Team:KU Leuven/Safety

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It is of course very important to stay safe. Here we have gathered information on how we stayed safe in our lab, what the rules of the government and the University of Leuven are and finally how we could keep our bacterium from spreading if it would actually be commercialised. We have also written an <a href="http://2013.igem.org/Team:KU_Leuven/Human_Practices/Ethics/Normative">ethical evaluation of our system</a> concerning some safety issues.</p>
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It is of course very important to stay safe. Here we have gathered information on how we stayed <a href="#general safety">safe in our lab</a>, what the <a href="#europe legislation">government rules</a> and the <a href="#KUL">rules of the KU Leuven</a> are. Finally, we also elaborate on how we could keep our <a href="#bacterial safety">bacterium from spreading</a> if it would eventually be commercialised and released into the <a href="#environmental safety">environment</a>. As well as which specific mechanisms have we implemented to keep our <a href="#auxotrophic">bacteria contained</a>, with for instance, a <a href="#kill switch">'kill switch'</a>. We have also written an <a href="http://2013.igem.org/Team:KU_Leuven/Human_Practices/Ethics/Normative">ethical evaluation of our system</a> concerning certain safety issues.</p>
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Safety forms were approved on September 29, 2013 by the iGEM Safety Committee.
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Safety forms were <b>approved</b> on September 29, 2013 by the iGEM Safety Committee.
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We have covered the safety aspect of our project in many ways. Within synthetic biology there are three main risks that have to be covered: exposition of the laboratory workers to the bacteria and chemicals, effects of a possible escape of the bacteria and the unintentional disturbance of the ecosystem. <br/>
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We have covered the safety aspect of our project in many ways. Within synthetic biology there should be three main risks that need to be covered: exposure of the laboratory workers to the bacteria and chemicals, effects of a possible escape of the bacteria and the unintentional disturbance of the ecosystem.
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We received extra safety training at the beginning of the project. On top of that we took courses regarding safety aspects, e.g. ‘Safe Microbiological Practices’. The safety training consisted of a summary of the important biosafety issues and the composition of the lab.
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In the past three months, we tried to add new branches to the pathways of <i>E. coli</i>. Our experiments were performed with non-pathogenic <i>E. coli</i> strains (BL21, DH5α, TOP10 and Rosetta(DE3)pLysS), which belong to risk group 1of the pathogenic organisms. Working with genetically modified organisms needs extra safety conditions, physical measures are taken to separatethe organism form the surrounding environment and vice versa.<br>
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In the past three months, we have added new branches into the pathways of <i>E. coli</i>. Our experiments were performed with <b>non-pathogenic <i>E. coli</i> strains</b> (BL21, DH5α, TOP10 and Rosetta™(DE3)pLysS), which belong to risk group 1 of the pathogenic organisms. Working with genetically modified organisms demands extra safety conditions; physical measures have been taken to separate the organism from the surrounding environment and vice versa.<br/>
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Our project wants to protect plants and crops against aphid infestations and reduce the use of harmful insecticides. Our genetically modified bacteria will produce (E)-β-farnesene (EBF) and methyl salicylate (MeS), which are natural occurring substances. EBF repels aphids and MeS attracts ladybugs, one of their natural enemies. The low concentrations needed to be effective are not toxic for humans. No harmful effects are described for EBF at high concentrations either. Contrarily, pure MeS could cause harm when it is ingested due to its similarities with acetylsalicylic acid (aspirin) and can cause irritation after contact with eyes and skin. Although, it is typically used in small amounts as a flavouring agent, in candy or as fragrance oils due to its mint flavour and wintergreen fragrance. The two chemicals being produced by our BioBricks could cause allergic reactions (swelling, redness, ...), but serious reactions are very rare.<br>
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Our project wants to protect plants and crops against aphid infestations and reduce the use of harmful insecticides. Our genetically modified bacteria (<b>BanAphids</b>) will produce (E)-β-farnesene (EBF) and methyl salicylate (MeS), which are naturally occurring substances. EBF repels aphids and MeS attracts ladybugs, one of the aphid's natural enemies. The low concentrations needed for these substances to be effective is not toxic for humans. No harmful effects are described for EBF at high concentrations either. Contrarily, pure MeS could cause harm when it is ingested due to its similarities with <b>acetylsalicylic acid (aspirin)</b> and can cause irritation after contact with eyes and skin. Nevertheless, it is typically used in small amounts as a flavouring agent in candy, or as fragrance oils due to its mint flavour and wintergreen fragrance. The two chemicals being produced by our <b>BanAphids</b> could cause allergic reactions (swelling, redness, ...), but serious reactions only occur in high concentrations and are very rare.<br/>
