Team:SDU-Denmark/safety-b

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== Biosafety ==
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=Project safety=
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=== Would any of our project ideas raise safety issues ===
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==Safety Concerns During Election of Project==
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[http://www.google.com [1]]
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=== Do any of the new BioBrick parts (or devices) that was made this year raise any safety issues? ===
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<p style="text-align: justify;">In electing a project there were numerous ideas to be considered. They were divided into the following categories: the environment, foods, health & disease, physics/chemistry/biochemistry and finally other which included some more artistic ideas along with a few humorous ones (like the jeopardy bacteria – knows all the right questions).  Especially while thinking about medical ideas for implementing in the body we knew there were serious risk issues that had to be considered because of the many ways bacteria may interact with the human body.</p>
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=== Is there a local biosafety group, committee, or review board at our institution? ===
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<p style="text-align: justify;">During our closer investigations of project ideas, we also considered which bacteria it was possible to use. Our focus was to try and find a project that was possible to carry out by using ''E. coli''. The reasons are that (cultivated strains of) ''E. coli'' are very well adapted to the laboratory environment since they are easy to keep alive, they can be fairly easy modified and unlike some wild strains of ''E. coli'' they no longer have the ability to thrive inside the intestines. Despite of these considerations we started out working on a project we called mE.chanic (because we wanted to make bacteria do mechanical work), and the idea was to have a culture of bacteria contract and relax, thereby making a pump-like movement creating mechanical work.</p>
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==== Which laws and guidelines we have to consider in Denmark ====
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[[Image:SDU-Denmark-2010-pili.png|600px|thumb|left|Our first project idea involved pili, but we decided it was unsafe, because of it's potential pathogenicity.]]
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Which laws and guidelines we have to consider in Denmark
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<p style="text-align: justify;">Now, we found out that we could use pili as some sort of ‘grappling hooks’ to make a connection between the bacteria. Pili from ''E.coli'' had been measured to have a pulling force of about 100 pN (enough to work a nano-machine), and so it seemed that we would have a good chance of making usable mechanical work if we continued this idea. We just needed to find out how to control the formation and retraction of the pili.</p>
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The scope of this part of the paper is to draw attention to some of the laws and guidelines, which we have to consider in Denmark, when we are dealing with genetically modified microorganisms (GMM's). Our project is defined as an 'contained use' project, which means that the organisms we are handling are contained from the environment at large. The following laws are based on the ”Bekendtgørelsen om Genteknologi og Arbejdsmiljø” (litt. The order on gene-technology and working environment) of 2008, which follows the rules laid down by the European Union in 1990 in the ”directive on the contained use of genetically modified micro-organisms”.
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<p style="text-align: justify;">The project turned out to be most likely to succeed if we used the pathogenic bacterium ''Pseudomonas aeruginosa'', because it is very good at making the type VI pili we wanted. But, that is unfortunately due to its pathogenicity since the pili are an important part of this, as they are used for the bacteria to stay put and not get washed away. So using ''P. aeruginosa'' was immediately out of the question because the risk of personal health (fx. getting a cystitis infection) for the researchers was too high, since they are students and not yet fully educated scientists. Also it would be a potential danger for the surrounding community if it spread. So instead we started to investigate the possibilities of using ''E. coli'', but then found that ''Pseudomonas putida'' was a non-pathogenic bacteria with close resemblance to ''P. aeruginosa'', and also with resemblance to ''E. coli''. So we started working with ''P. putida''. Eventually though, we had to discard this project idea on a safety basis: We had serious doubts that we could succeed with ''P. putida'', given that we basically didn’t believe the bacteria would function with all the genes we needed to provide it with (pili are virulence factors, and working with a non-pathogenic bacteria it would be less likely to make enough pili / keep its pili).</p>
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Risk-assessment
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<p style="text-align: justify;">Then our thought was to use ''E. coli'' and provide it with pili, but this started a long discussion about whether this would make the ''E. coli'' potentially pathogenic. The end of the discussion was to give our project a big make-over, keeping the concept of wanting to make mechanical work but changing the type of the actual mechanical work to be done.</p>
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One of the first, and indeed one of the weightiest points in the directive on GMM safety, is to ensure the public health and the preservation of the environment. And...
