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Team:Uppsala/Safety
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| + | document.getElementById("tab1").innerHTML = '<h2>Gene Design</h2><p>Bacteria are able to do some amazing things and with gene technology we can even program them to do what we want, like producing medicines. But this power also comes with responsibility since they could become dangerous if programmed in a certain way. When inserting a gene into a bacteria there are some things to consider:<br><br>Where does the gene come from? <br>What is its normal function?<br>What is the function of similar genes?<br><br>If the gene comes from a pathogen you should make sure you know what it’s effect are on virulence. And if it’s function is normally non-pathogenic but the gene is related to or can interact with some virulence factors you will have to take that into account.<br><br>This year Uppsala synthesised codon optimised genes from two class 2 bacteria. The YenR transcription factor from <i>Yersinia enterocolitica</i> and the Colicin Fy bacteriocin from <i>Yersinia frederiksenii</i>. YenR was not directly involved in causing virulence though it could play a role in regulating factors specific for <i>Y. enterocolitica.</i> Therefore we deemed it safe to use in <i>E. coli.</i> The colicin from <i>Y.frederiksenii</i> is stated to be specific towards <i>Y.enterocolitica</i>, however the documentation is poor. This might mean that the expressed colicin is at a higher risk of causing disease than normal lab strains of <i>E.coli</i>. Therefore lab work should be done extra carefully when working with the colicin expressing bacteria and colicin. We decided not to class this extra possible toxicity as a Class 2 bacteria due to its origin as a colicin, which are known to be specific to bacterias.</p><h2>Collagen-like fusion protein on the surface of <i>E.coli</i></h2><p>When designing a construct using genes expressing human-like proteins, great precautions need to be taken in consideration due to possible interactions with the human body. <br><br>While synthesizing a collagen-like protein <sup><a href="#reference1">[1]</a></sup> for the probiotic bacteria to express on its surface a great deal of investigation was spent on finding out what other mechanisms could get involved in interacting with the protein in our body apart from the pathogen we were targeting. Is it safe to express proteins resembling our own in a safety 1 lab? What could be the consequences of this modified bacteria entering our biological system? What was the ultimate problem with collagen that made us abandon the development of this construct?<br><br>At first glance, a mimicry of the adhesion system used by <i>Y. enterocolitica</i> to bind to our cells using collagen on the surface of its outer membrane would give an immerse advantage in targeting the pathogen. It would even inhibit the pathogen to bind to human cells as the bonding created in between the collagen-like protein to the invasin membrane protein on <i>Y. enterocolitica</i> is greater than the one formed with human collagen. <sup><a href="#reference2">[2]</a></sup> Activity in the stomach and gut would give a great advantage in our targeting system. But, the problem occurs as the bacteria could with a small risk enter the body in other places where our immune system is present.<sup><a href="#reference3">[3]</a></sup><br><br>Due to this risk we never fulfilled the idea of creating an adhesion system using the collagen-like protein.</p><h2>References:</h2><ul class="reference"><li><a id="reference1">[1]</a> Squeglia, Flavia, Beth Bachert, Alfonso De Simone, Slawomir Lukomski, and Rita Berisio. "The crystal structure of the streptococcal collagen-like protein 2 globular domain from invasive M3-type group A Streptococcus shows significant similarity to immunomodulatory HIV protein gp41." Journal of Biological Chemistry 289, no. 8 (2014): 5122-5133.</li><li><a id="reference2">[2]</a> Rutschmann, Christoph, Stephan Baumann, Jürg Cabalzar, Kelvin B. Luther, and Thierry Hennet. "Recombinant expression of hydroxylated human collagen in Escherichia coli." Applied microbiology and biotechnology 98, no. 10 (2014): 4445-4455.</li><li><a id="reference3">[3]</a> Professor Lars Hellman from Uppsala Universitet</li></ul>'; | ||
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| + | document.getElementById("tab2").innerHTML = '<h2>LAB SAFETY</h2><p>When working in any laboratory environment, safety must always be addressed. We held safety rounds in the lab and organized a safety-day for the team to focus on discussing safety questions. </p><h2>Disinfection and sterilization</h2><p>It is important to disinfect and sterilize to ensure both the health of the labcrew as well as for the environment. Working with class 1 bacteria means that the potential harm to the labcrew is limited, but there is a pressing issue about the spread of antibiotic-resistance from the standard plasmids used in iGEM. In our lab we use several precautions to make sure that no bacteria are spread outside the lab.