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The effect of our genetically modified bacteria on the environment is non-trivial and thorough field experiments must definitely be conducted. The maximum production rate of onebacterium has to be determined and scaling this model up to a whole field occupied by our bacteria will allow us to estimate the concentration in the surroundings. We can then determine the necessary parameters in order to minimise ecological disruption according to surrounding concentrations of MeS and EBF. We also thought about the interactions between aphids and ladybugs themselves. Aphids adapt readily when EBF is constantly present,therefore we included an oscillatory model. More information about this model can be found <a href="http://2013.igem.org/Team:KU_Leuven/Project/Oscillator">here</a>.  
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The effect of <b>BanAphids</b> on the environment is not trivial and thorough field experiments must definitely be conducted. The maximum production rate of a single bacterium needs to be determined so that this could be scaled up through a model. This way we would be able to estimate the concentration of EBF and MeS in the surroundings if a whole field would be occupied by <b>BanAphids</b>. We can then determine the necessary parameters in order to minimise ecological disruption according to these calculated estimates of surrounding EBF and MeS concentrations. We also thought about the interactions between aphids and ladybugs themselves. Aphids adapt readily when EBF is constantly present, therefore we included an oscillatory model. More information about this model can be found <a href="http://2013.igem.org/Team:KU_Leuven/Project/Oscillator">here</a>.  
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   <p align = "justify">Research concerning genetically modified organisms (GMOs) is subjected to strict safety legislation in Belgium. These laws are in place to protect the general public and the environment from the potential risks of GMOs. The European legislation forms the foundation of the more extensive Belgian laws. Firstly, the use of GMOs in Europe requires an environmental licence. In Belgium, an approval for the research activity is required on top of that. This way, the Belgian government controls all use of GMO’s and can assess risk analysis accurately. This is all recorded in the Decrees of the Flemish Government: Vlarem I and II section 51. The Belgian government describes four risk levels to which different restrictions are coupled. These restrictions are a combination of physical actions and safety regulations. For this purpose, the Belgian government appeals to the expertise of the Advisory Board of Biosafety (ABB) (section Biosafety and Biotechnology of the Scientific Institute of Public Health) which acts as the scientific secretary of the board. They evaluate the risk analysis in the licence request. At the University of Leuven, all the laboratories, including the iGEM lab, have an environmental licence and an approval for their activities.</p>
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In Belgium, the whole research about genetically modified organisms (GMOs) is subjected to strict safety legislation. These laws must protect the people and the environment against the potential risks of the GMOs. The European legislation forms the foundation of the more extensive Belgian laws. Firstly, the use of GMOs in Europe requires an environmental licence. In Belgium, an approval for the research activity is required on top of that. This way the Belgian government controls all use of GMO’s and can assess risk analysis accurately. This is all recorded in the Decrees of the Flemish Government: Vlarem I and II section 51. The Belgian government describes four risk levels to which different restrictions are coupled. These restrictions are a combination of physical actions and safety regulations. For this purpose, the Belgian government appeals to the expertise of the Advisory Board of Biosafety (ABB). The ABB (section Biosafety and biotechnology of the Scientific Institute of Public Health) forms the scientific secretariat of the board. They evaluate the risk analysis in the licence request. At the KU Leuven, all the laboratories, including the iGEM lab, have an environmental licence and an approval for their activities.</p>
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The University of Leuven has its own biosafety rules in addition to the national biosafety regulations, where Marianne Schoukens acts as the central contact point for Health, Safety and Environment (HSE) issues. All members of our team have received training in Safe Microbiological Techniques as well as general and specific lab safety required by the University of Leuven. At the beginning of our project, all members of the iGEM team have received an update from the KU HSE department. Via liaison Iris Govaerts, we followed a small seminar and filled in an orientation checklist concerning health and safety training prior to the start of project laboratory work. <br>
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The KU Leuven has its own biosafety rules in addition to the national biosafety regulations, where Marianne Schoukens acts as the central contact point for <b>Health, Safety and Environment (HSE)</b> issues. All members of our team have received training in Safe Microbiological Techniques as well as general and specific lab safety required by the KU Leuven. The safety training consisted of a summary of the important biosafety issues and the composition of the lab. At the beginning of our project, all members of the iGEM team have received an update from the KU Leuven's HSE department. Via liaison Iris Govaerts, we followed a small seminar and filled in an orientation checklist concerning health and safety training prior to the start of project laboratory work. <br/>
