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Team:Calgary/Project/HumanPractices/Killswitch/Regulation
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Regulation/Expression Platform
Rhamnose Inducible Promoter
Background
Given that oil sands tailings ponds contain significant amounts of magnesium and low levels manganese, and that the MgtA and MntP expression platforms are repressed by these conditions respectively, these systems are not appropriate for the bioreactor in a typical tailings pond site (FIND PAPER WHICH CITES IONs in TP). In order to ensure that the kill genes would be activated should bacteria escape from the bioreactor, we required a control element which would be expressed under conditions in typical tailings ponds. To this end, we selected a rhamnose inducible promoter from Eschericia coli as a potential method for regulating our kill gene combination.
The rhamnose inducible system is optimal in the bioreactor since the promoter is tightly repressed with the presence of glucose. We aim to supplement the bioreactor with low levels of glucose so that the kill genes downstream of the promoter would be repressed. In the event of escape into the tailings ponds, glucose levels would be insufficient for repression of the system, which would thus activate expression of the kill genes.
Although our team has also characterized a molydenum-repressed MOCO riboswitch as an additional control mechanism suitable to the bioreactor, we investigated the rhamnose promoter because of the lower cost and availability of the repression agent.
Native function of the rhamnose promoter
The rhamnose promoter (pRha) is responsible for regulating six genes related to rhamnose metabolism and contains a separate promoter on its leading and reverse strands (see Figure one). RhaR and RhaS are downstream on one side of the promoter and are the control proteins which regulate expression of the RhaB, RhaA, and RhaD genes on the opposite side of the promoter. Basal levels of RhaR transcription factor are activated by complexing with L-rhamnose so that expression of the rhaSR operon is up-regulated. In turn, the resulting RhaS activates the rhaBAD operon (Egan & Schleif, 1993). The RhaB, RhaA, and RhaD genes are directly involved in metabolism of rhamnose.
Although RhaS acts directly on pRha, Egan and Schleif (1993) proposed that extent of up-regulation of rhaBAD is dependent on two other factors: firstly, RhaS is more efficient when cAMP receptor protein (CRP-cAMP) is bound to the promoter; and secondly, RhaS causes higher expression of rhaBAD in the presence of rhamnose (see figure two).
Via the CRP-cAMP complex, glucose represses the rhaBAD operon through Eschericia coli's system of global catabolite represssion. In the presence glucose, membrane bound EIIA protein transfers phosphate to glucose. Under this condition, desphosphorylated EIIA is unable to activate adenyl cyclase resulting in lower levels of cAMP (CITE NATURE ARTICLE). In-turn, catabolite receptor protein (CRP) is unable to complex with cAMP, causing a down-regulation of the rhaBAD operon (see figure two, figure three (of CRP sites on promoter)).
Engineering pRha into a kill system
Our team has engineered the following rhamnose promoter kill system:
(Draw the final kill circuit)
In place of the rhaBAD operon, we have placed the CviAII endonuclease and S7 exoendonuclease for the active components of our kill system. We are capitalizing on glucose's global catabolite repression of these two genes as the controlled condition to repress cell destruction in the bioreactor. The rhamnose promoter as tightly controllable expression platform was inspired by two papers.
Giacalone et al. (2006) cloned the rhamnose promoter together with the rhaSR operon and proposed pRha as a viable system for expressing toxic proteins in E. coli. They tested the system in three different plasmids of varying copy number and replaced rhaBAD operon and characterized the system with GFP and TphoA protein. In their results, they found that 0.2% w/v D-glucose was significantly repressed GFP in the presence of L-rhamnose, and that it was completely repressed when only glucose was present. Likewise, they found that basal expression of TphoA was completely repressed when glucose was present in 0.2% w/v.
Jeske and Altenbuchner (2010) assembled a similar system with pRha, rhaSR operon, and GFP and found that florescence output was twenty-four times greater with rhamnose as opposed to rhamnose and glucose.
In designing this system for the bioreactor, we set out to replicate this tight glucose repression of the rhamnose promoter. As opposed to Giacalone et al. (2006) and Jeske and Altenbuchner (2010), we manipulated the rhaSR operon to better suit conditions in the bioreactor (see figure X).
The logic behind these changes is that rhamnose will not be present in the tailings ponds to activate the rhaSR operon cascade for induction of the promoter. Expression of these control genes are dependent on the rhamnose activation of RhaR. Given that rhamnose is not naturally present in tailings ponds, and that we are depending on the native composition of the waste water to activate the kill system, we had to modify the control system.
RhaS is the protein which directly activates the rhaBAD operon side of the pRha promoter. To bypass the natural induction by rhamnose, we put rhaS under control of a constitutive promoter (R0040) from the parts registry. In doing so, we intend to elevate expression of RhaS such that it will be able to activate pRha. When glucose levels are low, as is the case of the environment outside of the bioreactor, cAMP-CRP complexes bind to pRha so that the over-expressed RhaS may initiate the kill system.
Some may object to this system because Egan and Schleif (1993) suggested that RhaS was more efficient at activating pRha in the presence of rhamnose. This does not invalidate the methodology of our system though because Egan and Schleif (1993) only suggested that function of RhaS was optimized in the presence of rhamnose. They agreed that RhaS significantly upregulated pRha even when rhamnose was not available.
Assembly methods
For the construction of this system, our team had pRha commercially synthesized as it was described in Jeske and Altenbuchner (2010). SHOW PIC MAYBE
Additionally, we amplified rhaS and rhaR from Top Ten E. coli using Kapa Hi-Fi polymerase and PCR. Here follows the parts which we submitted for this system: (LIST THE PARTS)
Characterization
We followed similar protocols for characterizing pRha as Jeske and Altenbuchner (2010). In place of the rhaBAD operon, we inserted a gene for GFP with an LVA degradation tag. ASK WHAT WE DO REGARDING PROTOCOLS.
