To tackle our problem statement of methylmercury poisoning, we have proposed a dual plasmid system [Composite Bio-Brick 1 (Plasmid 1) and Composite Bio-Brick 2 (Plasmid 2)] with Escherichia coli Nissle 1917, a non-pathogenic, highly studied probiotic strain as the chassis.
Plasmid 1 includes a modified mer operon consisting of control, transport and enzymatic components as well as the partial control mechanism for Plasmid 2 and a reporter gene on a pSB3K5 vector. A constitutive promoter ensures continuous transcription of the homodimer MerR protein which acts like a repressor molecule in the absence of mercury. It binds to PmerT (a mercury responsive promoter) preventing transcription of the genes downstream to it. When the Hg cation is available (in the gut), it diffuses into the probiotic cell and binds to MerR which enables the transcription of all the downstream elements as the RNA polymerase can now bind to PmerT Brown et al., 2003 Nakaya et al., 1960 Park et al., 1992 Ralston & O'Halloran, 1990.
Figure 1: Plasmid 1 Diagram
Four transport elements, MerP a periplasmic protein component along with the transmembrane proteins MerE, MerT and MerC, helps in bringing the bulk of MeHg inside the cell Barkay et al., 2003. However, the efficiency of these genes in different combinations must be checked, considering the possibility of reduced genetic burden during production (For more details - refer to experimentation section). The dual enzyme system, consisting of MerB (Organomercurial Lyase) and MerA (Mercuric (II) Reductase) are present downstream to it. MerB facilitates the breaking of the bond between organic carbon and mercury after which MerA reduces the Hg cation formed to elemental mercury Mathema et al., 2011 Parks et al., 2009.
Plasmid 2 contains the anti-inflammatory genes that must be activated only in conditions of methylmercury induced inflammations. How do we make sure this happens?
Figure 2: Plasmid 2 Diagram
The SoxR gene which enables the transcription of SoxS promoter is present in Plasmid 1 and hence is transcribed only when Hg is present as it is present downstream to MerR. Nitric oxide, a pro-inflammatory signal that is released during inflammatory conditions binds to SoxR. This complex activates SoxS promoter on Plasmid 2 transcribing the genes downstream to it Hidalgo et al., 1998. The anti-inflammatory protein produced is Interleukin-10 (IL-10). In order to enable secretion, IL-10 has been fused with Hemolysin-A (HlyA) a transport signal peptide. Hemolysin-B (HlyB), Hemolysin-D (HlyD) and TolC enable the transport of any protein fused to HlyA (IL-10) outside the cell Gentschev et al., 2002.
Super Yellow Fluorescent Protein (SYFP) and Green Fluorescent Protein (GFP) would act as reporter genes enabling the assessment of the functioning of the Plasmid 1 and 2, respectively.
In unison, the two plasmids will break the bond of methylmercury, convert mercury cation to its elemental form and combat the side effect of mercury induced inflammation in the gut.