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The project we ultimately chose was the result of a whole series of brainstorm sessions. In this brainstorm sessions we talked about the feasibility of our ideas with our instructor (Dr. Ir. Ingmar Claes) and our coordinators (Prof.Dr. Johan Robben and Dr.Veerle De Wever). We also invited different PhD students, professors, ex-iGEMers and people from the industrial sector to our brainstorm sessions. These people have a lot more knowledge about the specific topics and could help us greatly with our decisions. Once the project was in progress, we also contacted a lot of people with questions and about possibilities. All these people had another view on our project and helped us taking into account different issues about the safety of our project: the molecules that our bacteria will produce, release into the environment, etc ...</p>
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The project we ultimately chose was the result of a whole series of brainstorm sessions. In these brainstorm sessions we discussed the feasibility of our ideas with our instructor (Dr. Ir. Ingmar Claes) and our coordinators (Prof. Dr. Johan Robben and Dr. Veerle De Wever). We also invited different PhD students, professors, ex-iGEMers and people from the industrial sector to our brainstorm sessions. They have a lot more knowledge about the specific topics and could help us greatly with our decisions. Once the project was in progress, we also contacted a lot of people with questions about the possibilities. For instance we talked to the company Perseus (Partner in Regulatory and Safety Services) to discuss the possible hurdles we would face and which safety aspects we need to incorporate. All these people had another view on our project and helped us taking into account different issues about the safety of our project: the molecules that our bacteria will produce, release into the environment, etc ...</p>
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If our project would be used around the country or in the whole world, many parameters would need to be determined. As stated earlier, the pheromones produced by the modified <i>E. coli</i> are not harmful for our health unless available in high concentrations. So when this modified <i>E. coli</i> would be spread across acres of land, the concentration of pheromones could attain a high level. The ceiling of this production would need to be determined to see whether it could be a harmful concentration for our health. Insects however do not need as high of a concentration as humans do to develop a reaction. Therefore the disruption of the ecosystem would need to be considered as well. As with most products released in the environment, its bioaccumulation needs to be at a minimum.</p>
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If our project would be used outside the laboratory, many parameters would need to be determined. As stated earlier, the pheromones produced by the <b>BanAphids</b> are not harmful for our health unless available in high concentrations. So if the <b>BanAphids</b> would be spread across acres of land, the concentration of pheromones could attain a high level. The ceiling of this production would need to be determined to see whether it could be a harmful concentration for our health. Insects however do not need as high of a concentration as humans do to develop a reaction. Therefore the disruption of the ecosystem would need to be considered as well. As with most products released in the environment, its bioaccumulation needs to be at a minimum.</p>
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     <img src="http://2013.igem.org/wiki/images/4/4c/Genetic_safeguard_strategies.jpg" alt="genetic safeguard strategies"><p align="justify"><b>Genetic safeguard strategies.</b><br/>
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Recombinant DNA (bright green) is introduced into the host chromosome (white wavy lines). Two pathways for engineered auxotrophy (A,B) kill synthetic organisms (blue) once they lose access to a supplement (+) in a controlled environment. The supplement either (A) suppresses a toxic gene product (−) or (B) provides nutrition to compensate for a genetic deletion (red X). The induced lethality system (C) produces a toxic gene product (−) in response to an inducer (i) such as IPTG, sucrose, arabinose, or heat. (Moe-Behrens <i>et al.</i>, 2013)</p>
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In all our experiments, we used non-pathogenic <i>E. coli</i> strains. These strains are not harmful for humans, except when they are inhaled or swallowed. The chemicals that are produced by our genetically modified <i>E. coli</i> strain are natural molecules and are not harmful for the environment. An accidental release of our bacteria into the environment could have unexpected effects on the ecological balance. As suggested by Moe-Behrens <i>et al.</i>, 2013, containment through engineered auxotrophy so that <b>BanAphids</b> are unable to synthesise an essential compound required for their survival, is the most reliable method currently for biocontainment. We also believe that providing a bacterium with an advantage eg. antibiotic resistance, provides bacteria with the opportunity to mutate into even stronger bacteria. We therefore propose the use of a tryptophan auxotrophic bacterium.<br/>
