestingly, IPTG induction performed in cells expressing both plasmids effectively blocked sfGFP production. Taken together, these data confirm that pTrc-mntR functions as an inducible regulator of the manganese homeostatic pathway and of our pSB3K3-pmntP-rs-sfGFP sensor.
Fig.2. pTrc-6X-HIS-mntR increases sfGFP expression by the pSB3K3-pmntP-rs-sfGFP sensor plasmid in response to MnCl2. Overnight cultures of MG1655 WT E.coli expressing the pSB3K3-pmntP-rs-sfGFP sensor alone of co-expressed with the pTrc-6X-HIS-mntR plasmid (“dual”) were back-diluted 1:10 and grown to an OD600 of 0.5. At that point, half of the “dual” culture was treated with 1 mM IPTG for 2 hours. All three cultures were then aliquoted into 3 mL cultures and treated with 0.01, 0.1, 1, or 2.5 mM MnCl2 for 4 hours. Fluor485/515 and A600 readings were taken and used to calculate the fold-change in culture density corrected fluorescence relative to the untreated (0mM MnCl2), control.