Team:Calgary/Project/HumanPractices/Killswitch

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<h2>Purpose:</h2>
<h2>Purpose:</h2>
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<p> Synthetic biology entails designing an organism to do a specific task. This involves genetic manipulation of the bugs and requires scientists to provide the bacteria with a selective advantage such as an antibiotic cassette which forces the bacteria to keep the gene of interest inside the cell. With such manipulation comes a valid “risk of accidental release” (Tucker and Zilinkas, 2006). Attempts have been made to address the concern regarding "accidental release". Some of these attempts include designing of lab strains, designing auxotrophes which cannot synthesize an important metabolite and designing killswitches. In order to contain our bug, we have designed a bioreactor which will have several in built safety mechanisms. Some of the methods of containment include creating a closed system for the bioreactor which minimizes the escape of bacteria. Additionally, we will also be treating the belt-skimmer with ultra-violet light which will ensure that there are no bacteria in the final product.</p>
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<p>The OSCAR component of our project aims to remediate toxins in the oil sands tailings ponds using synthetic bacteria. As we recognize in our project's safety practices(LINK), remediating tailings ponds creates the risk for our bacteria to escape into the environment, given the high volume of tailings water being circulated through our system. Although no evidence suggests that the OSCAR bacteria would proliferate or cause harm outside the bioreactor, industry leaders have voiced concerns to our team about synthetic biology's potential for environmental issues—safety is different in synthetic biology from more traditional engineering disciplines because bacteria are self-replicating entities. Thus, we cannot predict all possible consequences of OSCAR escaping the bioreactor.</p>
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<p>To counter potential safety issues, we engineered a genetic-based containment mechanism into our bacteria. Our kill switch system is designed to destruct the organism's synthetic genes upon escape from the bioreactor, thereby lessening the possibility of OSCAR spreading beyond the bioreactor or horizontally transferring genes to other organisms. We hope that our proactive approach to safety in OSCAR will promote adoption of our synthetic biology system for remediation of tailings ponds in the conventional oil sands industry.</p>
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<h2>Our System:</h2>
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<p>Two basic elements comprise our active kill system: firstly, two nucleases are used to degrade the genome; and secondly, a regulatory platform is used to control these two kill genes. The  nuclease enzymes ensure degradation of synthetic genes upon kill switch activation. Such a nuclease mechanism is superior to widely used lysis-based techniques which leave genetic material intact, allowing its release into the environment and potential horizontal gene transfer into other organisms. Our team submitted into the registry two novel kill enzymes (LINK). These are noteworthy because they are optimized for temperature conditions typical of tailing ponds. Furthermore, they cause finer degradation of the bacterial genome than existing nuclease mechanisms such as (SHOW ME ONE).</p>
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<p>In the rare instance that the bacteria escapes, we have designed a killswitch such that the bacteria is only able to survive in specific environments allowing them to perform the tasks of decarboxylation, denitrification and desulfurization in our bioreactor. However, in case of these bacteria escaping, the lack of a metabolite and or the presence of a particular metabolite will activate the “kill genes” which will cause the bacteria to self destruct. The killswitch mechanism was put in our system as a safety measure in addition to the bioreactor to contain the synthetic bacteria.</p>
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<p>Of course, introducing nucleases is insufficient to control the spread of our organism and its genes. Our system also required a means of regulating the expression of these nucleases, thereby allowing the organism to function as intended while within the bioreactor environment. To fulfill this need, we developed four novel regulatory elements for registry submission, significant because they are tightly regulated—these elements reduce inadvertent expression of the kill genes compared to existing regulatory mechanisms. Three are of particular interest because they are riboswitches that regulate translation at the post-transcriptional stage of gene expression. Of the four we developed, two proved suitable for tailings pond use.</p>
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<h2>History: </h2> <p>Scientists have been trying to develop methods to limit bacterial viability and growth outside of the lab environment. One of the most popular methods used to ensure the safety of bacteria used in the lab was the creation of lab strain bacteria such as DH5α and Top10. These bacteria are metabolically deficient and are unable to survive outside of the lab environment without very specific nutrients. Additionally, The Registry of Biological Parts also has several killswitches readily available that were submitted by previous iGEM teams. </p>
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<p>The different types of killswitches include:</p>
 