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In the second model we have designed, the bacterium would be free in the environment and thus not contained in an enclosed container with medium. Therefore, an auxotrophic mechanism would not be applicable. Here we turn to a ‘kill switch’ or induced lethality mechanism, which is less reliable because of spontaneous mutations that could inactivate or disable this ‘kill switch’.  Different kill switches are already being used in the iGEM competition and we find it very important that the work of previous iGEM teams do not go to waste which is why we decided to look into several 'kill switches' developed by previous iGEM teams.</p>
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In all our experiments, we used non-pathogenic <i>E. coli</i> strains. These strains are not harmful for humans, except when they are inhaled or swallowed. Nevertheless, they can cause irritation to the eyes and/or skin. The chemicals that are produced by our genetically modified <i>E. coli</i> strain are natural molecules and are not harmful for the environment. An accidental release of our bacteria into the environment could have unexpected effects on the ecological balance. As suggested in literature (reference), containment through engineered auxotrophy so that our genetically modified <i>E. coli</i> is unable to synthesise an essential compound required for their survival, is the most reliable method currently for biocontainment. We propose the use of a tryptophan auxotrophic bacterium.<br>
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When tryptophan auxotrophic bacteria will be used in our project, we must provide tryptophan in their medium. This is possible when the bacteria grow together on a petri dish or another closed environment, but not when we want to spray the bacteria on the plants and crops. Therefore we designed two models. In one model the bacteria are grown in a contained environment, a plastic sticker (Design of Groningen iGEM 2012), where the auxotrophic mechanism can be used. This plastic sticker is composed of a plastic film with pores that are too small for the bacteria, but through which our volatiles (EBF and MeS) can escape.<br>
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In the second model we have designed, the bacterium would be free in the environment and thus not contained in an enclosed container with medium therefore an auxotrophic mechanism would not be applicable. Here we turn to a ‘kill switch’ or induced lethality mechanism, which is less reliable because of spontaneous mutations that could inactivate or disable this ‘kill switch’. Different kill switches are already being used in the iGEM competition. When we would chose for this method, we have to examine all the available kill switches and select the best one.</p>
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<p align="justify"><b>Genetic safeguard strategies</b><br/>
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Recombinant DNA (green) is introduced into the host chromosome. Two pathways for engineered auxotrophy (A and B) kill synthetic organisms once they lose access to a supplement (+) in a controlled environment. The supplement either (A) suppresses a toxic gene product (−) or (B) provides nutrition to compensate for a genetic deletion. The induced lethality system (C) produces a toxic gene product (−) in response to an inducer (i) such as IPTG, sucrose, arabinose, or heat (Moe-Behrens <i>et al.</i>, 2013).</p>
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We, the KU Leuven iGEM team of 2013, propose the use of an auxotrophic mechanism as our main genetic safeguard, when we use our "sticker" or "oscillator" model. <br/>
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We, the KU Leuven iGEM team of 2013, propose the use of an auxotrophic mechanism as our main genetic safeguard, when we use our 'sticker system'. <br/>
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The basic mechanism of auxotrophy is based on the fact that, since auxotrophic organisms are unable to synthesize an essential compound required for their survival, they rapidly die once they escape the controlled environment where the compound is supplied.<br/>
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The basic mechanism of the <b>tryptophan auxotrophic bacteria</b> is based on the fact that they are unable to synthesise the essential compound, tryptophan, required for their survival, which means we must provide tryptophan in their medium. They therefore rapidly die once they escape from the controlled environment where the compound is supplied.<br/>
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Already in 1987, Molin and colleagues designed a DNA cassette that functions as a conditional suicide system in any healthy bacterial strain. In the absence of an artificially supplied growth supplement, the cassette produced a toxic protein that damages bacterial cell membranes and kills the cells. Also, stochastic activation of this toxic component could be used to kill a predetermined fraction of cells per unit of time. This helps to tune the level of lethality so that an optimal level of bioproduction is achieved.(Moe-Behrens <i>et al.</i>, 2013)<br/>
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Already in 1987, Molin and colleagues designed a DNA cassette that functions as a conditional suicide system in any healthy bacterial strain. In the absence of an artificially supplied growth supplement, the cassette produces a toxic protein that disrupts the bacterial cell's membrane and kills the cells. A stochastic activation of this toxic component could also be used to kill a predetermined fraction of cells per unit of time. This helps to tune the level of lethality so that an optimal level of bioproduction is achieved (Moe-Behrens <i>et al.</i>, 2013).<br/>
