Team:Calgary/Project/HumanPractices/Killswitch/Regulation

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<h1>Magnesium repressible system</h1>
 
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<p>This system is repressed in the presence of magnesium. This system has two control components – a promoter and a riboswitch. Normally the magnesium promoter (mgtA promoter) and the magnesium riboswitch (mgtArb) are activated if there is a deficiency of magnesium in the cell. The lack of magnesium activates other genes in <i>E. coli </i>to allow influx of magnesium into the cell. There are two proteins in the cascade that activate the system namely PhoP and PhoQ. 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.</p>
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<h2> Tight Regulation </h2>
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<p>Inducible kill systems are not new to iGEM. Looking through the registry, there are several constructs such as the inducible BamHI system contributed by Berkley in 2007 (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_I716462">BBa_I716462</a>) and <a href="http://partsregistry.org/Image:UoflBamHIdatasheet.png">tested by Lethbridge in 2011</a>.  This uses a <i>BamHI</i> gene downsteam of an arabinose-inducible promoter. Another example is an IPTG inducible Colicin construct (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K117009">BBa_K117009</a>) submitted by NTU-Singapore in 2008.  One major problem with these systems however is a lack of tight control.  As was demonstrated by the Lethbridge 2011 team, this part has leaky expression when inducer compound is not present. The frequently used lacI promoter has similar problems when not used in conjunction with strong plasmid-mediated expression of lacI.  This can be seen in our electrochemical characterization of the UidA hydrolase enzyme (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902002">BBa_K902002</a>) shown here. Tight control is not only a problem for kill switch application, but for any application requiring strict regulation.  As such, we decided that expanding the registry repertoire of control elements would be useful for our system as well as a variety of other applications. Therefore we added a new level of regulation in addition to the promoter, a riboswitch.</p>
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<h2> Introducing the Riboswitch </h2>
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<p>Riboswitches are small pieces of mRNA which bind ligands to modify translation of downstream genes. These sites are engineered into circuits by replacing traditional ribosome binding sites with riboswitches. The riboswitch is able to bind its respective ligand to inhibit or promote binding of translational machinery (Vitreschak <i>et al</i>, 2004). Riboswitches can be used in tandem with an appropriate promoter to enable tighter control of gene expression. Given this opportunity for control, and that ligands for riboswitches are often inexpensive small ions, these methods might be a feasible solution for controlling the kill switch in our industrial bioreactor.</p>
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</html> [[File:UofC_RIBOSWITCH.png|thumb|350px|centre|Figure 1: A simple diagram illustrating the riboswitch and the three metabolite, magnesium, manganese and molybdenum, we have tested.]] <html>
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[[File:Magnesium pathway Ucalgary.png|thumb|500px|center|Figure 2: MgtA pathway in <i>E. coli</i>. The phoQ protein is the transmembrane receptor which detects low magnesium concentration. PhoQ then phosphorylates PhoP which acts as a transcription factor on mgtA promoter and transcribes genes downstream necessary for bringing magnesium into the cell. There is a second level of control with the magnesium riboswitch. In the presence of high magnesium the riboswitch forms a secondary structure which does not allow the ribosome to bind to the transcript inhibiting translation. In the case of low magnesium however, the transcript is expressed and this allows influx of magnesium.]]<html>
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<h3><i>Test circuits for the magnesium system</i></h3>
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<p>We explored 3 different riboswitches, each responsive to a different metabolite (magnesium, manganese or molybdate co-factor) that would be inexpensive to implement into a bioreactor environment.  Additionally, we also investigated a repressible and inducible promoter, responsive to glucose and rhamnose respectively.</p>
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<p>The general approach taken to build the system was constructing the promoter with the respective riboswitch followed by the kill genes. </p>
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<h2>Magnesium riboswitch</h2>
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<p>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 (mgtA) promoter (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902009">BBa_K902009</a>) and the magnesium (mgtA) riboswitch (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902008">BBa_K902009</a>) are activated if there is a deficiency of magnesium in the cell (Groisman, 2001). The sequence of the <i>mgtA</i> promoter and riboswitch was obtained from Winnie and Groisman. A lack of magnesium activates other genes in <i>E. coli </i>to allow influx of magnesium into the cell. The two proteins in the cascade that activate the system are <i>PhoP</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902010">BBa_K902010</a>) and <i>PhoQ</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902011">BBa_K902011</a>). <i>PhoQ</i> is the trans-membrane protein which gets activated in the absence of magnesium and phosphorylates <i>PhoP</i>. <i>PhoP</i> in turn binds to the mgtA promoter and transcribes genes downstream (Groisman, 2001).</p>
