Difference between revisions of "Team:TU-Eindhoven/Safety"

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<h1>Lab Safety</h1>
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<p>There are four BioSafety levels, ranging from minimal risk to humans (level 1) to causing severe to fatal diseases (level 4). [1] Our lab is classified as a BioSafety Level 1. Most of the work done at the lab involves the recombinant expression of proteins in Escherichia coli (E. coli) or yeast expression systems and the application of bacteriophages for phage display. Furthermore, cloning and plasmid expansion is also part of the work done at this lab.</p>
  
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<p>When working with GMO's, it is important to work clean, carefully, hygienically and know safe microbiological techniques (SMT). Before starting an experiment, the risks should also be analyzed. Next to that, it is also necessary to check if the experiments are included into the license, this can be done with the PI or the biosafety officer. The biosafety officer at our department is Moniek de Liefde-van Beest. (https://www.tue.nl/universiteit/faculteiten/biomedische-technologie/de-faculteit/medewerkers/detail/ep/e/d/ep-uid/20060801/ep-tab/4/). She is the main responsible for biosafety at the university. The PI of our department is Maarten Merkx. (https://www.tue.nl/universiteit/faculteiten/biomedische-technologie/de-faculteit/medewerkers/detail/ep/e/d/ep-uid/20012354/).</p>
  
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<h1> Safety </h1>
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<p>The SMT ground rules are:</p>
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<p>Please visit <a href="https://2017.igem.org/Safety">the main Safety page</a> to find this year's safety requirements & deadlines, and to learn about safe & responsible research in iGEM.</p>
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<p>On this page of your wiki, you should write about how you are addressing any safety issues in your project. The wiki is a place where you can <strong>go beyond the questions on the safety forms</strong>, and write about whatever safety topics are most interesting in your project. (You do not need to copy your safety forms onto this wiki page.)</p>
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• Always work according to the rules
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• Keep doors and windows closed
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• Wear a lab coat
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• Gather everything and place it orderly
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• Avoid hand-body (e.g. face) contact
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• Do not eat or drink in the laboratory
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• Avoid aerosol formation
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• Do not pipette orally
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• Disinfect non-disposable materials, dispose other materials correctly
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• Clean up your workspace and disinfect the surface
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• Wash your hands when leaving the lab
  
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<p>The more elaborate rules can be found here. (https://static.igem.org/mediawiki/2013/2/2f/TU-Eindhoven_Attachments_LaboratoryChemicalBiology120103.pdf)</p>
  
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<h5>Safe Project Design</h5>
 
  
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<p>Does your project include any safety features? Have you made certain decisions about the design to reduce risks? Write about them here! For example:</p>
 
  
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�
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<li>Choosing a non-pathogenic chassis</li>
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Project Safety
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<li>Choosing parts that will not harm humans / animals / plants</li>
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Besides working safe in the lab, it is also important to think about the applications and risks of your project beforehand. This is called safe-by-design and together with the Dutch National Institute for Public Health and the Environment (RIVM) we looked into this and thought about ways to incorporate this into our project. More information about safe-by-design can be found here.
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<li>Substituting safer materials for dangerous materials in a proof-of-concept experiment</li>
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<li>Including an "induced lethality" or "kill-switch" device</li>
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For our project we use E. coli as a host organism, which is an organism of risk group 1. This organism is then transformed to express our proteins constructs: a scaffold protein (14-3-3) and its binding partner (CT52). Since E. coli is only used to produce the wanted proteins, we are not developing a bacterial system in which safety mechanisms like a kill switch can be induced. However, we did look at the safety of our proteins and at the safety of the network that is formed, more information can be found in the background and in safe-by-design. Furthermore, we also looked at eventual applications and what kind of problems may arise. For our system, we envision it as a gel that can encapsulate tumor cells. To make sure that this gel forms locally, a specific cleavage site can be incorporated in one of the protein constructs. More information on the application scenario can be found here.
  
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Fusicoccin
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<h5>Safe Lab Work</h5>
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Fusicoccin is a small molecule produced by the fungus Fusicoccum amygdali. In our project, it is used as a stabilizer for the interaction between the scaffolding construct and the binding construct. Research has shown that fusicoccin in a concentration 12 times larger than the minimum concentration did not cause measurable negative effects on HeLa and HEK293T mammalian cell lines. [1] However, more research on its toxicity is needed to ensure safe use of fusicoccin in bacterial or mammalian cells.
  
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<p>What safety procedures do you use every day in the lab? Did you perform any unusual experiments, or face any unusual safety issues? Write about them here!</p>
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Risks of our project
  
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Biosafety
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Organisms
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Different strains of E.Coli have been used in this project. For plasmid amplification, the plasmids were transformed into Nova Blue (K-12). When performing Gibson Assembly, NEB 5-alpha E.Coli was used. For protein expression, the plasmids were transformed into BL21 (DE3). All strains are attenuated E.Coli strains and can be handled at Biosafety Level 1.
  