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This year, we present two models in which the use of an auxotrophic mechanism can be easily manifested, especially for the "sticker" or "oscillator" model.<br/>
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This is a good candidate for the biocontainment of <b>BanAphids</b> in the 'sticker' <a href="http://2012.igem.org/Team:Groningen/Sticker">(Design of Groningen iGEM 2012)</a> or 'sticker system'. This plastic sticker is composed of a plastic film with pores that are too small for the bacteria, but through which our volatiles (EBF and MeS) can escape. However, we need a different mechanisms to contain the <b>BanAphids</b> of our 'spray' or 'honeydew' system.</p>
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However, auxotrophic mechanisms might be difficult to manage in our "spray" or "glucose" model. </p>
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The basic mechanism of a kill switch is that engineered organisms survive normally until an inducer signal (e.g., IPTG) is added, which kills the cells. This induced lethality could be used clean up synthetic microbe spills without harming other cells in the environment (Moe-Behrens <i>et al.</i>, 2013).<br/>
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The basic mechanism of a kill switch is that the engineered organisms survive until an inducer signal (e.g. IPTG) is added, and this will activate a cascade mechanism that will kill the cells. This induced lethality could, for instance, be used to clean up synthetic microbe spills without harming other cells in the environment (Moe-Behrens <i>et al.</i>, 2013).<br/>
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Inducible kill systems are not new to iGEM. There are already several constructs available in the database, such as the inducible BamHI system contributed by Berkeley in 2007 (<a href="http://parts.igem.org/Part:BBa_I716462">BBa_I716462</a>, tested by Lethbridge in 2011). Here, a <i>BamHI</i> gene was placed downstream of an arabinose-inducible promotor.<br/>
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Furthermore, as suggested by Balan <i>et al.</i> (2005), the use of a nuclease enzyme suicide system disfavours horizontal gene transfer compared to the above suggested suicide system that targets the cell membrane. If a bacterial cell is lysed, that bacterium might be dead but other bacteria in the environment are not dead (yet) and a suicide system that targets the disruption of the cell membrane does not guarantee the degradation of DNA. The lysed bacterium's DNA is now free and available to be taken up by neighbouring bacteria. For this reason we have chosen a 'kill switch' that activates a nuclease system. <br/>
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Unfortunately, the lethal gene is a central cause of safeguard failure. Spontaneous genetic mutations can both deactivate and activate lethal gene expression may worsen the failure of bio containment. As engineered cells are passaged in the laboratory, or as they propagate in large bioreactors, broken genetic safeguards can gradually accumulate in the population. If the utility of the bio containment mechanism is lost, then the synthetic organisms might survive in the environment after disposal or accidental release.(Moe-Behrens <i>et al.</i>, 2013)<br/>
+
Inducible kill systems are not new to iGEM, there are already several constructs available in the database, such as the inducible <i>BamHI</i> system contributed by Berkeley in 2007 (<a href="http://parts.igem.org/Part:BBa_I716462">BBa_I716462</a>, tested by Lethbridge in 2011). Here, a <i>BamHI</i> gene was placed downstream of an arabinose-inducible promotor.<br/>
-
Next, a lack of tight control is also a big problem within these systems. Leaky systems cannot be controlled strictly. Therefore, the Calgary team of 2012 added a riboswitch, which are small pieces of mRNA that bind ligands and modify translation of downstream genes; this way the kill switch can be more tightly controlled. For further details, we would gladly invite you to read the extensive literature study on the wiki of the <a href="http://2012.igem.org/Team:Calgary/Project/Hum
+
Unfortunately, spontaneous genetic mutations in this lethal <i>BamHI</i> gene is a central cause of safeguard failure. Spontaneous genetic mutations can deactivate lethal gene expression causing failure of biocontainment. As these engineered cells propagate in the laboratory, or in large bioreactors, disfunctional genetic safeguards can gradually accumulate in the population. If this accumulation is high enough, then the synthetic organisms might survive in the environment after disposal or accidental release (Moe-Behrens <i>et al.</i>, 2013).<br/>
 +
Next, a lack of tight control is also a big problem within these systems. Leaky systems cannot be controlled strictly. Therefore, the Calgary team of 2012 added a riboswitch, which is a small piece of mRNA that binds ligands and modifies translation of downstream genes; this way the kill switch can be more tightly controlled. For further details, we would gladly invite you to read the extensive literature study on the wiki of the <a href="http://2012.igem.org/Team:Calgary/Project/HumanPractices/Killswitch">Calgary 2012 Team</a>.</p>
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Moe-Behrens, G. H. G., Davis, R., Haynes, K., Preparing synthetic biology for the world, Frontiers in Microbiology 2013, 4(5): 1-10. <br/>
 +
Balan, A., Schenberg, A. C. G., A conditional suicide system for Saccaromyces cerevisiae relying on the intracellular production of the Serratia marcescens nuclease, Yeast 2005; 22; 203-212.
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Latest revision as of 19:44, 28 October 2013

