Team:SDU-Denmark/safety-b

From 2010.igem.org

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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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<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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=== 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;">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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==== analysis of parts and devices ====
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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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'''SopII'''  – No homogenity when blasted. The gene is mentioned in Halobacterium salinarum R1 [1] and in Natronomonas pharaonis DSM 2160[2] as being protein coding. The protein is a light dependant iontransporter, and is often seen with the halophilic bacteria (halophilic: organisms that thrive in high concentrations of salt). So it might make non extremophiles more competitive in environments with high salt concentrations. The word: “might“ should be underlined, as it of course takes several genes / proteins to make it more durable in high salt concentrated areas . This clearly is a risk. If a bacterium were to gain the advantage, to survive in high salt concentrations, it would mean we could create diverse bacteria which more easily can survive, and proliferate in completely different environment, than we usually meet them. The outcome of “old” bacteria proliferating in another environment than usual is not easy to foresee.
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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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'''HtrII''' – (sequence?). It contains three domains, which gives a chemotaxis-like property towards Methyl, Aspartate and related amino acids. One of the domains (cl01054, which is the one out of three) is commonly observed in bacteria. [3] The fact that it is often represented in bacteria creates a lower safety risk, as it will less likely transfer its genes to bacteria which don’t have this specific chemotaxis already.
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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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'''Tsr''' - The protein is often present in various cell-types. It serves as a methyl-accepting chemotaxis protein. In some certain bacteria it is required for morphogenesis of rhabdomere [4]. As in the case with htrII, the same lack of issues arises; many different bacteria already have this property and/or specific gene.
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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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'''CheW''' – No homogeneity when blasted. The CheW protein is yet another chemotaxis protein. In halobacterium it is only to link with CheA (which can enable the flagella-motor), but, at the moment, we were not able to figure out whether or not this specific protein is able to interfere with other pathways. By the looks of it, it will not interfere with other pathways [5]. This uncertainty creates a few problems. The fact that not every pathway is known, could lead to unknowingly give ‘wild’ bacteria an advantage. If this were to happen, people could criticize us, for not doing science – as we are not aware of what we are doing, and should not toy with something which we cannot foresee the consequences of.
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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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'''CheA''' – is another chemotaxis protein, and is known to appear in many different bacteria. The same safety issue, or lack of such, which is described for the CheW-gene is also present here [6].
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==Risk-assessment for individual parts==
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'''CheY''' – contains signal receiving domains. Present in bunch of bacteria with flagella. The same safety issue, or lack of such, which is described for the CheW-gene is also present here [7].
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Here we consider the safety of each of our parts.
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[http://www.ncbi.nlm.nih.gov/gene/5953098[1]]
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===Method===
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[http://www.ncbi.nlm.nih.gov/gene/3703211[2]]
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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://www.ncbi.nlm.nih.gov/pubmed?Db=gene&Cmd=retrieve&dopt=full_report&list_uids=3702851[3]]
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===[http://partsregistry.org/wiki/index.php?title=Part:BBa_K343001 Monooxygenase (Part K343001)]===
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[http://www.ncbi.nlm.nih.gov/gene/37841[4]]
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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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[http://www.ncbi.nlm.nih.gov/gene/1447697[5]]
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====Potential pathogenicity====
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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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[http://www.ncbi.nlm.nih.gov/gene/5953633[6]]
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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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*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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*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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[http://www.ncbi.nlm.nih.gov/gene/5953632[7]]
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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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=== Is there a local biosafety group, committee, or review board at our institution? ===
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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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==== Which laws and guidelines we have to consider in Denmark ====
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====Environmental impact====
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'''Laws and guidelines to be considered in Denmark'''
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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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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” (eng. 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 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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'''Risk-assessment'''
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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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====Possible malign use====
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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;">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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''To that end [...to avoid adverse effects on human health and the environment which might rise 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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===[http://partsregistry.org/wiki/index.php?title=Part:BBa_K343000 Hyperflagellation (Part K343000)]===
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Article 4.2
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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 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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It is required of us to make a 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 a list of the minimum elements required:
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====Potential pathogenicity====
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1.   Assessment of potential harmful effects, defined as
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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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a)    Disease in human beings animals or plants
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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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b)    Harmful effects resulting from inability to cure disease
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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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c)    Harmful effects resulting from organisms establishing itself in nature
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====Environmental impact====
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d)    Harmful effects resulting from the organism, through natural processes confers part of its genome, such as heightened resistance, to other organisms in nature
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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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2.   Resulting from
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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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a)    The host-organism to be modified
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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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b)    The parts inserted into or otherwise used to alter the organism
 
