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Regulation/Expression Platform

Tight Regulation


Introducing the Riboswitch




Magnesium riboswitch

The magnesium riboswitch that we looked at is repressed in the presence of magnesium ions. This system has two control components – a promoter and a riboswitch. Normally the magnesium promoter (mgtA promoter) and the magnesium riboswitch (mgtA riboswitch) are activated if there is a deficiency of magnesium in the cell. The lack of magnesium activates other genes in E. coli to allow influx of magnesium into the cell. There are two proteins in the cascade that activate the system namely PhoP (BBa_K902010) and PhoQ (BBa_K902011). PhoQ is the trans-membrane protein which gets activated in the absence of magnesium and phosphorylates PhoP. PhoP in turn binds to the mgtA promoter and transcribes genes downstream.

Manganese riboswitch

Manganese is an essential micronutrient. It is an important co-factor for enzymes and it also reduces oxidative stress in the cell (Waters et al, 2011). Despite being an important micronutrient it is toxic to cells at high levels. MntR detects the level of manganese in the cell and acts as a transcription factor to control the expression of manganese transporter such as mntH, mntP and mntABCDE. In order to regulate these genes mntR (BBa_K902030) binds to the mntP promoter (mntP promoter). The manganese homeostatsis is also controlled by the manganese riboswitch mntPrb (BBa_K90274)


The Moco Riboswitch

The molybdenum cofactor (moco) riboswitch (BBa_K902023) is an RNA element which responds to the presence of the metabolite molybdenum cofactor (Regulski et al, 2008). This RNA element is located in the E.coli genome just upstream of the moaABCDE operon (BBa_K902024), which contain the important moco synthesis genes. Moco is an important cofactor in many different enzymes. The moco riboswitch has 2 regions: an aptamer domain and the expression platform. When moco is present in the cell it will bind to the aptamer region in the riboswitch which will cause an allosteric change. This allosteric change affects the expression platform by physically hiding the ribosome binding site thus preventing translation from occurring and hence adding a translational level of gene expression regulation. Therefore, the moco riboswitch when activated by moco inhibits gene expression.

Figure 2: This picture depicts the Moco RNA motif which is upstream of the moaABCDE operon.

Building the Systems

Using these riboswitches, we wanted to eventually design a system where we would place our kill genes downstream, and then supplement our bioreactor with the appropriate ions to keep the systems turned off. We biobricked and submitted DNA for the the mgtaP (BBa_K902009) and mntP promoters (BBa_K902073) as well as their respective riboswitches (BBa_K902008) (BBa_K902074) and the moco riboswitch (part). In addition, we also biobricked some of the regulatory proteins: PhoP (BBa_K902010), PhoQ (BBa_K902011), mntr (BBa_K902030) and the Moa Operon (BBa_K902024) . Our final system would inovolve constitutive expression of these necessary regulatory elements upstream of our riboswitches and kill genes. An example of the manganese system is shown in figure 3.


Characterizing the riboswitches

GFP testing


In order to test the control of these promoters and riboswitches, we constructed them independently and together upstream of GFP (BBa_K082003) with an LVA tag. Figure 4 shows these circuits for the mgta system. Identical circuits were deisgned for all three systems, however only the top two were needed for the mocoriboswitch system.

We then tested the aforementioned circuits by growing cells containing our circuits with differing concentrations of their respective ions. Our detailed protocols can be found here. We then measured fluorescent output, normalizing to a negative control not expressing GFP.


So far, we have been able to obtain results for our magneisum system, as can be seen in figure 5).


As the graph shows, there is a much larger decrease in the GFP output when the mgtA promoter and riboswitch are working together as compared to the mgtA riboswitch alone under the control of TetR promoter (BBa_J13002). This suggests that having both the promoter and the riboswitch together provides a tighter control over the genes expressed downstream. This also suggests that magnesium riboswitch alone is sufficient in reducing gene expression downstream of a constitutive promoter.


Although the magnesium system is highly regulated, it is not a suitable system for the purposes of our bioreactor. The tailings are composed of very high concentration of magnesium- up to 120mM(Kim et al, 2011). As can be seen, this would inhibit the system. Therefore, if our bacteria escapes into the tailings, the kill genes would not be activated and the bacteria would be able to survive. We feel howeever that this could still be an incredibly useful system for other teams for both killswtitch and non-killswitch-related applications, amking it still a valuable contribution to the registry.

Kill Gene Testing

In tandem to building our systems with GFP in order to test their control, we also constucted them with our kill genes. This was delayed substantially however due to problems in their synthesis. Specifically, the micrococcal nuclease that arrived from IDT had a 1bp mutation which changed a isoleucine residue into a lysine. Initially, our systmes resulted in no killing of cells. Therefore we had to mutate this resiude using site-directed mutagenesis. Once completed, we were able to begin testing. With our GFP data collected, we moved on to characterizing the mgtA control system upstream of our S7 killgene (BBa_K902019). To test the circuits, we incubated cells expressing our construct with differing concentrations of magnesium. We then measured both Colony forming units and OD 600. For a deatiled protocol, see here.




An alternative: a glucose repressible system

Based on the problem with the magneisum and system in relation to tailings ponds, we wanted to find an alterntative (other than the manganese and moco systems, which require further testing) We found a promoter in the literature that was both induced by rhamnose and repressed by glucose. This seemed to be a very suitable candidate for controlling the kill switch in the the bioreactor since the promoter is shown to be tightly repressed. and glucose and rhamnose are fairly inexpensive and not found in high concentrations in tailings ponds. We could supplement the bioreactor with glucose to inhibit xpression of the kill genes in the bioreactor. Escape of bacteria into the tailings ponds will cause expression of the kill genes since glucose levels in the surrounding environment would be insufficient for deactivate the promoter.

This promoter, known as pRha (BBa_K902065), is responsible for regulating six genes related to rhamnose metabolism and contains a separate promoter on its leading and reverse strands (see Figure 7). RhaR (BBa_K902069) and RhaS (BBa_K902068) serve to regulate expression of the rhamnose metabolism genes rhaB, rhaA, and rhaD on the opposite side of the promoter. The RhaR transcription factor is activated by L-rhamnose to up-regulate expression rhaSR operon. In turn, the resulting RhaS activates the rhaBAD operon to generate the rhamnose metabolism genes (Egan & Schleif, 1993).

Figure 7: The rhamnose metabolism genes as they exist in Top Ten E. coli


Our kill system is different from the native rhamnose system with the rhaR and rhaS control genes. We have constituitively expressed RhaS to overcome dependency on rhamnose to cause activation of the kill switch. While RhaS is continuously present, global catabolite repression prevents its activation the kill genes in the bioreactor; in the outside environment, glucose levels are lower such that RhaS is able to activate the kill genes.

Building the system

Our team had pRha promoter (BBa_K902065) commercially synthesized as per the sequence given by Jeske and Altenbuchner (2010). The rhaS (BBa_K902068) and rhaR (BBa_K902069) genes were amplified via PCR from Top Ten E. coli using Kapa Hi-Fi polymerase.

Given the control gene modifications which we have engineered into our system to optimize it for the tailings ponds, we are working to determine whether glucose repression of our modified system can match patterns shown by Giacalone et al. (2006) and Jeske and Altenbuchner (2010). To this end, we have constructed the rhamnose promoter with GFP (BBa_K902066) and are finalizing construction with constituitively expressed RhaS, so that we can characterize this promoter and test it in combination with our kill genes.