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Vectors
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<h5>Safe Shipment</h5>
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The pET28a vector was used in this project. A lac promotor is built into this plasmid, making controlled protein expression possible. The pSB1C3 vector was used for creating the BioBricks.
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Antibiotics
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The use of anti-microbial resistance factors are a useful tool to quickly determine which cells received the correct plasmid. In our project, kanamycin was used to check for the presence of the pET28a vector. For BioBricking, chloramphenicol was used as a resistance factor to check for the presence of the pSB1C3 vector.
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[1] Skwarczynska, M., Molzan, M. and Ottmann, C. (2012). Activation of NF- B signalling by fusicoccin-induced dimerization. Proceedings of the National Academy of Sciences, 110(5), pp.E377-E386.
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[1] Laboratory Biosafety Manual, World Health Organization, Third Edition
  
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<p>Did you face any safety problems in sending your DNA parts to the Registry? How did you solve those problems?</p>
 
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Revision as of 12:07, 28 September 2017

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Lab Safety

There are four BioSafety levels, ranging from minimal risk to humans (level 1) to causing severe to fatal diseases (level 4). [1] Our lab is classified as a BioSafety Level 1. Most of the work done at the lab involves the recombinant expression of proteins in Escherichia coli (E. coli) or yeast expression systems and the application of bacteriophages for phage display. Furthermore, cloning and plasmid expansion is also part of the work done at this lab.

When working with GMO's, it is important to work clean, carefully, hygienically and know safe microbiological techniques (SMT). Before starting an experiment, the risks should also be analyzed. Next to that, it is also necessary to check if the experiments are included into the license, this can be done with the PI or the biosafety officer. The biosafety officer at our department is Moniek de Liefde-van Beest. (https://www.tue.nl/universiteit/faculteiten/biomedische-technologie/de-faculteit/medewerkers/detail/ep/e/d/ep-uid/20060801/ep-tab/4/). She is the main responsible for biosafety at the university. The PI of our department is Maarten Merkx. (https://www.tue.nl/universiteit/faculteiten/biomedische-technologie/de-faculteit/medewerkers/detail/ep/e/d/ep-uid/20012354/).

The SMT ground rules are:

• Always work according to the rules • Keep doors and windows closed • Wear a lab coat • Gather everything and place it orderly • Avoid hand-body (e.g. face) contact • Do not eat or drink in the laboratory • Avoid aerosol formation • Do not pipette orally • Disinfect non-disposable materials, dispose other materials correctly • Clean up your workspace and disinfect the surface • Wash your hands when leaving the lab

The more elaborate rules can be found here. (https://static.igem.org/mediawiki/2013/2/2f/TU-Eindhoven_Attachments_LaboratoryChemicalBiology120103.pdf)

� Project Safety Besides working safe in the lab, it is also important to think about the applications and risks of your project beforehand. This is called safe-by-design and together with the Dutch National Institute for Public Health and the Environment (RIVM) we looked into this and thought about ways to incorporate this into our project. More information about safe-by-design can be found here. For our project we use E. coli as a host organism, which is an organism of risk group 1. This organism is then transformed to express our proteins constructs: a scaffold protein (14-3-3) and its binding partner (CT52). Since E. coli is only used to produce the wanted proteins, we are not developing a bacterial system in which safety mechanisms like a kill switch can be induced. However, we did look at the safety of our proteins and at the safety of the network that is formed, more information can be found in the background and in safe-by-design. Furthermore, we also looked at eventual applications and what kind of problems may arise. For our system, we envision it as a gel that can encapsulate tumor cells. To make sure that this gel forms locally, a specific cleavage site can be incorporated in one of the protein constructs. More information on the application scenario can be found here. Fusicoccin Fusicoccin is a small molecule produced by the fungus Fusicoccum amygdali. In our project, it is used as a stabilizer for the interaction between the scaffolding construct and the binding construct. Research has shown that fusicoccin in a concentration 12 times larger than the minimum concentration did not cause measurable negative effects on HeLa and HEK293T mammalian cell lines. [1] However, more research on its toxicity is needed to ensure safe use of fusicoccin in bacterial or mammalian cells. Risks of our project � Biosafety Organisms Different strains of E.Coli have been used in this project. For plasmid amplification, the plasmids were transformed into Nova Blue (K-12). When performing Gibson Assembly, NEB 5-alpha E.Coli was used. For protein expression, the plasmids were transformed into BL21 (DE3). All strains are attenuated E.Coli strains and can be handled at Biosafety Level 1. Vectors The pET28a vector was used in this project. A lac promotor is built into this plasmid, making controlled protein expression possible. The pSB1C3 vector was used for creating the BioBricks. Antibiotics The use of anti-microbial resistance factors are a useful tool to quickly determine which cells received the correct plasmid. In our project, kanamycin was used to check for the presence of the pET28a vector. For BioBricking, chloramphenicol was used as a resistance factor to check for the presence of the pSB1C3 vector. [1] Skwarczynska, M., Molzan, M. and Ottmann, C. (2012). Activation of NF- B signalling by fusicoccin-induced dimerization. Proceedings of the National Academy of Sciences, 110(5), pp.E377-E386. [1] Laboratory Biosafety Manual, World Health Organization, Third Edition Footer







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