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<p> To test the magnesium regulatory elements we built each of the elements with a reporter gene. We chose Bba_K082003 which is GFP with an LVA tag as our choice of reporter. We did not choose BBa_E0040, the stable GFP, because we wanted a real time indication of the system's control. Stable GFP has a half life of 8 hours and would still fluoresce when the system is shut off.</p>
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<h2>Manganese riboswitch</h2>
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<p> Manganese is an essential micronutrient. It is an important co-factor for enzymes and it also reduces oxidative stress in the cell (Waters <i>et al</i>. 2011). Despite being an important micronutrient, it is toxic to cells at high levels. MntR protein 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 <i>mntR</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902030">BBa_K902030</a>) binds to the mntP promoter (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902073">BBa_K902073</a>). The manganese homeostasis is also controlled by the manganese riboswitch <i>mntPrb</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902074">BBa_K90274</a>). The sequences of the <i>mntP</i> promoter and the <i>mntP</i> riboswitch was obtained from the Waters et al, 2011.</p>
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<p>We build these circuits to test the control elements of the system, namely the <i>mgtA</i> promoter and the <i>mgtA</i> riboswitch.</p>
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[[File:MgtA circuits Ucalgary1.png|thumb|200px|left|Figure 3: In these set of circuits, <i>TetR</i>-RBS-K082003 serves as a positive control and the <i>mgtAp-mgtArb</i> serves as a negative control.]]<html>
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[[File:Ucalgary2012 KillswitchstuffsystemsAandB.png|thumb|800px|left|Figure 2: '''A)''' MgtA pathway in <i>E. coli</i>. <i>PhoQ</i> is the transmembrane receptor which, upon detecting low magnesium concentrations, phosphorylates <i>PhoP</i> which acts as a transcription factor, transcribing genes downstream of the MgtA promoter necessary for bringing magnesium into the cell. There is a second level of control with the magnesium riboswitch. In the presence of high magnesium the riboswitch forms a secondary structure which does not allow the ribosome to bind to the transcript, thus inhibiting translation. '''B)''' In the presence of manganese, the <i>MntR</i> protein represses the <i>mntH</i> transporter, preventing the movement of manganese and also upregulating the putative efflux pump. Genes downstream of the mntP promoter are thus transcribed in the presence of manganese.  The addition of the <i>MntR</i> protein in this system allows for tighter regulation of the system.]]<html>
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<h3><i>Characterization of these circuits</i></h3>
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<h2> The Moco Riboswitch </h2>
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We tested the aforementioned circuits in different concentrations of magnesium. For detailed protocol see INSERT LINK HERE. The values were normalized to the negative control which is the magnesium promoter and riboswitch alone.</p>
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<p>The molybdenum cofactor riboswitch (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902023">BBa_K902023</a>) is an RNA element which responds to the presence of the metabolite molybdenum cofactor (MOCO) (Regulski et al, 2008). This RNA element is located in the <i>E.coli</i> genome just upstream of the <i>moaABCDE</i> operon (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902024">BBa_K902024</a>), containing the MOCO synthesis genes. MOCO is an important co-factor 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 causing an allosteric change. This allosteric change affects the expression platform by physically hiding the ribosome binding site which prevents translation.</p>
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[[File:Magmesium graph ucalgary2.png|thumb|500px|center|Figure 3: This graph represents the relative flourescence units from the mgtA promoter riboswitch construct as well as the riboswitch construct under the TetR promoter (BBa_R0040). We can see a decrease in the level of GFP output with increasing concentrations of magnesium. There is much steeper decrease in the GFP output in the construct with the magnesium promoter and riboswitch compared to the construct with just the riboswitch alone.]]<html>
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<p>There is a much larger drop in the GFP output when the <i>mgtA</i> promoter and riboswitch are working together compared to the <i>mgtA</i> riboswitch under the control of TetR promoter. 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 inhibiting gene expression downstream of a constitutive promoter.</p>
 