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c)   The vector
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===[http://partsregistry.org/wiki/index.php?title=Part:BBa_K343003 Photosensor (Part K343003)]===
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d)    The donor-organism         
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====General use====
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e)    The resulting modified organism
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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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3.    Characteristics for the organism's activity
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====Potential pathogenicity====
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4.    How potent the potential harmful effects are
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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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5.   The likelihood of harmful effects being realized
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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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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. See appendix I for the risk-assessment we made for our project.
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====Environmental impact====
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'''Personal safety'''
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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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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. All access to the lab by non-members of this group or the lab-staff is to be restricted.
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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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All members of our team have in the time prior to the work in the laboratory received a lab-safety-course, thus fulfilling the requirement. See appendix II for the actual safety guidelines laid down by our local work-safety group.
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====Possible malign use====
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'''Substitution'''
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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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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, favoring safety above economical issues.
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===References===
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As we're working with relatively harmless strains of E. coli (mg1655 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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<p style="text-align: justify;">
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==== Assessment by local bio-safety group ====
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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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</p>
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'''The Group'''
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“Arbejdsmiljøgruppen” (eng. The Working Environment Group) 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, its 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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<p style="text-align: left;">
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</p>
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'''If they perceived an increased risk due to work being performed by relatively inexperienced students'''
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<br>
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__TOC__
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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 Working Environment Group, 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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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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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 a level 1 GMM, which due to its 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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We have at all times upheld the laws and regulations imposed upon us by UN and by the Working Environment Group. We have performed a risk-assessment of our project as required by the UN, as well as following the laws regarding to personal safety and to substitution of potentially harmful host and donor organisms. The work safety group has assessed our project to be a class 1 project, as they have perceived no risk associated with our work.
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They see no apparent way of weaponizing or in any other way using our project for malign purposes. Thus our project should not pose any threat to the security of the world at large. Although most of the genes inserted into our bacteria are harmless, hyper-flagellation is in fact something that increased pathogenicity, due to heightened mobility. Further the bacteria we work with are unable to survive and reproduce outside of laboratory conditions. Should it accidentally be released into the wild it would lose its plasmids within a very short time span and thus return to a non-GMO state. And as the bacteria we have been working with, namely E. coli mg.1065, is a naturally occurring bacteria, it should not pose any threat to the environment at all.
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As long as the normative work safety protocols were followed they could not perceive any danger due to the work being performed by relatively inexperienced students.
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Thus they perceived no security nor safety 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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[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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==== watermarking standard ====
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Watermarking
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To increase public safety we propose to introduce a water-marking standard
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Following the example of J. Craig Venter, who, in may 2010, created the first watermark in a bacteria, containing several readable messages, we propose to create a watermarking standard to increase the safety of the environment, as well as the safety of the community at large.
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'''Why we should consider watermarking'''
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But why should we consider creating and using a watermarking standard for work in synthetic biology? Let us consider the following example.
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A company has created a synthetic organism capable of absorbing harmful substances from the ground. The government, eager to help clean polluted ground, releases the bacteria into the wild, confident that the bacteria will not pose any threat to the environment. These bacteria do not, however, react as planned. Instead of absorbing only the harmful substances from the ground, they transfer the substances to other organisms, or simply run amok spreading themselves in an uncontrolled manner, causing harm instead of good. Let us imagine that this happens close to the border. The government in the neighboring state finds itself with a unexplained biological phenomenon, possibly causing great harm. Should the rogue bacteria contain engineered, watermarked parts, it would be a small matter to have the parts sequenced, thus the government would be able to easily access all the relevant information on the rogue bacteria, contact the manufacturers and would know how to stop it.
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'''The watermark'''
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'''We believe that a watermark should contain the following:'''
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  <nowiki>  I.        A 12 nucleotide ‘license’</nowiki>
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We have set the following criteria for a good watermark:
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<nowiki>
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    I.        It should contain the creating team’s ‘license’
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  II.        It should not interfere with the other functions of the part
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  III.        It should be persistent in the plasmid, i.e. not be removed from the plasmid due to natural evolution
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IV.        It should be easy to find, easy to read and easy to insert by the developing team
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</nowiki>
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We have set the following criteria to the team page in the parts-registry:
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  <nowiki>                                       
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    I.        information on the creating team
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  II.        the name of the part
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  III.        a description of the part
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IV.        the risk-assessment performed by the creating team
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  V.        information on how to neutralize organism, and, if available, kill-code
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</nowiki>
+
-
       
