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| - | =='''Project Overview''' == | + | |
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| | + | =='''StickE. Coli : Single Protein Attachment of Escherichia coli''' == |
| | [[File:TU-Delft_websiteopvulplaatje4.png|right|x200px]] | | [[File:TU-Delft_websiteopvulplaatje4.png|right|x200px]] |
| - | Our aim of competing in the 2011 iGEM competition is to engineer a bacterial strain to give it new properties in order to achieve controllable adhesion of microbial reactors. The main theme to achieve this aim is to control cell-cell attachment and cell-surface attachment in such a way that it is applicable for industry and fundamental research.
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| - | In our project we strive for full control of the attachment and detachment of cells. In nature attachment consists of a complex network involving an extracellular matrix containing a wide variety of compounds. This complexity has hindered easy regulation. We will give ''Escherichia coli'' a much simpler but equally effective way of binding: mussel glue. Expressing the strongest protein responsible for the attachment of mussels to rocks, we can allow ''E. coli'' to strongly attach to even glass and plastic, whenever we want it, and subsequently releasing it again. This system should be viewed in the same category as “ the wheel”, by itself it is just a neat trick, but combination is key. Combining it for example with an ''E. coli'' capable biocatalytic conversion , one can create microbial production lines, use attachment for temporary rapid settling of biomass before product removal, or achieve fundamental premiers like bacterial cells forming a micro circle on command.
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| | + | Natural attachment of micro-organisms relies on a complex network of varying compounds known as biofilms. This complexity hinders an easy control and regulation of attachment and detachment. We will give Escherichia coli a simple, effective and controllable mechanism for biofilm formation, based on the strong glue from mussel feet. E. coli, expressing the strongest-binding mussel foot protein Mfp5 on the outer cell surface, can robustly attach to a wide variety of surfaces, including glass, plastic and itself.<br/> |
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| | + | Using highly sensitive TIRF microscopy and atomic force measurements we visualize and characterize the localization and attachment of cells. Combining these results with our mathematical models allows us to predict the attachment speed and stability as well as cell clustering and settling. The controllable, strong attachment opens up new possibilities for the use of bacterial machines in environmental applications, medicine and industry. |
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| | ===Workflow=== | | ===Workflow=== |
| - | Our workflow has been designed in such a way that each project is individual and independent. All the projects however contribute to one greater project. | + | Our workflow has been designed in such a way that each project is individual and independent. All the projects however can enhance each-other and contribute to one greater project. |
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| | [[File:TUDelft-Horizontal_Workflow.jpg]] | | [[File:TUDelft-Horizontal_Workflow.jpg]] |
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| - | In March we started by gathering project ideas with the team members, advisors, and supervisors. In April we did a lot of research on bioadhesion and mussel foot proteins. In May we started with sponsoring and working on our wiki. In June we designed our first BioBrick. We did some modeling and worked out our lab plan. Also, we made a plan to make the Dutch society aware of synthetic biology. Now, in July, we go on with al the subprojects we started. By the end of September, we hope to have succeeded in all our separate projects and reached our goal on scientific level. We hope to have a good modeling plan, well characterized Biobricks, a fruitful collaboration with other iGEM teams and the Rathenau Institute and wonderful exposition in the Science Centre. | + | In March we started by gathering project ideas with the team members, advisors, and supervisors. In April we did a lot of research on bio-adhesion and mussel foot proteins. In May we started with sponsoring and working on our wiki. In June we designed our first BioBrick. We did some modelling and worked on our lab plan. Also, we made a plan to make the Dutch society aware of synthetic biology. Now, in July, we continue with all the subprojects we started. By the end of September, we hope to have succeeded in all our separate projects and reached our goal on a scientific level. We hope to have a good modeling plan, well characterized Biobricks, a fruitful collaboration with other iGEM teams and the Rathenau Institute and a wonderful exposition in the Science Centre. |
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| - | =='''Safety proposal''' ==
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| - | '''
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| - | ''Biosafety regulations''
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| - | Since 1997 Dr. L.A. (Lesley) Robertson is the Biological Safety Officer (BSO) of the Faculty of Applies Sciences of Delft University of Technology, she manages biological safety issues. She has knowledge of the laboratory practices and procedures within the faculty and oversees the biological safety program of the faculty.
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| - | Our faculty has ML-1 laboratories and one ML-2 laboratory, but we are only allowed to work in ML-1 labs for which the basic microbiology laboratory rules apply. Internationally, there are strict rules on genetically modified organisms (GMO). A license is required to be allowed to work with GMO’s. Only if the risks to humans and the environment are minimal to negligible, the government grants a license. The rules for working with GMO’s have to be followed by the laboratory researchers. For each laboratory one person is responsible for the compliance to the rules on that specific lab. Dr. L.A. Robertson has the final responsibility for all the labs. This means that she always knows what biological material is used, where and by whom.
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| - | Before you are allowed to work in a laboratory you have to pass a Biosafety Test, which includes identifying of possible hazards in a lab, and doing a so called “washing test”. This includes all the basic rules for researches to be allowed to work on the lab. All team members passed this test successfully!!! Afterwards she explained us under which permits our project is conducted and which permits we should verify to be sure it is allowed.