<br><br>All liquid bacterial cultures are gathered in glass containers. This also includes LB which has been in contact with bacteria, such as the supernatant of centrifuged cultures. The liquid is then treated with “Jodopax”, an iodine rich substance that is used in lower concentrations as a wound disinfector. In high concentrations Jodopax could cause rashes or smaller allergic reactions. To limit the exposure of Jodopax we have set it up in “spray bottles” so it could be inserted directly into the glass container. This is also done close to the sink so if skin is exposed to Jodopax it can easily be rinsed off. After at least one hour of treatment the solution is disposed in the sink. The glass container is then “flushed out” and then cleaned as normal glassware. All glass pipettes are put into buckets with “Virkon” solution to ensure sterilization. They are left in the buckets for at least one day. <br><br>Since our class one bacteria is not dangerous upon skin contact we have put our focus towards not letting the bacteria enter the body through e.g. the mouth. Therefore we do not use gloves. If you use gloves, you will probably not notice when you get bacteria on your gloves, later on you will spread the bacteria in the lab by touching things with your infected glove and you might even touch yourself in the eye or mouth. Instead we make sure to have 70% ethanol at all benches in the lab and disinfect frequently. As soon as we feel any drop or after working with bacteria we disinfect with ethanol. This way the bacteria is killed off straight away and its risk of spreading both in the environment and in us is reduced. <br><br>All one time use materials such as pipette tips, tubes and plastic loops are disposed in special hazardous waste containers. Swedish law states that the waste must be packaged such that it can not be re-opened and re-closed without notice. No material should be able to escape the box. To ensure this our bins consists of thick cardboard boxes with two thick plastic bags inside. In the inner bag an adsorption medium is placed to ensure that no liquid can escape. The boxes are changed each day and are then stored a maximum of five days in a cold room before transport or movement to a freezer storage for contagious waste. The waste is then transported via ADR certified trucks to local wasteincinerators “Vattenfall Värme AB” in Uppsala or “SVA:s förbränningsanläggning” in Ultuna. <br><br>To make sure that our working solutions of medium and plates are sterile we use autoclavation. Since autoclavation is a machine that builds up pressure and temperature it can be dangerous if handled incorrectly. To limit mistakes four persons from our team, that had previous experience from last years team, were trained to use the autoclave. They were the only ones that were allowed to use the autoclave from there on. </p><h2>Hazardous chemicals</h2><p>It is not only bacteria that pose a threat to the lab workers, there are also several chemicals that especially in a Class 1 lab are often more dangerous than the bacteria itself. In our lab we are working with EtBr, Acrylamide, Antibiotics and acids & bases. We have taken precautions when working with all these.<br><br><u>Etbr:</u><br>EtBr is a substance used to stain nucleotides , this means that it might hurt our own DNA or RNA if it can penetrate the cell membrane. At first we used gelred as a substitute to lower the risk. Gelred is a EtBr molecule that has a carbon chain attached to it to limit its possibilities to enter through the membrane. However the gelred ran out and the personnel responsible for ordering reagents were on vacation, and thus we had to use EtBr. To limit exposure of EtBr we decided to stain via shaking gels that have finished running in water with EtBr. All work with EtBr was done with nitrile gloves, lab coats and goggles in a fume hood to limit exposure and spread. From the part when the gel had finished running we always worked in pairs. One of them, that touched EtBr, was not allowed to touch anything outside the cabinet, the other person helped by being able to touch things. Before disposal of the shaking bath with EtBr we neutralized it with “teabags” that degrade the EtBr for more than 24 hours.<br><br><u>Acrylamide:</u><br>Acrylamide is a cancerogenic substance that is used for the gel in SDS page. To protect us from acrylamide, we used nitrile gloves, lab coats and safety glasses. Only the person who worked with SDS page was allowed at the bench with acrylamide. Similarly the work was done in pairs as with the EtBr. All materials that have been in contact with the Acrylamid was disposed in the contagious waste. <br><br><u>Antibiotics:</u><br>Antibiotics are at risk of increasing the resistance of antibiotics if released into the environment. This might affect health care leading to decreased effective treatments for everyday diseases. Swedish law classify it as a “Drug” waste. Antibiotics can also be somewhat toxic in high concentrations, so gloves, labcoats and glasses were worn at all times when creating stock solutions of antibiotics. <br><br>Concentrated antibiotics together with one-time use material was sorted into contagious waste boxes as mentioned above while small liquid cultures can be treated with “Jodopax” to destruct the chemical.<br><br><u>Acids & bases:</u><br>All members of the team had taken at least two courses in chemistry, which of one was “Organic Chemistry”, a course known to handle concentrated strong acids and bases. In our lab we use NaOH pellets to get correct pH for our TBE and Boric Acid. They are handled with care and lab coats, gloves and goggles and always in a fume hood. The fume hood is very important when working with boric acid since it can be poisonous through inhalation of gas.