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<p style="text-align: justify;">We finally decided on the Flow-E project, and the safety issues in relation to this project will be presented in the following. These include issues of researchers’ safety, public safety and environmental safety. We will also look at safety in relation to the specific BioBricks we use and make, and will have a chapter on what the safety-staff at our university think of this project. Now, let us start at the lab.</p>
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    To that end [to avoid adverse effects on human health and the environment which might arise from the contained use of GMM’s], the user shall carry out an assessment of the contained uses as regards the risks to human health and the environment that those contained uses may pose, using as a minimum the elements of assessment and the procedure set out in Annex III, Sections A and B.
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==Risk-assessment for individual parts==
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Article 4.2
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Here we consider the safety of each of our parts.
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It is required of us to make an throughout risk-assessment, so that we may judge if our use of GMM's poses a threat towards the well being or safety of human beings, animals, plants, or the environment. To help perform this assessment, the UN has laid down a minimum standard of elements required to make an adequate assessment of the potential harm of an accident resulting in the release of the GMM's into the environment. The following is an list of the minimum required elements:
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===Method===
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<p style="text-align: justify;">We have used our [https://2010.igem.org/Team:SDU-Denmark/safety-d#Procedure risk-assessment guidelines] to describe the safety of each of our contributed parts. The genes were [http://blast.ncbi.nlm.nih.gov/Blast.cgi BLASTed] to find their function and known homologs. This function was then reasearched (mainly via [http://www.ncbi.nlm.nih.gov/PubMed PubMed]), described and considered according to the questions asked in the risk-assessment paper.</p>
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===[http://partsregistry.org/wiki/index.php?title=Part:BBa_K343001 Monooxygenase (Part K343001)]===
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    -Assessment of potential harmful effects, defined as:
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====General use====
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        -disease in human beings animals or plants
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<p style="text-align: justify;">This BioBrick poses no treat to the welfare of people working with it, as long as this is done in at least a level 1 safety lab by trained people. No special care is needed when working with this BioBrick. </p>
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        -harmful effects resulting from inability to cure disease
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        - harmful effects resulting from organisms establishing itself in nature
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        -harmful effects resulting from the organism, through natural processes confers part of it's genome, such as heightened resistance, to other organisms in nature
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    -resulting from:
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====Potential pathogenicity====
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        -the host-organism to be modified
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<p style="text-align: justify;">The BioBrick’s product is not in itself toxic, but we do not recommend using this BioBrick for any type of system in humans or animals for the following reasons:
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        -the parts inserted into or otherwise used to alter the organism
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        -the vector
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        -the donor-organism
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        -the resulting modified organism
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    -characteristics for the organism's activity
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*Retinoic acid, which retinal can degrade into, can affect gene expression and function of almost any cell, including cells of the immune system; it also plays a fundamental role in cellular functions by activating nuclear receptors ([https://2010.igem.org/Team:SDU-Denmark/safety-b#References 1]).
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    -How potent the potential harmful effects are
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*Vitamin A toxicity can lead to hepatic congestion and fibrosis ([https://2010.igem.org/Team:SDU-Denmark/safety-b#References 2]).
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    -The likelihood of harmful effects being realised
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*Vitamin A and its derivatives have been implicated as chemopreventive and differentiating agents in a variety of cancers ([https://2010.igem.org/Team:SDU-Denmark/safety-b#References 3]). </p>
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<p style="text-align: justify;">These effects are not directly associated with the enzyme itself, but have been observed in humans. It is highly unlikely that high enough doses can be reached with this BioBrick. Please see references for more information about the diseases. </p>
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Based on this risk-assessment it is possible to rank the project according to the risk, ranking from level 1 to 4, in accordance to the procedure giving by the UN.