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| - | Therefore, a research proposal was provided to Dr. L.A. Robertson. The research proposal included a table containing the micro-organisms, plasmids and existing BioBricks we intended to use. To ensure that we’ll work within the permit, there are two lists which state all the allowed vectors and hosts. These lists are provided, just like the permit, by the Dutch Ministry of Housing, Spatial planning and the environment. During the writing of our research proposal, we constantly verified in these lists if our plans were allowed. After Dr. L.A. Robertson read or research proposal and verified all the vectors and hosts, she gave us the permission for our project. Now we can finally start our project, informing our BSO Dr. L.A. Robertson regularly on our progression in the lab and what we are planning to do in the near future.
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| - | '''''“Safety Issues” related to our Project'''''
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| - | =====Our Parts:=====
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| - | Although, working for iGEM is a great opportunity to design your “amazing” new organism and have fun doing that, as in every scientific project, there are also safety issues that have to be taken seriously into account. It was very important for us, during the whole design of our iGEM project, to take into consideration all the safety parameters that our project and especially our parts could raise.
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| - | Because, by regulation we are permitted to work only in ML-1 laboratories, we were aware that we will not work with hazardous and infectious host organisms and genes. Specifically, all the genes and devices that we use should be subject to regulations laid down by the Dutch Government. In our faculty of Applied Sciences and more specifically in Kluyver Laboratory of TU Delft, our team is allowed to work only in ML-1 laboratories with organisms labelled H1 and parts which are originated from non-infectious organisms by the regulation. All of them are already commercially used systems. As a result, all the parts we will construct can be considered as harmless. All our ideas and plans are verified and approved by our BSO. (Biological Safety Officer)
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| - | =====Researchers’ safety:=====
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| - | Another safety aspect that we have to be aware of is our own safety in the laboratory, the so called “researchers’ safety”. In this case, we had to verify the safety of our biological material but also the safety of all the techniques and chemicals that we intended to use.
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| - | As mentioned earlier, all our organisms are classified as Risk Group 1, which contains microorganisms with no recogna. While working in ML-1 laboratories, we have to deal with strains which are indicated that they have low existent virulence; however it is not non-existent virulence. These microorganisms are considered not to be hazardous for healthy persons. However, organisms labelled H1 have been shown some infectiveness for immunocompromized persons. This seems not to be a problem in our case, because luckily all team members can be considered healthy adults.
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| - | On the other hand, regarding the laboratory techniques and chemicals that we are planning to use, there are some aspects that we have to be careful about. For our own safety in the lab we need to be careful with materials such as Bisacrylamide (cross-linking agent for the preparation of polyacrylamide gels) and ethyl bromide (chemical compound of the haloalkanes group). These materials are regarded as “dangerous, potentially carcinogenic substances”. Nevertheless, if everybody works according to a good laboratory practice, there will be no risks involved.
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| - | =====Environmental and public safety:=====
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| - | Last but not least, the environmental and public safety was one of the most serious considerations of ours. In our project, the most important issue we had to consider about was the choice of the host organisms that we decided to use.
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| - | The strain that we use is the E.coli strain K12, which is a specifically weakened laboratory strain. This strain is well-adapted to the laboratory environment, and unlike wild type strains, has lost its ability to compete with natural organisms outside of the laboratory and in human organism. Therefore our E.coli strain does not pose a threat to the public or the environment.
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| - | Finally because our project is basically targeted in fundamental and industrial purposes, the organisms are not designed to be released in the environment! The organisms are designed to be used in a closed system, which of course involve specific rules. This makes our current contribution in synthetic biology lacking of environmental risk.
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| - | '''''Biosafety for the “FUTURE”'''''
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| - | The iGEM competition should keep working with well-known organisms. Off course projects, where the toxicity of organisms can be studied and be regulated, may be also very interesting and with huge contribution in the scientific field that we represent. However, it should be well contained and guided by experienced supervisors and scientists. Students should be well informed on lab regulations, especially when working as interdisciplinary teams.
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| - | A categorized and government controlled system of lab experienced certificates should be implemented. This is already in place for nuclear laboratories, but not yet for the biohazard laboratories. This would give more clarity about who is capable to work in such environments, creating also the awareness that these hazards deserve.
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| - | An obvious solution is to confirm Event Tree Analysis (ETA) and Fault Tree Analysis (FTA) as a compulsory part of the iGEM-designing phase. This will significantly limit the risks of all iGEM projects and student will learn to take Biosafety issues into account already before they are going to work in the laboratory. Also for the Biosafety point of view and as mentioned above, in engineering of organisms it is recommended well-known microorganism to be used. The reason is because there is more knowledge on how internal systems work and intertwine. This will make an ETA and an FTA more effective and therefore the risks will be reduced.
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| - | The last years it is well known the existence of a serious debate about the field of synthetic biology, and generally about genetic engineering, and what this area can offer. The way that public addresses that issue, is based in the personal knowledge and opinion that every one of us has for the idea of GMOs but it also depends on the media exposure of the topic. It is a novel and “exotic” technique, something that is not naturally occurred. This fact seems to be the source of the majority of the conflicts. People believe in catastrophic consequences and in the lack of benefits that this area can offer.
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| - | The most of the times, this is the result of the limited and sometimes negative overall media coverage of the topic. The iGEM contest, representing the new generation of scientist working on the field of Genetic Engineering, should make synthetic biology issues more clear to the public.
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| - | It is our task while working on iGEM to send out the correct view to the public about the field of synthetic biology and a very straight and easy way to do this is the proper communication between the media and scientists. As a result, it is very important the media and the public to be approached carefully and be informed properly. Without reason negative media attention should be prevented, but it should also be our duty never to cover up the truth and be aware of the consequences that we can create as scientists!
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