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<p> It is important to consider however that the control elements of the system namely<i> PhoP</i> and<i> PhoQ</i> were not present in the circuits tested. We believe that would give us much better control. Although the data suggests that there is enough production of PhoP and PhoQ proteins from the genome to control expression of the genes downstream of a high copy plasmid.</p>
 
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<p> Although the magnesium system is a brilliant system which 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- upto 30mM(REFERENCE). As can be seen from figure 3, 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. </p>
 
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<p> In contrast, it is important to note that this system adds important regulatory elements to the registry such as an inducible promoter and a riboswitch which can be used by other teams to control both killswitches as well as other regulatory pathways which do not pertain using tailings. </p>
 
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<p> We also wanted to test the magnesium system with our kill genes. The micrococcal nuclease that arrived from IDT had 1bp mutation which changed a isoleucine residue into a lysine. Therefore we had to mutate it. We strategized it such that we built our circuits with S7 first and then mutated the S7. We plated the mutated plasmid on plates with magnesium and without magnesium.</p>
 
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[[File:S7 mutagenesis for the magnesium system ucalgary.png|thumb|400px|center|Figure : Plating of S7 mutagenesis on plates with both magnesium and without magnesium. The plate with magnesium has more colonies compared to the plates without magensium.]]<html>
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[[File:Moco_riboswitchCalgary2012.jpg|thumb|750px|center|Figure 3: This picture depicts the MOCO RNA motif which is upstream of the <i>moaABCDE</i> operon. ]]<html>  
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<p>There are less colonies on the plates without magnesium which indicates that the mutagenesis was successful as well as the fact that the S7 is sufficiently controlled by Mg2+ thus allowing colony growth on Mg2+ treated plates.</p>
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<h1>Manganese regulation</h1>
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<h2> Building the Systems </h2>
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<p> Manganese is found to be an essential nutrient in most bacteria and larger organisms. Due to the properties of manganese this metal can work as a catalyst for many chemical reactions in the body of organisms as well as playing a key role in the structure of macromolecules. Therefore it is not surprising to believe that the cells will need a way to regulate the amount of manganese in its system. Native to the Escherichia coli K-12 strain is the mntR metalloregulatory protein. This transcriptional regulator is found to observe the level of manganese and responds accordingly. The structure of this protein consists of a metal-binding domain that will respond in the presence of manganese and repress the small RNA gene mntS. Lack of mntS will then repress the manganese ion transporter (mntH) as 42-amino-acid protein forms. When this protein membrane is repress the bacteria cannot gained the needed manganese. It is also found that the mntR positively regulates the mntP a putative efflux pump that regulates the intracellular level of manganese.
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<p> Using these riboswitches, we wanted to 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 <i>mgtaP</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902009">BBa_K902009</a>) and mntP promoter (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902073">BBa_K902073</a>) as well as their respective riboswitches (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902008">BBa_K902008</a>) (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902074">BBa_K902074</a>) and the MOCO riboswitch (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902023">BBa_K902023</a>).  In addition, we also biobricked some of the regulatory proteins: <i>PhoP</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902010">BBa_K902010</a>), <i>PhoQ</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902011">BBa_K902011</a>), <i>mntR</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902030">BBa_K902030</a>) and the Moa Operon (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902024">BBa_K902024</a>). 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 4. </p>
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</html>[[File:U.Calgary.2012_09.28.2012_Membrane_Final.png|thumb|500px|center|Figure 4: The manganese system in the presence of manganese found in the tailing ponds will initially trigger the mntR regulator. As one of its function, the regulator will repress the mntH transporter preventing the movement of manganese. As a second function, the mntR will upregulate the putative efflux pump. The manganese system itself responds to the manganese metal allowing the transcription of the gene downstream. The addition of the mntR in this system is generally used to better regulate the manganese system.]]<html>
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</html>[[File:U.Calgary.2012_10.02.2012_Final_Construct_1.png|thumb|600px|center|Figure 4: Final construct for the manganese system. The circuit includes a TetR promoter, RBS, mntR, double terminator, mntP promoter, mntP riboswitch, <i>S7</i>, mntP riboswitch and <i>CViAII</i>.]]<html>
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<h3><i>Our manganese system</i></h3>
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<a name="killswitch"></a><h2> Characterizing the riboswitches </h2>
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<p>For our system we use the mntR, mntP promoter and mntP riboswitch. As shown in figure _ our system in the present of high Mn2+ will activate the mntR which upregulates the mntP promoter. The mntP promoter in the presence of manganese then initiates the transcription of mntP riboswitch. Likewise in the presence of manganese the mntP riboswitch will express the genes downstream of it or in our case the kill genes S7 and CViAII. In order to control this system in our bioreactor there will have to be low concentrations of Mn2+ in the structure, however when the exposed to the environment or tailing ponds there has to be fairly high concentrations of Mn2+. </p>
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<h3> GFP testing</h3>
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<h3><i>Test circuits for the manganese system</i></h3>
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</html>[[File:MgtA circuits Ucalgary1.png|thumb|150px|right|Figure 5: In these sets of circuits, <i>TetR</i>-RBS-K082003 serves as a positive control and the <i>mgtAp-mgtArb</i> serves as a negative control.]]<html>
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<p>The manganese will use a GFP LVA tag.  The following are the control circuits built in order to characterise the MntA promoter and the MntA riboswitch.</p>
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<p> In order to test the control of these promoters and riboswitches, we constructed them independently and together upstream of GFP (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K082003">BBa_K082003</a>) with an LVA tag.  Figure 5 shows these circuits for the mgtA system.  Identical circuits were designed for all three systems, however only the top two were needed for the MOCO riboswitch system.</p>
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</html>[[File:U.Calgary.2012_09.28.2012_Construct_2.png|thumb|500px|center|Figure 5: These set of circuits are used to characterise both the mntP promoter and the mntP riboswitch. The negative control composes of mntP promoter-mntP riboswitch while the positive control is the circuit TetR-RBS-K08200.]]<html>
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<p>We then tested the aforementioned circuits by growing cells containing our circuits with varying concentrations of their respective ions.  Our detailed protocols can be found <a href="http://2012.igem.org/Team:Calgary/Notebook/Protocols/mgcircuit">here</a>. We then measured fluorescent output, normalizing to a negative control not expressing GFP.</p>
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<h3><i>Future Use</i></h3>
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<h3> Results </h3>
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<p>Even though this system is a relatively good way to regulate our kill genes, there is some limitation to this system. The main problem why this circuit will not work for our system is because when tailing pond water is added to the bioreactor there is fairly high concentration of Mn2+ in the contaminated water ~48mg/L or ~170µM/L. This concentration is 17 times the amount of Mn2+ needed to trigger the system (10 µM) therefore an additional chemical such as EDTA (a chelator) will have to be added to lower the manganese levels in the bioreactor. This however brings up another situation since EDTA is fairly expensive and will have to be constantly supplied to the bioreactor. Although this system may not be feasible for our system, this regulator system may be used in another pathway.</p>
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<p>So far, we have been able to obtain results for our magnesium system, as can be seen in Figure 6. </html>
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[[File:Magmesium graph ucalgary2.png|thumb|500px|left|Figure 6: This graph represents the relative fluorescence units from the mgtA promoter riboswitch construct as well as the riboswitch construct under the TetR promoter (BBa_R0040). We can see a decrease in the level of GFP output with increasing concentrations of magnesium. There is much steeper decrease in the GFP output in the construct with the magnesium promoter and riboswitch compared to the construct with just the riboswitch alone.]]<html></p>
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<h1>Glucose repressible system</h1>
 