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<p style="text-align: justify;">The BioBrick has many homologs that have the same function as this BioBrick and is highly conserved in bacteria and eukaryotes. The BioBrick does not affect the immune system in humans.</p>
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====Environmental impact====
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Personal safety
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<p style="text-align: justify;">To our knowledge, retinal should not play a significant role in environmental processes or would disrupt natural occurring symbiosis. </p>
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To be allowed to work in a level 1 laboratory, it is required that there at all times is a suitable instructed person present. At level 2, all personnel in the laboratory is required to have been suitable instructed in lab safety and procedure. Al access to the lab by non-members of this group or the lab-staff is to be restricted.
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All members of our team has in the time prior to the work in the laboratory received a lab-safety-course, thus fulfilling the requirement. See appendix x for the actual safety guidelines laid down by our local work-safety group.
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<p style="text-align: justify;">The BioBrick should not increase its host’s ability to spread, survive outside the laboratory, and will most likely decrease its ability to replicate.</p>
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<p style="text-align: justify;">Beta-carotene monooxygenase is found in a wide variety of different bacteria, insects and animals ([https://2010.igem.org/Team:SDU-Denmark/safety-b#References 4]). As such we would be cautious as to letting a system containing this BioBrick into the wild, since it's function might conflict with existing systems. On the other hand, one might argue that since its function is already available in nature, the function is widely available. </p>
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Substitution
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<p style="text-align: justify;">The product of the BioBrick, retinal, also plays an important function in nature and animals. For this reason we fell that the BioBrick could be used in controlled settings, but not in the wild. </p>
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Further, it is not allowed to work with any host, donor or vector-system, should another, safer, system, containing the same basic features, be available. If it is possible to find a suitable system, compatible with the intended work, that is safer for humans, animals and plants, or the environment at large, it must always substitute the other, more dangerous system. It is in other words prohibited to take unnecessary risks, or use unnecessarily risky setups. Should a possible substitute system be unreasonably difficult or expensive to acquire, then the risks and benefits must be weighted out against each other, favouring safety above economical issues.
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As we're working with an relatively harmless strains of E.coli (mg16 and TOP10), it has not been necessary to locate a safer, compatible host, donor or system, but we have nonetheless attempted to locate such systems for wholesomeness, although without luck.
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==== Assessment by local bio-safety group ====
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====Possible malign use====
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Assessment by local bio-safety group
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<p style="text-align: justify;">There is not reason to believe this BioBrick could be used for malign uses; it does not increase the hosts ability to vaporize, create spores, regulate the immunesystem or should be pathogenic. </p>
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Arbejdsmiljøgruppen is the local bio-safety group associated with the University of Southern Denmark. During an interview with a representative from this group we explained the project, it's scope, parts and procedure. The following is a number of questions concerning the safety and security issues relating to our project, and the essence of their replies
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If they perceived an increased risk due to work being performed by relatively inexperienced students
 
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The project is not considered any more dangerous due to the fact that most of the work in the lab is performed by relative inexperienced students. As long as the lab's safety protocol is followed, and the fact that the risk-assessment of the work safety group put our project firmly on level 1, they believe that there should be little to no risk to lab personnel or the outside environment. As all students participating in the lab has successfully completed the lab safety course provided by the arbejdsmiljøgruppe, they perceived no increased risk.
 
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If they perceived any danger should the bacteria get out of the lab
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===[http://partsregistry.org/wiki/index.php?title=Part:BBa_K343000 Hyperflagellation (Part K343000)]===
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They perceived no danger to the environment or the well being of animals, plants or human being should the bacteria be released into the environment. This is due to the extremely fragile nature of the E. coli strain that we are using in our project. Should it somehow find its way outside of the lab, it would die within a very short time.
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If there exists an emergency safety protocol in case of accident (i.e. unintentional release of GMM's into environment)
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====General use====
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The emergency protocol is still a work in progress, but although it is unfinished it should not pose a breach in safety, as we're only working with an level 1 GMM, which due to it's extremely fragile nature cannot survive outside of laboratory environment. This coupled with adherence to the standard laboratory safety protocol, should at all times ensure the safety of the environment.