</p><h2>Class 2 organisms</h2><p>We were able to work with the real <i>Y.enterocolitica</i> at “Livsmedelsverket”, ‘Food Administration of Sweden’, in their class 2 lab. Since they work with <i>Y.enterocolitica</i> every day, well thought out routines were held. A difference from WHO recommendations are the use of gloves. At Livsmedelsverket we almost never wore gloves. This is to ensure that you notice when you get bacteria on yourself so you disinfect straight away. No fire disinfection was used to limit the creation of aerosols, instead plastic one time use material was used. <br><br>To access the lab several restricted access area doors had to be passed, and the door was marked with a biohazard symbol and “Class 2 lab” logo. The lab is authorized by Swedish lab security agency. </p><h2>Lab security</h2><p>Most things in a laboratory can be used for malicious purposes, especially chemicals and basic synthetic biology. It is therefore important to address the issue of lab security. The main concern for our project would be the genes we had synthesised and in what ways they could be used in the wrong hands. We dropped the idea of collagen early on when discovering it could be harmful. Other genes such as USP45, the anchor protein and antibiotic resistance could probably be used with synthetic biology combined with other genes to create something dangerous. We also store chemicals as stated above that are potentially dangerous. They are stored in alphabetical order, which makes it easier for someone that might want to use them maliciously. However our lab is a restricted access area and only us, and the lab officers have access to the area. Further more, whatever we might have in the lab there are probably a lot of easier ways for these kind of individuals to get their hands on it, including the real <i>Y.enterocolitica</i> at livsmedelsverket.</p><h2>Sources and inspiration</h2><p>Our methods in safety and security are mostly based upon oral information from our supervisors and lab officers. Uppsala University has a well described document for lab safety which we follow, called “Riskhantering vid Uppsala Universitet” loosely translated to ‘Handling of hazards at Uppsala University’. In that document references to Swedish laws and regulations are also made. To make sure that we were also following international guidelines we decided to read the “WHO biosafety manual”, this gave us inspiration and thoughts about the safety issues with designing a system that should be addressed.</p>'; | ||
| - | < | + | document.getElementById("tab3").innerHTML = '<h2>Product safety</h2><p>When developing a product through lab work you have to consider not only the lab safety, but also what implications the product might cause. To discuss the safety risks of our product the iGEM Uppsala team had a safety day during which we sat in groups, discussed and answered questions together.</p><h2>Safety concerns</h2><p>So, suppose that our project has been developed successfully, then we have a bacteria that is able to find and kill the pathogen <i>Yersinia</i> selectively. If the bacteria is produced as large scale the risk of mutation increases. If this bacteria mutates, to generate a new variant of bacteriocin, so that it can go around and kill good bacteria it could become dangerous. Since the bacteria leaves our body through feces, this harmful version would easily be spread to the environment without being detected. The larger stocks of bacteria makes possible leaks more devastating.<br><br>Even if it doesn’t mutate we will also still have the issue where it can disturb the ecosystem in environments we have not thought of. Our organism is engineered to work in our intestines and gut but there is a risk that there is some other niches where it could survive. If our bacteria kills <i>Y.enterocolitica</i> effectively in the environment aswell the microbial balance might be disturbed. The microbial ecosystem is still very unknown and therefore great precaution must be taken.<br><br>The signal peptide and anchor protein could be used together with something dangerous. Since we’re designing a targeting, sensing and killing system, those systems could be redesigned for malicious purposes. For example improved export peptide might be used to secrete toxins in the environment. For each step towards more complex systems in synthetic biology is a step towards more unpredictable bioweapons.</p><h2>Precautions</h2><p>Our project currently doesn’t include any features to reduce risks. We would however, need to to invoke some kind of kill switch if our organism comes in contact with an environment where it is not supposed to be present. Such a switch could be a temperature-dependent-switch. The toilet is seldom 37 dC like the human body. This could make it tricky with culturing, bacteria should be at 37 dC all the time. This might be challenging when freezing and thawing cells, but not unovercomable. Another way could be to make it react to a substance very common in global environment that’s not present our gut and intestines, or the other way around. A third option is to modify the system so that the Bactissiles will start producing a lysis protein at the same time as they produce the toxin. However this is poor in that it does not stop the bacteria from growing if no <i>Y.enterocolitica</i> is present.<br><br>Another important aspect is to have thorough product control to check for mutations in the system. If continuous mutation checks are done in a broad range screening then mutated toxicology of colicin can be reduced. A frozen stock of the original unmutated bacteria should be kept so cultures can be restarted.<br><br>To reduce the spread in nature and effect on microbial ecosystem the advantage of the modified bacteria can be tested with a simple competition test. This should be done routinely to ensure no mutations.</p>'; |
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Latest revision as of 13:00, 17 October 2014
Stephanie Herman
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