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<h2>Background</h2>
 
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<p>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.</p>  
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<p>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 <i>mgtA</i> riboswitch alone under the control of TetR promoter (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_J13002">BBa_J13002</a>). This suggests that having both the promoter and the riboswitch together provides a tighter control over the genes expressed downstream. This also suggests that the magnesium riboswitch alone is sufficient in reducing gene expression downstream of a constitutive promoter.</p>
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<p>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.</p>
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<p> It is important to consider however that the control elements of the system, <a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902010"><i>PhoP</i> </a> and <a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902011"> <i>PhoQ</i></a>, that were described above were not present in the circuits tested and therefore there is GFP expression in at the inhibitory concentration (10 mM MgCl<sub>2</sub>). We believe that having the regulatory elements would give us better control and limit the leakiness.</p>
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<p>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.</p>
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<p>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, as high as 120 mM (Kim <i>et al</i>. 2011). As can be seen, this would inhibit the system. Therefore, if our bacteria were to escape into the tailings, the kill genes would not be activated and the bacteria would be able to survive.  However, we feel that this could still be an incredibly useful system for other teams for both killswitch and non-killswitch-related applications, making it still a valuable contribution to the registry. </p>
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<p></p>
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<h3> Kill Gene Testing </h3>
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<h2>Native function of the rhamnose promoter</h2>
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<p>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.</p>
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<p> While building our systems with GFP in order to test their control, we also constructed 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 point mutation which changed an isoleucine residue into a lysine.  Initially, our systems resulted in no killing of cells. Therefore we had to mutate this residue using <a href="http://2012.igem.org/Team:Calgary/Notebook/Protocols/mutagenesis"> site-directed mutagenesis</a>. 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 <i>S7</i> kill gene (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902019">BBa_K902019</a>). To test the circuits, we incubated cells expressing our construct with varying concentrations of magnesiumWe then measured both Colony Forming Units (CFU) and OD 600. For a detailed protocol, see <a href="http://2012.igem.org/Team:Calgary/Notebook/Protocols/mgtacircuit">here</a>.</p>
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<p>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).</p>
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<h3> Results </h3>
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<p>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)).</p>
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</html>[[File: 24 hour assay with mgtap-rb-S7-1.png|thumb|750px|center| Figure 7: This shows the OD600 values of mgtA circuits with S7 both mutated and unmutated. The negative control consists of <i>mgtAp-mgtArb</i>.]]<html>
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<p> Figure 7 shows that the mgtAp-mgtArb-S7 (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902018">BBa_K902018</a>) starts acting approximately 4 hours after induction. However, it also shows that 10mM MgCl<sub>2</sub> is not enough salt to inhibit the entire system because there is no difference in OD600 measurement at 4hr time point between 10mM and the 0mM concentrations. This test needs to be repeated with higher concentrations of Mg<sup>2+</sup> however this data suggests that the mutagenesis was successful and <i>S7</i> is active and killing the cells at approximately 4hr which does not necessarily reflect solely upon the activity of <i>S7</i> but also on the response time of the mgtA system.</p>
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<p></p>
 