+
-
 
+
-
'''Placing the watermark'''
+
-
 
+
-
The initial thought was to insert the watermark into the genome of the bacteria, as to increase the stability of the watermark in the bacteria. However, as all parts inserted into the bacteria are placed in plasmids, it would make no sense to insert the watermark into the genome. The bacteria could transfer their plasmids to other bacteria, and retain their watermark, and the watermark would have become useless, persisting in bacteria that have no modified material at all.
+
-
 
+
-
Also the thought was to mark the bacteria, and not the parts. Thus we could have a single watermark to cover the entire modified organism. However, this presents us with many of the above problems. Should anyone encounter a rogue bacteria which has lost some of its plasmids, the watermark would be useless. It would only cover the entire modified system, and should some, or all, the plasmids have been discarded by the bacteria, the watermark would be useless. We therefore propose to mark every plasmid, before and after the coding sequence. The watermark will therefore be split into two separate components. The team creates a watermark for every part, and inserts it into the plasmid, before and after the coding sequence.
+
-
 
+
-
We chose to divide the code into two separate parts for two reasons. Firstly, being split into two equally long parts at each end of the coding sequence ensures symmetry, which again should help make insertion of a watermark easy, as this will reduce the complexity of the primers we need to design to insert them. Secondly this ensures that identification is done easily, since the combination of nucleotides of the sequence from E to X and from S to P is known. Thus we effectively increase the sequence we search for from 6 to 26 nucleotides.  Also this means that the risk of a naturally occurring combination of nucleotides, identical to the synthetically created one decreases.
+
-
<nowiki>
+
-
 
+
-
E      X        Start                                              End              S          P
+
-
 
+
-
|------|----------|-------------------------------------------|---------------|---------|
+
-
 
+
-
    watermark 1        coding sequence              watermark 2         
+
-
 
+
-
    license part 1                                                                                      license part 2
+
-
 
+
-
</nowiki>
+
-
Sequencing the part should therefore yield the watermark, which could then be accessed, read and understood.
+
-
 
+
-
'''Alternative placement'''
+
-
 
+
-
We also thought about the possibility of placing the watermark between the cutting sites of E and X, and S and P respectively. Since this area is a spacing area, it might be a good spot to insert the nucleotide combination we want. Difficulties this might cause include disturbing ... (hvad præcist? – LC snakkede om at kombinationen blev brugt til at forstærke et eller andet..). Another idea was to expand the restriction sites E-X and S-P, but we are uncertain whether this might disturb the functionality of the part.
+
-
 
+
-
 
+
-
 
+
-
'''Size and design'''
+
-
 
+
-
 
+
-
Placing the watermark after the restriction sites, it should be relatively small, as to not interfere with the functionality of the part into which it is inserted. We propose that the watermark should be 12 nucleotides, divided in two groups of six which will allow for 4096 combinations each.
+
-
 
+
-
The watermark must not contain any restriction-enzymes or stop codons. If the watermark accidentally contained a restriction-enzyme or a stop codon, the watermark would interfere with the function of the part into which it is inserted, and would likely render the part useless. This severely restricts the number of combinations we can use.
+
-
 
+
-
Ideally we would have liked to use more nucleotides, as we would have been able to generate more combinations. But the watermark should be as small as possible as not to interfere with the functions (i.e. cause a frame shift) of the part and not make the design of primers unduly complicated. We could make relatively long watermarks to satisfy our need for a very large number of possible combinations, but it would make the design of the necessary primers extremely complicated, and would go against our goal of making the insertion of watermarks as small and easy a procedure as possible. We believe that the best compromise between the amount of combinations and the ease of insertion would be at around 12 nucleotides. [RÆSONNEMENT? udregninger?]
+
-
 