iGem

Secret garden

Congratulations! You've found our secret garden! Follow the instructions below and win a great prize at the World jamboree!


  • A video shows that two of our team members are having great fun at our favourite company. Do you know the name of the second member that appears in the video?
  • For one of our models we had to do very extensive computations. To prevent our own computers from overheating and to keep the temperature in our iGEM room at a normal level, we used a supercomputer. Which centre maintains this supercomputer? (Dutch abbreviation)
  • We organised a symposium with a debate, some seminars and 2 iGEM project presentations. An iGEM team came all the way from the Netherlands to present their project. What is the name of their city?

Now put all of these in this URL:http://2013.igem.org/Team:KU_Leuven/(firstname)(abbreviation)(city), (loose the brackets and put everything in lowercase) and follow the very last instruction to get your special jamboree prize!

tree ladybugcartoon

Journals

Follow our activities week-by-week.

Protocols

No magic involved - unfortunately

Safety

You are here!

It is of course very important to stay safe. Here we have gathered information on how we stayed safe in our lab, what the government rules and the rules of the KU Leuven are. Finally, we also elaborate on how we could keep our bacterium from spreading if it would eventually be commercialised and released into the environment. As well as which specific mechanisms have we implemented to keep our bacteria contained, with for instance, a 'kill switch'. We have also written an ethical evaluation of our system concerning certain safety issues.

Safety forms were approved on September 29, 2013 by the iGEM Safety Committee.

We have covered the safety aspect of our project in many ways. Within synthetic biology there should be three main risks that need to be covered: exposure of the laboratory workers to the bacteria and chemicals, effects of a possible escape of the bacteria and the unintentional disturbance of the ecosystem.

Laurens Labo
some_text

(Max Gibson)

In the past three months, we have added new branches into the pathways of E. coli. Our experiments were performed with non-pathogenic E. coli strains (BL21, DH5α, TOP10 and Rosetta™(DE3)pLysS), which belong to risk group 1 of the pathogenic organisms. Working with genetically modified organisms demands extra safety conditions; physical measures have been taken to separate the organism from the surrounding environment and vice versa.
Our project wants to protect plants and crops against aphid infestations and reduce the use of harmful insecticides. Our genetically modified bacteria (BanAphids) will produce (E)-β-farnesene (EBF) and methyl salicylate (MeS), which are naturally occurring substances. EBF repels aphids and MeS attracts ladybugs, one of the aphid's natural enemies. The low concentrations needed for these substances to be effective is not toxic for humans. No harmful effects are described for EBF at high concentrations either. Contrarily, pure MeS could cause harm when it is ingested due to its similarities with acetylsalicylic acid (aspirin) and can cause irritation after contact with eyes and skin. Nevertheless, it is typically used in small amounts as a flavouring agent in candy, or as fragrance oils due to its mint flavour and wintergreen fragrance. The two chemicals being produced by our BanAphids could cause allergic reactions (swelling, redness, ...), but serious reactions only occur in high concentrations and are very rare.
The effect of BanAphids on the environment is not trivial and thorough field experiments must definitely be conducted. The maximum production rate of a single bacterium needs to be determined so that this could be scaled up through a model. This way we would be able to estimate the concentration of EBF and MeS in the surroundings if a whole field would be occupied by BanAphids. We can then determine the necessary parameters in order to minimise ecological disruption according to these calculated estimates of surrounding EBF and MeS concentrations. We also thought about the interactions between aphids and ladybugs themselves. Aphids adapt readily when EBF is constantly present, therefore we included an oscillatory model. More information about this model can be found here.