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<h3><i>Toxin-antitoxin systems: </i></h3><p>These systems usually insert antitoxin in the plasmid and toxin in the genome. Ideally if the bacteria lost the plasmid then the bacteria dies. The advantages of these types of system is that__________ and the caveat with these systems is that they do not prevent the bacteria from horizontally transferring the genetic material. </p>
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<h2>Justification of our approach</h2>
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<p>An apparent weakness of our system lies in the potential for the kill switch mechanism to mutate, rendering it ineffective and allowing the synthetic organism or its genetic material to escape into the environment. We have, however, taken several approaches to mitigate this risk. Firstly, we engineered redundancy into our system—with two kill genes, both would have to be rendered inoperable for the kill switch mechanism to malfunction. Knudsen et al. (1995) proposed that active kill switches containing a single kill element were subject to a mutation rate of 10^-6 per cell per generation, but that a second redundant kill gene reduces this value by two orders of magnitude. Secondly, the kill switch mechanism is, of course, only a failsafe measure for controlling our organism's spread. The primary means of preventing its escape is through the multiple layers of mechanical security provided by our bioreactor (LINK TO SAFETY). Only when these measures fail will the kill switch be required to function.</p>
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<h3><i>Auxotrophic marker</i></h3><p> Auxotrophes are bugs that are unable to survive in the absence of a metabolite. These bugs are used widely in the lab. An auxotrophe is unable to synthesize an essential metabolite, often an amino acid. Therefore, it requires the presence of the said metabolite in order to survive. Often these amino acids are unavailable in the environment. Therefore, these bugs are unable to survive outside the laboratory environment. The limitations of using an auxotrophe for the purpose of our bioreactor is the cost associated with it. Given the size of our envisioned bioreactor, 100L+, auxotrophes would add a large cost to the system.</p>  
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<h3><i>Inducible systems</i></h3><p>Inducible systems generally consist of a regulatory element such as a promoter which is activated in the presence or absence of a metabolite. There is several kill genes inducible kill genes in the registry. Some of them include BamHI under the control of AraC promoter. The literature uses LacI promoter and the LacUV promoter as control elements. Some of the limitations of using an inducible system are the escaper bacteria mutating out either the kill gene or the regulatory element associated with the kill gene such as the promoter thereby blocking the expression of the kill gene. In order to combat this, researchers often create plasmids with multiple copies of the kill systems. This reduces the chances of mutation and also provides backup copies in case one of the promoters is mutated. Knudsen and Karlstorm suggest the use of a tightly controlled promoter to reduce the chances of mutation. </p>
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<p>At first blush, an auxotrophic bacterial strain would seem a superior choice for our application. Such a strain would have a critical metabolic gene knocked out, requiring the organism be supplemented externally with an intermediate metabolite. As a mutation restoring the metabolite would be sufficiently complex as to be rendered improbable, an auxotrophic mechanism seems ideal. This system, however, is impractical due to its cost. Metabolic supplements are considerably more expensive than the glucose and metal ions that our system requires. As we learned in our discussions with industry leaders, cost is a paramount factor in permitting adoption at industrial scales, and so our system has a greater likelihood of being implemented outside the laboratory. More worrisome is that an auxotrophic control mechanism would kill the organism without degrading its genetic material, thereby making possible horizontal gene transfer to other organisms. As the overarching goal of our kill switch is not to kill the organism but to prevent the escape of its engineered genetic elements, our kill switch design is superior to one relying on auxotrophy.</p>
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<h2>Design considerations:</h2>
 