+
-
We talked about whether or not we should mark every part or only create a watermark for each composite part. [HVAD BLEV VI LIGE ENIGE OM, HVIS NOGET?]
+
-
 
+
-
 
+
-
'''License'''
+
-
 
+
-
 
+
-
Every team, and lab / company which works with synthetic biology will be assigned a specific “license plate”, unique to them and being their ‘ID’ on parts-registry. An extension to parts registry’s search function should then be added. If its database contains information on each “license”, one could easily find information on a foreign organism, in case they found it in the wild (that is, anywhere else than the lab), and had it sequenced afterwards. Upon entering the parts-registry, one should then be able to enter the license and gain access to information on the parts that the team / lab / company has created. Anyone should be able to enter the license code into the parts-registry and gain access to all information on the creating team, the parts created by the team, and of course some contact information, in order to seek advice.
+
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+
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'''Procedure'''
+
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+
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Retrieval of the watermark should be easy, in the event of a rogue bacteria spreading havoc in the environment. Standardized placement of the watermark within the part should make it possible to easily retrieving the watermark through sequencing. Thus, if a rogue bacteria is discovered, it should be a simple matter to sequence the part, and obtain the license. Then it is just a matter of consulting the parts-registry to access all available information on the part and on how to contact the creating team. Getting a bacteria sequenced is a technology which is easy to access, and a lot of companies worldwide offers sequencing of bacteria, in addition it is a relative speedy process, which further makes the identification of a malign bacteria, and its properties relatively easy.
+
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+
-
 
+
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''A full description of the modified organism''
+
-
 
+
-
A full description of the modified organism should ideally contain the following information
+
-
 
+
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A. characteristics of the host and donor organisms
+
-
1. Name(s) of the organism(s) in question
+
-
 
+
-
2. Origin of organism(s) in question
+
-
 
+
-
3. Information on the reproductive cyclus of the parental organisms as well as the host
+
-
 
+
-
4. Description of any previous genetic modification
+
-
 
+
-
5. Stability
+
-
 
+
-
6. Details concerning pathogenesis, virulence, infectivity or toxicity
+
-
 
+
-
7. characteristics of endogene vectors:
+
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+
-
-sequence
+
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+
-
-mobilisation
+
-
 
+
-
-specificity
+
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+
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-the presence of resistance-genes
+
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+
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8. host spectrum,
+
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+
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9. potentially significant physiological traits and the stability of these traits
+
-
 
+
-
10. natural habitat
+
-
 
+
-
11. significant role in environmental processes 
+
-
 
+
-
12. Competition or symbiosis with other naturally occurring organisms
+
-
 
+
-
13. Ability to create survival structures (i.e. the ability to create spores)
+
-
 
+
-
 
+
-
 
+
-
B. characteristics of the genetically modified organism
+
-
 
+
-
1. origin of the genetic material used to modify the organism, as well as the intended functions of this material
+
-
 
+
-
2. Description of the modification, including the method of vector insertion in the host organism, as well as the
+
-
method used to create the genetically modified production-organism
+
-
 
+
-
3. the function of the genetic modification
+
-
 
+
-
4. origin and characteristics of the vector
+
-
 
+
-
5. structure and size of vector in the genetically modified production-organism
+
-
 
+
-
6. stability of the organism with respect to genetic traits
+
-
 
+
-
7. mobiliseringshyppigheden of the inserted vector and/or the organism’s ability to transfer genetic material
+
-
 
+
-
8. activity of the expressed protein
+
-
 
+
-
 
+
-
 
+
-
C. Health concerns
+
-
 
+
-
1. Toxic or allergenic properties
+
-
 
+
-
2. Product risks
+
-
 
+
-
3. The genetic modified organism’s pathogenic properties compared with the donor – or the host organisms or possibly
+
-
the donor organism
+
-
 