Legislation in Belgium & Europe

Research concerning genetically modified organisms (GMOs) is subjected to strict safety legislation in Belgium. These laws are in place to protect the general public and the environment from the potential risks of GMOs. The European legislation forms the foundation of the more extensive Belgian laws. Firstly, the use of GMOs in Europe requires an environmental licence. In Belgium, an approval for the research activity is required on top of that. This way, the Belgian government controls all use of GMO’s and can assess risk analysis accurately. This is all recorded in the Decrees of the Flemish Government: Vlarem I and II section 51. The Belgian government describes four risk levels to which different restrictions are coupled. These restrictions are a combination of physical actions and safety regulations. For this purpose, the Belgian government appeals to the expertise of the Advisory Board of Biosafety (ABB) (section Biosafety and Biotechnology of the Scientific Institute of Public Health) which acts as the scientific secretary of the board. They evaluate the risk analysis in the licence request. At the University of Leuven, all the laboratories, including the iGEM lab, have an environmental licence and an approval for their activities.

some_text

(JRC - European Commission)

KU Leuven "rules"

The KU Leuven has its own biosafety rules in addition to the national biosafety regulations, where Marianne Schoukens acts as the central contact point for Health, Safety and Environment (HSE) issues. All members of our team have received training in Safe Microbiological Techniques as well as general and specific lab safety required by the KU Leuven. The safety training consisted of a summary of the important biosafety issues and the composition of the lab. At the beginning of our project, all members of the iGEM team have received an update from the KU Leuven's HSE department. Via liaison Iris Govaerts, we followed a small seminar and filled in an orientation checklist concerning health and safety training prior to the start of project laboratory work.
The project we ultimately chose was the result of a whole series of brainstorm sessions. In these brainstorm sessions we discussed the feasibility of our ideas with our instructor (Dr. Ir. Ingmar Claes) and our coordinators (Prof. Dr. Johan Robben and Dr. Veerle De Wever). We also invited different PhD students, professors, ex-iGEMers and people from the industrial sector to our brainstorm sessions. They have a lot more knowledge about the specific topics and could help us greatly with our decisions. Once the project was in progress, we also contacted a lot of people with questions about the possibilities. For instance we talked to the company Perseus (Partner in Regulatory and Safety Services) to discuss the possible hurdles we would face and which safety aspects we need to incorporate. All these people had another view on our project and helped us taking into account different issues about the safety of our project: the molecules that our bacteria will produce, release into the environment, etc ...

If our project would be used outside the laboratory, many parameters would need to be determined. As stated earlier, the pheromones produced by the BanAphids are not harmful for our health unless available in high concentrations. So if the BanAphids would be spread across acres of land, the concentration of pheromones could attain a high level. The ceiling of this production would need to be determined to see whether it could be a harmful concentration for our health. Insects however do not need as high of a concentration as humans do to develop a reaction. Therefore the disruption of the ecosystem would need to be considered as well. As with most products released in the environment, its bioaccumulation needs to be at a minimum.

(Norcal)

In all our experiments, we used non-pathogenic E. coli strains. These strains are not harmful for humans, except when they are inhaled or swallowed. The chemicals that are produced by our genetically modified E. coli strain are natural molecules and are not harmful for the environment. An accidental release of our bacteria into the environment could have unexpected effects on the ecological balance. As suggested by Moe-Behrens et al., 2013, containment through engineered auxotrophy so that BanAphids are unable to synthesise an essential compound required for their survival, is the most reliable method currently for biocontainment. We also believe that providing a bacterium with an advantage eg. antibiotic resistance, provides bacteria with the opportunity to mutate into even stronger bacteria. We therefore propose the use of a tryptophan auxotrophic bacterium.
In the second model we have designed, the bacterium would be free in the environment and thus not contained in an enclosed container with medium. Therefore, an auxotrophic mechanism would not be applicable. Here we turn to a ‘kill switch’ or induced lethality mechanism, which is less reliable because of spontaneous mutations that could inactivate or disable this ‘kill switch’. Different kill switches are already being used in the iGEM competition and we find it very important that the work of previous iGEM teams do not go to waste which is why we decided to look into several 'kill switches' developed by previous iGEM teams.