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<h2>Engineering pRha into a kill system</h2>
 
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<p>Our team has engineered the following rhamnose promoter kill system:</p>
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<h2>An alternative: a glucose repressible system</h2>
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<p>(Draw the final kill circuit)</p>
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<p>Based on the problem with the magnesium system in relation to tailings pond conditions, we wanted to find an alternative. We found a promoter that was induced by rhamnose and repressed by glucose.  This seemed to be a very suitable candidate for controlling the kill switch in the bioreactor since the promoter was shown to be tightly repressed by glucose. We could supplement the bioreactor with glucose to inhibit expression of the kill genes in the bioreactor. Escape of bacteria into the tailings ponds would cause expression of the kill genes due to lack of glucose in the surrounding environment.
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</p>
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<p>This promoter, known as <i>pRha</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902065">BBa_K902065</a>), is responsible for regulating genes related to rhamnose metabolism and contains a separate promoter on its leading and reverse strands (see Figure 8). <i>RhaR</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902069">BBa_K902069</a>)  and <i>RhaS</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902068">BBa_K902068</a>) serve to regulate expression of the rhamnose metabolism operon <i>rhaBAD</i>. The <i>RhaR</i> transcription factor is activated by L-rhamnose to up-regulate expression <i>rhaSR</i> operon. In turn, the resulting <i>RhaS</i> activates the <i>rhaBAD</i> operon to generate the rhamnose metabolism genes (Egan & Schleif, 1993).</p>
-
<p>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.</p>
+
</html>[[File:NativeRhamnosePromoter_Calgary2012.jpg|thumb|600px|center|Figure 8: The rhamnose metabolism genes as they exist in Top Ten <i>E. coli</i>]]
 +
<html>
-
<p>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.</p>
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</html>[[File:PrhaFinal.png|thumb|600px|center|Figure 9: The rhamnose metabolism genes native to <i>E. coli</i>]]
 +
<html>
 +
<p>Our kill system is different from the native rhamnose system with the <i>rhaR</i> and <i>rhaS</i> control genes. We have constitutively expressed <i>RhaS</i> to overcome dependency on rhamnose to cause activation of the kill switch. While <i>RhaS</i> is continuously present, the system is shut off in the presence of glucose. However, in the outside environment glucose levels are lower such that <i>RhaS</i> is able to activate the kill genes.</p>
-
<p>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.</p>
+
<a name="Prha_results"></a>
-
<p>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).</p>
+
<h3>Building the system</h3>
 +
<p>Our team had <i>pRha</i> promoter (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902065">BBa_K902065</a>) commercially synthesized as per the sequence given by Jeske and Altenbuchner (2010). The <i>rhaS</i>  (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902068">BBa_K902068</a>) and <i>rhaR</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902069">BBa_K902069</a>)  genes were amplified via PCR from Top 10 <i>E. coli</i> using Kapa HiFi polymerase. </p>
 +
<p>We tested the unoptimized rhamnose system using a fluorescent output. </html> [[File:Calgary_RhaGFPFinal.png|thumb|600px|centre|Figure 10: This has Prha-RBS-GFP that was incubated under different conditions. We can see a large increase with 0.2% rhamnose, 0.5% rhamnose whereas there is no GFP expression in the cells incubated with glucose.]] <html> </p>
-
<p>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.</p>
+
<p> Figure 10 shows that the rhamnose system works as expected. The system is turned off with 0.2% glucose whereas GFP is significantly upregulated with 0.2% rhamnose and even more with 0.5% rhamnose. In this system we do not have the RhaS constitutively expressed and therefore GFP may not be expressed in the the control without either glucose or rhamnose. But, we are currently working on building this circuit and will be characterizing the RhaS with Prha and Prha by itself using GFP as a reporter.
-
<p>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.</p>
 
-
 
-
<p>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.</p>
 
-
 
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<p></p>
 
-
<h2>Assembly methods</h2>
 
-
 
-
<p>For the construction of this system, our team had pRha commercially synthesized as it was described in Jeske and Altenbuchner (2010). SHOW PIC MAYBE</p>
 
-
 
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<p>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)</p>
 
-
 
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<p></p>
 
-
<h2>Characterization</h2>
 
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<p>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.</p>
 
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<h1>The Moco Riboswitch - In progess</h1>
 
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<h2>Background</h2>
 
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<p>
 
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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.
 