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Overall assessment
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<p style="text-align: justify;">This BioBrick poses no treat to the welfare of people working with it, as long as this is done in at least a level 1 safety lab by trained people and in non-pathogenic hosts such as ''E. coli'' TOP10 or MG1655. No special care is needed when working with this BioBrick. </p>
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The work safety group [ufærdig]
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They see no apparent way of weaponizing or in any other way using our project for malign purposes. None of the modifications made to the bacteria has in any way made it more pathogenic. Further the bacteria we work with are unable to survive and reproduce outside of laboratory conditions. Thus they perceived no security issues with our project.  
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[http://www.bmwf.gv.at/fileadmin/user_upload/forschung/gentechnik/2009-41-EC.pdf [1]]
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====Potential pathogenicity====
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[https://www.retsinformation.dk/Forms/R0710.aspx?id=121099 [2]]
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=== Our ideas on how to deal with safety issues that could be useful for future iGEM competitions! How could parts, devices and systems be made even safer through biosafety engineering! ===
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<p style="text-align: justify;">This BioBrick increases the potential of its host to move. Increased motility has been associated to the bacteria’s ability to invade humans ([https://2010.igem.org/Team:SDU-Denmark/safety-b#References 5]); on the other hand it has also been shown that bacteria that lose the function of the ''FlhDC'' operon, are considerably better at colonizing the intestine ([https://2010.igem.org/Team:SDU-Denmark/safety-b#References 6]), and so an increased expression might decrease a host's ability to colonize and invade humans. It is impossible to ensure, that this plasmid is not transferred to pathogenic bacteria since the ''FlhDC'' operons is used in a wide array of other bacteria that are known to be pathogenic.</p>
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<p style="text-align: justify;">A number of effector cells in the human immune system react specifically to bacteria’s flagella, and so a hyperflagellated host will most likely induce a stronger immune response. </p>
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<p style="text-align: justify;">These different considerations lead us to conclude that we do not recommend using this BioBrick in humans.</p>
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====Environmental impact====
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<p style="text-align: justify;">The BioBrick might increase its host's ability to find foods, but we do not think it will be able to outmatch naturally occurring bacteria. It will not increase the host's ability to replicate, but will increase its ability to spread, which might increase its ability to survive. </p>
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<p style="text-align: justify;">The BioBrick itself will most likely not make an environmental impact, since it only regulates internal systems of the bacteria.</p>
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====Possible malign use====
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<p style="text-align: justify;">There is no reason to believe this BioBrick could be used for malign uses; it does not increase the host’s ability to vaporize, create spores or ability to survive under storage conditions. The fact that this BioBrick will likely increase the immune systems response to hosts carrying it makes it a bad candidate for malign use.</p>
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===[http://partsregistry.org/wiki/index.php?title=Part:BBa_K343003 Photosensor (Part K343003)]===
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====General use====
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<p style="text-align: justify;">This BioBrick poses no treat to the welfare of people working with it, as long as this is done in at least a level 1 safety lab by trained people. No special care is needed when working with this BioBrick. </p>
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====Potential pathogenicity====