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<p>In our design we had considered all three of the possibilities however considering the large increase in cost in the bioreactor if auxotrophic systems were used, we decided to explore different inducible systems.  We considered using the AraC promoter (Bba_I0500) as well as the LacI promoter (Bba_R0010) with our kill genes. However, data suggests that both AraC as well as LacI promoters are both leaky. PUT DIAGRAM. Therefore, we explored four inducible systems which are new to the registry and are induced by inexpensive ligands such as magnesium, manganese, molybate salts and glucose. In order to make sure the systems are controlled well and the kill switch regulation is not leaky, we have added an additional control using the riboswitch.</p></html>[[File:Riboswitch-Ucalgary.png|thumb|200px|right|Figure X: this diagram suggest that in the presence of the <b>aptamer</b>, the ligand which binds to the riboswitch, the mRNA cannot be translated thereby reducing the level of protein in the cell. ]]<html>
 
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<p>A <b>riboswitch </b>provides post-transcriptional control of gene expression. A riboswitch is a small stretch of mRNA which binds to a ligand which increases or decreases the expression of the gene downstream. </p>
 
<h2>Our Kill Genes</h2>
<h2>Our Kill Genes</h2>

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A Killswitch for Increased Security

Purpose:

The OSCAR component of our project aims to remediate toxins in the oil sands tailings ponds using synthetic bacteria. As we recognize in our project's safety practices(LINK), remediating tailings ponds creates the risk for our bacteria to escape into the environment, given the high volume of tailings water being circulated through our system. Although no evidence suggests that the OSCAR bacteria would proliferate or cause harm outside the bioreactor, industry leaders have voiced concerns to our team about synthetic biology's potential for environmental issues—safety is different in synthetic biology from more traditional engineering disciplines because bacteria are self-replicating entities. Thus, we cannot predict all possible consequences of OSCAR escaping the bioreactor.

To counter potential safety issues, we engineered a genetic-based containment mechanism into our bacteria. Our kill switch system is designed to destruct the organism's synthetic genes upon escape from the bioreactor, thereby lessening the possibility of OSCAR spreading beyond the bioreactor or horizontally transferring genes to other organisms. We hope that our proactive approach to safety in OSCAR will promote adoption of our synthetic biology system for remediation of tailings ponds in the conventional oil sands industry.

Our System:

Two basic elements comprise our active kill system: firstly, two nucleases are used to degrade the genome; and secondly, a regulatory platform is used to control these two kill genes. The nuclease enzymes ensure degradation of synthetic genes upon kill switch activation. Such a nuclease mechanism is superior to widely used lysis-based techniques which leave genetic material intact, allowing its release into the environment and potential horizontal gene transfer into other organisms. Our team submitted into the registry two novel kill enzymes (LINK). These are noteworthy because they are optimized for temperature conditions typical of tailing ponds. Furthermore, they cause finer degradation of the bacterial genome than existing nuclease mechanisms such as (SHOW ME ONE).

Of course, introducing nucleases is insufficient to control the spread of our organism and its genes. Our system also required a means of regulating the expression of these nucleases, thereby allowing the organism to function as intended while within the bioreactor environment. To fulfill this need, we developed four novel regulatory elements for registry submission, significant because they are tightly regulated—these elements reduce inadvertent expression of the kill genes compared to existing regulatory mechanisms. Three are of particular interest because they are riboswitches that regulate translation at the post-transcriptional stage of gene expression. Of the four we developed, two proved suitable for tailings pond use.

Justification of our approach

An apparent weakness of our system lies in the potential for the kill switch mechanism to mutate, rendering it ineffective and allowing the synthetic organism or its genetic material to escape into the environment. We have, however, taken several approaches to mitigate this risk. Firstly, we engineered redundancy into our system—with two kill genes, both would have to be rendered inoperable for the kill switch mechanism to malfunction. Knudsen et al. (1995) proposed that active kill switches containing a single kill element were subject to a mutation rate of 10^-6 per cell per generation, but that a second redundant kill gene reduces this value by two orders of magnitude. Secondly, the kill switch mechanism is, of course, only a failsafe measure for controlling our organism's spread. The primary means of preventing its escape is through the multiple layers of mechanical security provided by our bioreactor (LINK TO SAFETY). Only when these measures fail will the kill switch be required to function.