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</p>
</p>
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</html>
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<p>Additionally, we also tested the rhamnose system with micrococcal nuclease in the presence of glucose and rhamnose in both Top10 cells as well as glyA knockout from the Keio knockout collection on the <a href="http://2012.igem.org/Team:Calgary/Project/Synergy">Synergy Page</a> to compare the GlyA knockout alone, GlyA knockout with killswitch, Top10 with killswitch. </p>
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[[File:Moco_riboswitchCalgary2012.jpg|thumb|500px|center|Figure (#): This picture depicts the Moco RNA motif upstream of the moaABCDE operon ]]<html>  
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<h2>Molybdate in TPW</h2>
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<h2>The Killswitch Design</h2>
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<p>
<p>
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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.
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<h2> The Glycine Auxotroph </h2>
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<p> The idea of using an auxotropic system was initially considered, however due to the pricing of this system we felt it to be inappropriate for a large scale bioreactor. Auxotrophic systems that we had looked into included the 5-fluoro-orotic acid and histidine, which were both found to be expensive. This idea was reconsidered when our <a href="http://2012.igem.org/Team:Calgary/Project/OSCAR/FluxAnalysis">Flux Variability Analysis</a> showed that the Petrobrick system can be optimized with glycine added to the media. The production of hydrocarbons increased by a factor of 3 with our glycine media when compared to Washington’s production media. This finding justified our introduction of a glycine auxotrophic system as the increased efficiency of the Petrobrick in addition to another safety feature far outweighed the cost of implementing the system. This is feasible because glycine is not readily found in the environment and is relatively inexpensive to supplement on a large scale. </p> <p> We used a knockout strain JW2535-1 from the Keio collection in which the gene responsible for the synthesis of glycine was knocked out. The bacteria become dependent on glycine in the environment. The JW2535-1 knockout strain used works directly on glyA which is a component of the glycine hydroxymethyltransferase by mutating the glyA into Kan which overall prevents the bacteria’s growth. A glycine assay was set up to determine concentrations of glycine needed for the survival of the bacteria. The bacteria were grown on minimal media plate with glycine concentrations ranging from 1 nM to 100 mM. When zero glycine was added to the media there was some bacterial growth over time. This system will therefore need to work in conjunction with the kill switch system as another layer of security to reduce possibility of escapers. Please see our <a href="http://2012.igem.org/Team:Calgary/Project/Synergy">Synergy Page</a> for more information. </p>
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</p>
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</html>
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[[File:RaioperonCalgary2012.png|thumb|500px|center|Figure #: Moa Operon]]<html>
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<p>
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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. </p>
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<p>
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Concentrations of Mo in the TPW:
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• [Mo] in the Syncrude and Suncor TPW  in 1990:
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– 0.183 mg/L  in the surface region (1m-10m)
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– 0.045 mg/L  in the sludge region (11m-20m)
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</p>
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<p>
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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. </p>
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<h2>The Solution </h2>
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<p>
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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. </p>
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<h2>Characterization</h2>
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<p>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. </p>
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<p> Biobricks being built: </p>
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[[File:Raistuff1Calgary2012.png|thumb|500px|center]]<html>
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[[File:Raiconstructs2Calgary2012.png|thumb|500px|center]]<html>
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Latest revision as of 04:23, 17 December 2012

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Regulation/Expression Platform

Tight Regulation

Inducible kill systems are not new to iGEM. Looking through the registry, there are several constructs such as the inducible BamHI system contributed by Berkley in 2007 (BBa_I716462) and tested by Lethbridge in 2011. This uses a BamHI gene downsteam of an arabinose-inducible promoter. Another example is an IPTG inducible Colicin construct (BBa_K117009) submitted by NTU-Singapore in 2008. One major problem with these systems however is a lack of tight control. As was demonstrated by the Lethbridge 2011 team, this part has leaky expression when inducer compound is not present. The frequently used lacI promoter has similar problems when not used in conjunction with strong plasmid-mediated expression of lacI. This can be seen in our electrochemical characterization of the UidA hydrolase enzyme (BBa_K902002) shown here. Tight control is not only a problem for kill switch application, but for any application requiring strict regulation. As such, we decided that expanding the registry repertoire of control elements would be useful for our system as well as a variety of other applications. Therefore we added a new level of regulation in addition to the promoter, a riboswitch.

Introducing the Riboswitch

Riboswitches are small pieces of mRNA which bind ligands to modify translation of downstream genes. These sites are engineered into circuits by replacing traditional ribosome binding sites with riboswitches. The riboswitch is able to bind its respective ligand to inhibit or promote binding of translational machinery (Vitreschak et al, 2004). Riboswitches can be used in tandem with an appropriate promoter to enable tighter control of gene expression. Given this opportunity for control, and that ligands for riboswitches are often inexpensive small ions, these methods might be a feasible solution for controlling the kill switch in our industrial bioreactor.