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<p style="text-align: justify;">This BioBrick consists of three different parts: The first 224 amino acid residues come from the ''NpSopII'' gene from ''Natronomonas pharaonis'', encoding a blue-light photon receptor with 15 residues removed at the C-terminal. The following 9 amino acids are a linker. The last part is ''HtrII'' fused with ''Tar'' from ''E. coli''. The complex' first 125 amino acid residues come from ''HtrII'' and the remaining 279 from ''Tar'' ([https://2010.igem.org/Team:SDU-Denmark/safety-b#References 7]). ''NpHtrII'' is thought to function in signal transduction and activation of microbial signalling cascades ([https://2010.igem.org/Team:SDU-Denmark/safety-b#References 8]).  </p>
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<p style="text-align: justify;">A single article has been written about haloarchaea in humans indicating that these played a role in patients with inflammatory bowel disease ([https://2010.igem.org/Team:SDU-Denmark/safety-b#References 9]), but there is no evidence that the genes this BioBrick is made from or any near homologs are involved in any disease processes, toxic products or invasion properties. They do not regulate the immune system in any way.</p>
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====Environmental impact====
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<p style="text-align: justify;">The BioBrick does not produce a product that is secreted into the environment, nor is it’s gene product itself toxic. It would not produce anything that distrupt natural occurring symbiosis.</p>
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<p style="text-align: justify;">The BioBrick might increase a bacteria’s ability to find nutrients and as such ease its ability to replicate and spread in certain dark environments. On the other hand the BioBrick is very large and this will naturally slow down its replication rate. Generally we do not believe this BioBrick will make its host able to outcompete natural occurring bacteria, simply because it’s function is not something that will give its host a functional advantage.  </p>
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====Possible malign use====
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<p style="text-align: justify;">This BioBrick will not increase its hosts ability to survive in storage conditions, to be aerosoled, to be vaporized or create spores. None of its proteins regulate or affect the immune system or are pathogenic towards humans and animals.</p>
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===References===
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<p style="text-align: justify;">
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# Spiegl N, Didichenko S, McCaffery P, Langen H, Dahinden CA. [http://www.ncbi.nlm.nih.gov/pubmed Human basophils activated by mast cell-derived IL-3 express retinaldehyde dehydrogenase-II and produce the immunoregulatory mediator retinoic acid]. Blood. 2008 Nov 1;112(9):3762-71.
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# Russell RM. [http://www.ncbi.nlm.nih.gov/pubmed/10731492 The vitamin A spectrum: from deficiency to toxicity]. American Journal of Clinical Nutrition, Vol. 71, No. 4, 878-884, April 2000.
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# Pasquali D, Thaller C, Eichele G. [http://www.ncbi.nlm.nih.gov/pubmed/8964849 Abnormal level of retinoic acid in prostate cancer tissues]. J Clin Endocrinol Metab. 1996 Jun;81(6):2186-91.
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# Bryant DA, Frigaard N. [http://www.ncbi.nlm.nih.gov/pubmed Prokaryotic photosynthesis and phototrophy illuminated]. Trends Microbiol. 2006 Nov;14(11):488-496.
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# Young GM, Badger JL, Miller VL. [http://www.ncbi.nlm.nih.gov/pubmed Motility is required to initiate host cell invasion by Yersinia enterocolitica]. Infect. Immun. 2000 Jul;68(7):4323-4326.
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# Gauger EJ, Leatham MP, Mercado-Lubo R, Laux DC, Conway T, Cohen PS. [http://www.ncbi.nlm.nih.gov/pubmed Role of Motility and the flhDC Operon in Escherichia coli MG1655 Colonization of the Mouse Intestine]. Infect. Immun. 2007 Jul 1;75(7):3315-3324.
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# Jung KH, Spudich EN, Trivedi VD, Spudich JL. [http://www.ncbi.nlm.nih.gov/pubmed An archaeal photosignal-transducing module mediates phototaxis in Escherichia coli]. J. Bacteriol. 2001 Nov;183(21):6365-6371.
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# Mennes N, Klare JP, Chizhov I, Seidel R, Schlesinger R, Engelhard M. [http://www.ncbi.nlm.nih.gov/pubmed Expression of the halobacterial transducer protein HtrII from Natronomonas pharaonis in Escherichia coli.] FEBS Lett. 2007 Apr 3;581(7):1487-1494.
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# Oxley APA, Lanfranconi MP, Würdemann D, Ott S, Schreiber S, McGenity TJ, et al. [http://www.ncbi.nlm.nih.gov/pubmed Halophilic archaea in the human intestinal mucosa]. Environ Microbiol [Internet]. 2010 Apr 23 [cited 2010 Oct 26].
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