At first blush, an auxotrophic bacterial strain would seem a superior choice for our application. Such a strain would have a critical metabolic gene knocked out, requiring the organism be supplemented externally with an intermediate metabolite. As a mutation restoring the metabolite would be sufficiently complex as to be rendered improbable, an auxotrophic mechanism seems ideal. This system, however, is impractical due to its cost. Metabolic supplements are considerably more expensive than the glucose and metal ions that our system requires. As we learned in our discussions with industry leaders, cost is a paramount factor in permitting adoption at industrial scales, and so our system has a greater likelihood of being implemented outside the laboratory. More worrisome is that an auxotrophic control mechanism would kill the organism without degrading its genetic material, thereby making possible horizontal gene transfer to other organisms. As the overarching goal of our kill switch is not to kill the organism but to prevent the escape of its engineered genetic elements, our kill switch design is superior to one relying on auxotrophy.

Our Kill Genes

The principal mechanism behind our active killswitch system are exo and endonucleases which work in tandem to cause substantial degradation of the bacterial genome. The chance event of bacterial escape from the bioreactor into tailings ponds triggers the transcription of S7 micrococcal nuclease and CviAII endonuclease.

Nuclease assay to evaluate the nucleases present in the registry (BglII and BamHI):

To compare S7 and CviAII to the nucleases already present in the registry we did a nuclease assay with commercially available enzymes from New England Biolabs and an E. coli genomic prep. To see detailed protocol please link see here/link. As can be seen in Figure X, S7 starts acting almost immediately. Within 45 minutes both S7 and CviAII have chewed up the E. coli genome into small fragments whereas BamHI and BglII have sheared the genome into large fragments. Additionally, in 90 minutes, S7 and CviAII have sheared the genome into pieces <200 bp in size whereas there is no difference in the lanes with BglII and BamHI at 90 minutes compared to 45 minutes.

Figure X:

S7 micrococcal nuclease

S7 nuclease is native to Staphylococcus aureus. S. aureus uses this enzyme to destroy extracellular DNA when it infects humans. S7 has both endo and exonuclease activity. This enzyme has a preference for -AT rich regions as opposed to -GC rich regions. However, this enzyme digests the DNA into <200 bp fragments. Ideally this enzyme will be present both intracellularly and extracellularly. We synthesized this enzyme from IDT. However this came with a mutation which altered a lysine residue to an isoleucine thereby making the enzyme dysfunctional.

CviAII restriction enzyme

CviAII is a restriction endonuclease that was sourced from the Chlorella virus PBCV-1 (REF PAPER). Our team selected this enzyme for three reasons.

Firstly, this enzyme recognizes the small four-base restriction site CATG wherein it cuts a staggered end between the A and C on the forward and reverse strands. This is advantageous for the design of our system because of the frequency of this short cut site in the E. coli genome. As opposed to the six base cutter BamHI system submitted by the 2007 Berkely team (BBa_I716462), the CviAII restriction site is to be 16 times more prevalent in the E. coli genome, which translates in finder degradation of the genetic material.

Secondly, the CATG cut site has the probability of being present in start codons of one quarter of genes in the E. coli genome. As such, coding genes will preferentially be selected with activation of CviAII; at this point, the exonuclease activity of S7 micrococcal nuclease can complete degradation of the gene element. Additionally, CviAII is able to cut Dam and Dcm methylated sites in the E. coli genome, and this translates into decreased selectivity of the enzyme.

Finally, the optimum temperature for CviAII activity is 23 degrees Celsius (REF PAPER). This value is relatively low and better suited to operation the cooler tailings water compared to other systems in the registry. For example, the 2007 Berkely BamHI system is optimized for 37 degrees Celsius and thus would be non-functional the tailings ponds. FIND SOME DATA OF TAILING WATER TEMP.

Regulation of our kill genes:

We have explored four different systems in our project. All of these systems fall under the umbrella of inducible kill gene systems. They are: Glucose repressible system, magnesium repressible system , manganese inducible system and the molybdate repressible system.

Molybdate co-factor protein regulation