+
-
4. Colonization ability
+
-
 
+
-
5. If the organism is pathogenic to humans, who are immune competent:
+
-
 
+
-
a) Cause illness and the pathogenic mechanism, including invasiveness and virulence
+
-
 
+
-
b) infectivity
+
-
 
+
-
c) infective dose
+
-
 
+
-
d) host range, possibility of change
+
-
 
+
-
e) possibility for survival outside the human host
+
-
 
+
-
f) The presence of vectors or other distribution areas
+
-
 
+
-
g) Biological stability
+
-
 
+
-
h) resistance patterns against antibiotics
+
-
 
+
-
i) allergencity
+
-
 
+
-
j) chance for suitable disease treatment
+
-
 
+
-
 
+
-
 
+
-
D. Environmental concerns
+
-
 
+
-
1. factors that might affect the organism’s ability for survival, reproduction and it’s ability to spread in the
+
-
environment.
+
-
 
+
-
2. techniques for detection, identification and surveillance of the modified organism
+
-
 
+
-
3. techniques for detection of transfer of genetic material to other organisms
+
-
 
+
-
4. known and expected habitats of the modified organism
+
-
 
+
-
5. description of ecosystems into which the organism could spread in the event of an accident
+
-
 
+
-
6. expected result of interaction between the modified organism and naturally occurring bacteria that would be
+
-
affected in the event of an accident
+
-
 
+
-
7. known and expected effects on animals and plants, with regards to pathogenesis, virulence, infectivity, toxicity,
+
-
allergenicity, colonisation
+
-
 
+
-
8.known or expected contribution to bio-geo-chemic processes 
+
-
 
+
-
9. methods for decontamination of the area in the event of an accident
+
-
 
+
-
 
+
-
[https://www.retsinformation.dk/Forms/R0710.aspx?id=12325 [3]]
+
-
 
+
-
Once again please note that this is only intended as a guideline on how to characterize the part in the most wholesome manner.
+
-
 
+
-
'''Risk-assessment in conjunction with the use of this part in a particular organism.'''
+
-
 
+
-
Should the part, or a number of parts, be inserted into an organism the team should perform a risk-assessment and make it available on the parts-registry. In some countries, it is mandatory to submit a risk-assessment prior to engaging in a project involving synthetic biology, so we believe that any risk-assessments should be made public through parts-registry.
+
-
 
+
-
'''Inclusion of copyright information?'''
+
-
 
+
-
We do not believe in any form of copyright prohibition. We believe in an open-source approach to the field of synthetic biology, as in iGEM. Any copy-right prohibitions would only stall the progress in this most vital field of science. We believe that any and all information on created parts, and experience with these parts in particular organisms, should be shared freely.
+
-
 
+
-
'''Information on how to neutralize bacteria'''
+
-
 
+
-
This clause is intended as security measure. Should the bacteria be released into the environment, the parts-registry site should contain information on how to neutralize the bacteria. If the bacteria has an kill-code inserted, the site should describe how to enact the self-destruct mechanism.
+
-
 
+
-
Anticipated problems
+
-
 
+
-
'''Code deterioration'''
+
-
 
+
-
The code will deteriorate over time due to mutation. This could prove to be a serious problem should watermarking become an integrated part of synthetic biology. Should the genetic watermark deteriorate to the point where one is no longer able to read it, it would not constitute any kind of safety measure, being able to tell that the part was likely to have been made artificially being the only thing we would be able to tell.
+
-
 
+
-
Knowing that data will deteriorate, it may be impossible to determine whether the watermark found in a rogue bacteria is authentic or a degenerate. The deterioration is however slow and arbitrary. Our code is so small, that the change that any nucleotide associated with the watermark is going to mutate is very limited. The chances of finding the authentic code intact should be very good.
+
-
 
+
-
'''The open source approach'''
+
-
 
+
-
Sharing all this information on creating new synthetic parts that can be inserted into living organisms, also means that people with harmful agendas have access to this knowledge. This naturally means that wrongful actions are possible by use of synthetic biology, but we do not think that this should stand in the way of all the possibilities synthetic biology holds. Also, the more we know about how to create synthetic DNA strands, the better we are equipped if any harmful incident should occur. As of now, it might also just be easier to drop a bomb or send out letters of anthrax.
+
-
 