Genetic safeguard strategies
Recombinant DNA (green) is introduced into the host chromosome. Two pathways for engineered auxotrophy (A and B) kill synthetic organisms once they lose access to a supplement (+) in a controlled environment. The supplement either (A) suppresses a toxic gene product (−) or (B) provides nutrition to compensate for a genetic deletion. The induced lethality system (C) produces a toxic gene product (−) in response to an inducer (i) such as IPTG, sucrose, arabinose, or heat (Moe-Behrens et al., 2013).

Auxotrophic mechanism

We, the KU Leuven iGEM team of 2013, propose the use of an auxotrophic mechanism as our main genetic safeguard, when we use our 'sticker system'.
The basic mechanism of the tryptophan auxotrophic bacteria is based on the fact that they are unable to synthesise the essential compound, tryptophan, required for their survival, which means we must provide tryptophan in their medium. They therefore rapidly die once they escape from the controlled environment where the compound is supplied.
Already in 1987, Molin and colleagues designed a DNA cassette that functions as a conditional suicide system in any healthy bacterial strain. In the absence of an artificially supplied growth supplement, the cassette produces a toxic protein that disrupts the bacterial cell's membrane and kills the cells. A stochastic activation of this toxic component could also be used to kill a predetermined fraction of cells per unit of time. This helps to tune the level of lethality so that an optimal level of bioproduction is achieved (Moe-Behrens et al., 2013).
This is a good candidate for the biocontainment of BanAphids in the 'sticker' (Design of Groningen iGEM 2012) or 'sticker system'. This plastic sticker is composed of a plastic film with pores that are too small for the bacteria, but through which our volatiles (EBF and MeS) can escape. However, we need a different mechanisms to contain the BanAphids of our 'spray' or 'honeydew' system.

stickermodel


some_text

Kill Switch

The basic mechanism of a kill switch is that the engineered organisms survive until an inducer signal (e.g. IPTG) is added, and this will activate a cascade mechanism that will kill the cells. This induced lethality could, for instance, be used to clean up synthetic microbe spills without harming other cells in the environment (Moe-Behrens et al., 2013).
Furthermore, as suggested by Balan et al. (2005), the use of a nuclease enzyme suicide system disfavours horizontal gene transfer compared to the above suggested suicide system that targets the cell membrane. If a bacterial cell is lysed, that bacterium might be dead but other bacteria in the environment are not dead (yet) and a suicide system that targets the disruption of the cell membrane does not guarantee the degradation of DNA. The lysed bacterium's DNA is now free and available to be taken up by neighbouring bacteria. For this reason we have chosen a 'kill switch' that activates a nuclease system.
Inducible kill systems are not new to iGEM, there are already several constructs available in the database, such as the inducible BamHI system contributed by Berkeley in 2007 (BBa_I716462, tested by Lethbridge in 2011). Here, a BamHI gene was placed downstream of an arabinose-inducible promotor.
Unfortunately, spontaneous genetic mutations in this lethal BamHI gene is a central cause of safeguard failure. Spontaneous genetic mutations can deactivate lethal gene expression causing failure of biocontainment. As these engineered cells propagate in the laboratory, or in large bioreactors, disfunctional genetic safeguards can gradually accumulate in the population. If this accumulation is high enough, then the synthetic organisms might survive in the environment after disposal or accidental release (Moe-Behrens et al., 2013).
Next, a lack of tight control is also a big problem within these systems. Leaky systems cannot be controlled strictly. Therefore, the Calgary team of 2012 added a riboswitch, which is a small piece of mRNA that binds ligands and modifies translation of downstream genes; this way the kill switch can be more tightly controlled. For further details, we would gladly invite you to read the extensive literature study on the wiki of the Calgary 2012 Team.

Moe-Behrens, G. H. G., Davis, R., Haynes, K., Preparing synthetic biology for the world, Frontiers in Microbiology 2013, 4(5): 1-10.
Balan, A., Schenberg, A. C. G., A conditional suicide system for Saccaromyces cerevisiae relying on the intracellular production of the Serratia marcescens nuclease, Yeast 2005; 22; 203-212.