Figure 1: A simple diagram illustrating the riboswitch and the three metabolite, magnesium, manganese and molybdenum, we have tested.

We explored 3 different riboswitches, each responsive to a different metabolite (magnesium, manganese or molybdate co-factor) that would be inexpensive to implement into a bioreactor environment. Additionally, we also investigated a repressible and inducible promoter, responsive to glucose and rhamnose respectively.

The general approach taken to build the system was constructing the promoter with the respective riboswitch followed by the kill genes.

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 (mgtA) promoter (BBa_K902009) and the magnesium (mgtA) riboswitch (BBa_K902009) are activated if there is a deficiency of magnesium in the cell (Groisman, 2001). The sequence of the mgtA promoter and riboswitch was obtained from Winnie and Groisman. A lack of magnesium activates other genes in E. coli to allow influx of magnesium into the cell. The two proteins in the cascade that activate the system are 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 (Groisman, 2001).

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 protein 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 (BBa_K902073). The manganese homeostasis is also controlled by the manganese riboswitch mntPrb (BBa_K90274). The sequences of the mntP promoter and the mntP riboswitch was obtained from the Waters et al, 2011.

Figure 2: A) MgtA pathway in E. coli. PhoQ is the transmembrane receptor which, upon detecting low magnesium concentrations, phosphorylates PhoP which acts as a transcription factor, transcribing genes downstream of the MgtA promoter necessary for bringing magnesium into the cell. There is a second level of control with the magnesium riboswitch. In the presence of high magnesium the riboswitch forms a secondary structure which does not allow the ribosome to bind to the transcript, thus inhibiting translation. B) In the presence of manganese, the MntR protein represses the mntH transporter, preventing the movement of manganese and also upregulating the putative efflux pump. Genes downstream of the mntP promoter are thus transcribed in the presence of manganese. The addition of the MntR protein in this system allows for tighter regulation of the system.

The Moco Riboswitch

The molybdenum cofactor riboswitch (BBa_K902023) is an RNA element which responds to the presence of the metabolite molybdenum cofactor (MOCO) (Regulski et al, 2008). This RNA element is located in the E.coli genome just upstream of the moaABCDE operon (BBa_K902024), containing the MOCO synthesis genes. MOCO is an important co-factor 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 causing an allosteric change. This allosteric change affects the expression platform by physically hiding the ribosome binding site which prevents translation.

Figure 3: This picture depicts the MOCO RNA motif which is upstream of the moaABCDE operon.

Building the Systems

Using these riboswitches, we wanted to 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 promoter (BBa_K902073) as well as their respective riboswitches (BBa_K902008) (BBa_K902074) and the MOCO riboswitch (BBa_K902023). 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 4.

Figure 4: Final construct for the manganese system. The circuit includes a TetR promoter, RBS, mntR, double terminator, mntP promoter, mntP riboswitch, S7, mntP riboswitch and CViAII.

Characterizing the riboswitches

GFP testing

Figure 5: In these sets of circuits, TetR-RBS-K082003 serves as a positive control and the mgtAp-mgtArb serves as a negative control.

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 5 shows these circuits for the mgtA system. Identical circuits were designed for all three systems, however only the top two were needed for the MOCO riboswitch system.

We then tested the aforementioned circuits by growing cells containing our circuits with varying 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.

Results

So far, we have been able to obtain results for our magnesium system, as can be seen in Figure 6.

Figure 6: This graph represents the relative fluorescence units from the mgtA promoter riboswitch construct as well as the riboswitch construct under the TetR promoter (BBa_R0040). We can see a decrease in the level of GFP output with increasing concentrations of magnesium. There is much steeper decrease in the GFP output in the construct with the magnesium promoter and riboswitch compared to the construct with just the riboswitch alone.




























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 the magnesium riboswitch alone is sufficient in reducing gene expression downstream of a constitutive promoter.

It is important to consider however that the control elements of the system, PhoP and PhoQ, that were described above were not present in the circuits tested and therefore there is GFP expression in at the inhibitory concentration (10 mM MgCl2). We believe that having the regulatory elements would give us better control and limit the leakiness.

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, as high as 120 mM (Kim et al. 2011). As can be seen, this would inhibit the system. Therefore, if our bacteria were to escape into the tailings, the kill genes would not be activated and the bacteria would be able to survive. However, we feel that this could still be an incredibly useful system for other teams for both killswitch and non-killswitch-related applications, making it still a valuable contribution to the registry.

