The Moco Riboswitch The molybdenum cofactor (moco) riboswitch is an RNA element which responds to the presence of the metabolite molybdenum cofactor. This RNA element is located in the E.coli genome just upstream of the moaABCDE operon, which contain the important moco synthesis genes. Moco is an important cofactor in many different enzymes ranging from to this. 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. The Killswitch Design This riboswitch system can be used to design a killswitch mechanism to regulate the expression of our kill genes, CviAII and S7. The basic design of this system includes the kill genes downstream of the riboswitch all of which is under the control of a constitutive promoter. Downstream of this construct is the moa operon constitutively expressed. This entire construct will be present in a low copy plasmid in our bacteria. While moco synthesis is a normal process in the bacteria we wanted to constitutively express the moa operon for two reasons. First, we wanted to up regulate the expression of moco to ensure high enough concentrations of moco capable of inactivating the kill switch when the bacteria are in the bioreactor. Second, the moa operon in the genome is under regulation by the bacteria to maintain equilibrium and therefore we think it might not be reliable in producing the required concentration. The two moa operons will express our metabolite moco, which will activate the riboswitch and represses the kill genes. Molybdate in TPW Molybdenum, Mo, is a trace element that is required by the bacteria for moco synthesis. Bacteria cannot uptake molybdenum in the elemental form and so uptakes molybdenum in its oxyanion form molybdate, MoO4, using the molybdate transport system. We wanted a system where molybdate is present in the bioreactor permitting moco synthesis (inactivate killswitch) and absent in the tailings pond water (TPW) preventing moco synthesis (activate killswitch). We discovered that molybdenum is indeed present in the TPW. Concentrations of Mo in the TPW: [Mo] in the Syncrude and Suncor TPW in 1990: 0.183 mg/L in the surface region (1m-10m) 0.045 mg/L in the sludge region (11m-20m) Molybdate forms when molybdenum is in contact with both water and oxygen. If the bacteria escape they will first enter the surface region where it is possible for the bacteria to encounter molybdate. If molybdate is present in the TPW, then there is a possibility that moco can be synthesized in the escaped bacteria inactivating the kill switch. This dilemma would defeat the purpose of this killswitch mechanism. The Solution Upon further literature research we found that molybdate is transported using a molybdate transport system which is coded by the modABCD operon. It has been shown that knocking out mod C, a gene encoding the ATPase of the transporter, enables the transport system dysfunctional and prevents moco synthesis. However, if molybdate is supplemented in high enough concentrations to the bacteria, the bacteria are able to use its sulphate transport system to transport molybdate. This gave rise to the idea of having a mod C knock out strain of bacteria in our system and supplementing it with molybdate allowing moco synthesis (inactivate killswitch) inside the bioreactor. The paper tried 10mM sodium molybdate supplementation in the media whereas that the litreture level states less than 1 uM. Therefore, in the tailings ponds, the concentration is not high enough and molybdate is transported into the cell preventing moco synthesis. This activates the kill switch. Sodium molybdate is expensive and it can be said that on a large bioreactor scale it is impractical to provide. However, there are inexpensive and effective filtration mechanisms available to filter most of it out and reuse it. The filtration also prevents dumping a load of molybdate ions in the TPW. Characterization There are two experiments that we want to run with this system. Firstly, the paper did not characterize the concentration of molybdate needed to enter through the sulphate system. So we want to supply e.coli and mod c knockout strain with various concentations of molybdate and measure the optical density and do a cfu assay. Secondly we want to characterize the following system. Flourescence assay for K08 and od and cfyu for kill genes. At the moment the system is still being built. Biobricks being built Moco Operonΰchlor** Ψ J13002-Mocooperon** Ψ Moaribo in chlor** Ψ Moaribo-S7** Ψ Moaribo-k082003** Ψ R0010-Moaribo-k082003** Ψ R0010-moaribo-k082003-j13002-mocooperon** Ψ Mocoribo-s7-mocoribo** Ψ Moaribo-S7-moaRibo-cviaii** Ψ R0010-moaribo-S7** Ψ R0010-mocoribo-s7-mocoribo-cviaii R0010-moaribo-k082003-j13002-mocooperon** R0010-mocoribo-s7-mocoribo-cviaii j13002-mocooperon