+
-
Should a bacteria be used for a malign purpose it would be quite easy to insert a false watermark to blame others. So we need to keep in mind that a plot could be made against someone. However, if anyone was interested in harming as many as possible, this person probably wouldn’t care about watermarking at all. This also means that we cannot expect watermarking to play a part in any legal case.
+
-
 
+
-
==== kill-code ====
+
-
 
+
-
-Why should we consider inserting kill-codes in genetically modified organisms
+
-
-What should an efficient kill-code contain
+
-
-Which bacteria should have a kill-code inserted
+
-
-When should one enact a kill
+
-
-Should it be mandatory or optional
+
-
-Availability of the kill-code
+
-
 
+
-
''Why should we consider kill-codes''
+
-
 
+
-
Why should we consider inserting an self-destruct device into modified bacteria?
+
-
No system is completely safe. Accidents, no matter how statistically unlikely, will occur. This is especially true when human beings are involved.
+
-
 
+
-
An efficient kill-code should
+
-
 
+
-
    -be activated by an efficient signal
+
-
    -be persistent
+
-
    -terminate the bacteria within a very short time span
+
-
    -not interfere with other functions in the bacteria
+
-
 
+
-
''Efficient signal''
+
-
 
+
-
What is to be considered an efficient signal? It should be a signal that
+
-
 
+
-
-We can control
+
-
 
+
-
-We can induce at will
+
-
 
+
-
-That is unlikely to affect other organisms in any harmful way
+
-
 
+
-
Would it be sensible to use a naturally occurring signal? Would be beneficial should the bacteria be released into the environment, where the naturally occurring signal would help destroy the rogue bacteria within the shortest possible time span. Of course, it would only be usable if the laboratory does not itself emit, or at least is able to shield the organism, from the activating signal.
+
-
An example could be that the kill-code is activated by light of a certain wave-length. If the sun emits this wave-length of light, it should destroy any bacteria that might have been released into nature. It is very easy to shield the organism from the light of the sun in the laboratory, and thus we will not accidentally destroy controlled organisms. Thus we could satisfy the three criteria I have listed above: we can control light of a certain wave-length, at least in a laboratory environment. We can induce this light at will and, thirdly, this light will not harm any other organisms.
+
-
One of the major cons of using a naturally occurring signal is that it would be almost impossible to use the organism, in case it would serve any environmental purposes.       
+
-
 
+
-
''Persistence''
+
-
 
+
-
The kill-code would be left useless should the bacteria dispose of the code through natural evolution within a very short time. Should the bacteria accidentally or, being subject to malign use, intentionally be released into the environment, we would be unable to enact the built-in kill-code, if the code is not linked in some manner to a vital part of the bacterium's genome. If the code is linked to an essential part of the bacterium's genome, it should be unable to dispose of the code without self-termination, thus ensuring persistence. Without the requirement for persistence, the kill-code would give a false sense of security, not knowing if the code is still present in the organism in question.
+
-
 
+
-
''Termination within a short time span''
+
-
 
+
-
The shorter the amount of time before the signal is enacted, 'till the rogue bacteria is destroyed, the less harm it is likely to cause.
+
-
''
+
-
Non-interference with other functions''
+
-
 
+
-
''Which type of bacteria should contain kill-codes''
+
-
 
+
-
We suggest level 3 and 4 bacteria would be edible for insertion of a kill-code. Level 1 bacteria pose little to no threat to human beings or the environment, and insertion of a kill-code would not be relevant. Level 2 organisms too would not pose any notable threat, and insertion of a kill-code would be overkill.
+
-
Level 3 and 4 organisms however pose moderate to serious threat to human beings, animals, plants and the environment at large. Should any of these organisms escape into the outside world, they would cause considerable harm to the milieu.
+
-
 
+
-
 
+
-
== Watermark generator ==
+
-
[http://jingz.dk/converter/index.php Hej]
+
-
 
+
-
</div>
+
-
<div id="rightcolumn">
+
-
Here you will find what ever your philosophic and science merged heart, may dream of.
+
-
</div>
+
</div>
</div>

Latest revision as of 19:01, 27 October 2010

Retrieved from "http://2010.igem.org/Team:SDU-Denmark/safety-b"