Kill Gene Testing

While building our systems with GFP in order to test their control, we also constructed 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 point mutation which changed an isoleucine residue into a lysine. Initially, our systems resulted in no killing of cells. Therefore we had to mutate this residue 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 kill gene (BBa_K902019). To test the circuits, we incubated cells expressing our construct with varying concentrations of magnesium. We then measured both Colony Forming Units (CFU) and OD 600. For a detailed protocol, see here.

Results

Figure 7: This shows the OD600 values of mgtA circuits with S7 both mutated and unmutated. The negative control consists of mgtAp-mgtArb.

Figure 7 shows that the mgtAp-mgtArb-S7 (BBa_K902018) starts acting approximately 4 hours after induction. However, it also shows that 10mM MgCl2 is not enough salt to inhibit the entire system because there is no difference in OD600 measurement at 4hr time point between 10mM and the 0mM concentrations. This test needs to be repeated with higher concentrations of Mg2+ however this data suggests that the mutagenesis was successful and S7 is active and killing the cells at approximately 4hr which does not necessarily reflect solely upon the activity of S7 but also on the response time of the mgtA system.

An alternative: a glucose repressible system

Based on the problem with the magnesium system in relation to tailings pond conditions, we wanted to find an alternative. We found a promoter that was induced by rhamnose and repressed by glucose. This seemed to be a very suitable candidate for controlling the kill switch in the bioreactor since the promoter was shown to be tightly repressed by glucose. We could supplement the bioreactor with glucose to inhibit expression of the kill genes in the bioreactor. Escape of bacteria into the tailings ponds would cause expression of the kill genes due to lack of glucose in the surrounding environment.

This promoter, known as pRha (BBa_K902065), is responsible for regulating genes related to rhamnose metabolism and contains a separate promoter on its leading and reverse strands (see Figure 8). RhaR (BBa_K902069) and RhaS (BBa_K902068) serve to regulate expression of the rhamnose metabolism operon rhaBAD. 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 8: The rhamnose metabolism genes as they exist in Top Ten E. coli
Figure 9: The rhamnose metabolism genes native to E. coli

Our kill system is different from the native rhamnose system with the rhaR and rhaS control genes. We have constitutively expressed RhaS to overcome dependency on rhamnose to cause activation of the kill switch. While RhaS is continuously present, the system is shut off in the presence of glucose. However, 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 10 E. coli using Kapa HiFi polymerase.

We tested the unoptimized rhamnose system using a fluorescent output.

Figure 10: This has Prha-RBS-GFP that was incubated under different conditions. We can see a large increase with 0.2% rhamnose, 0.5% rhamnose whereas there is no GFP expression in the cells incubated with glucose.

Figure 10 shows that the rhamnose system works as expected. The system is turned off with 0.2% glucose whereas GFP is significantly upregulated with 0.2% rhamnose and even more with 0.5% rhamnose. In this system we do not have the RhaS constitutively expressed and therefore GFP may not be expressed in the the control without either glucose or rhamnose. But, we are currently working on building this circuit and will be characterizing the RhaS with Prha and Prha by itself using GFP as a reporter.

Additionally, we also tested the rhamnose system with micrococcal nuclease in the presence of glucose and rhamnose in both Top10 cells as well as glyA knockout from the Keio knockout collection on the Synergy Page to compare the GlyA knockout alone, GlyA knockout with killswitch, Top10 with killswitch.

The Glycine Auxotroph

The idea of using an auxotropic system was initially considered, however due to the pricing of this system we felt it to be inappropriate for a large scale bioreactor. Auxotrophic systems that we had looked into included the 5-fluoro-orotic acid and histidine, which were both found to be expensive. This idea was reconsidered when our Flux Variability Analysis showed that the Petrobrick system can be optimized with glycine added to the media. The production of hydrocarbons increased by a factor of 3 with our glycine media when compared to Washington’s production media. This finding justified our introduction of a glycine auxotrophic system as the increased efficiency of the Petrobrick in addition to another safety feature far outweighed the cost of implementing the system. This is feasible because glycine is not readily found in the environment and is relatively inexpensive to supplement on a large scale.

We used a knockout strain JW2535-1 from the Keio collection in which the gene responsible for the synthesis of glycine was knocked out. The bacteria become dependent on glycine in the environment. The JW2535-1 knockout strain used works directly on glyA which is a component of the glycine hydroxymethyltransferase by mutating the glyA into Kan which overall prevents the bacteria’s growth. A glycine assay was set up to determine concentrations of glycine needed for the survival of the bacteria. The bacteria were grown on minimal media plate with glycine concentrations ranging from 1 nM to 100 mM. When zero glycine was added to the media there was some bacterial growth over time. This system will therefore need to work in conjunction with the kill switch system as another layer of security to reduce possibility of escapers. Please see our Synergy Page for more information.