Team:KULeuven/Project

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__NOTOC__
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== Dr. Coli,  the bacterial drug delivery system ==
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Our team’s project is Dr. Coli, an ''E. coli'' bacterium that produces a drug when and where it is needed in the human body. It does this in an intelligent way, such that the drug production meets the individual patient’s needs. And when the patient is cured, Dr. Coli eliminates itself from the body. To achieve this, a molecular timer registers the time since the last disease signal sensed. Then after a certain time, Dr. Coli self-destructs. However, when the disease flares up again – above a certain noise level - the timer is reset and new drug is produced. Finally, the timer will not start counting during the production of Dr. Coli, thanks to its disease-memory.
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[[Image:kulbanner.jpg|960px]]
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Dr. Coli thus has several advantages over classical drugs, and, if proven successful, could have many medical applications. One example could be the delivery of a vasoactive intestinal peptide as a potential treatment for Crohn's disease.
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=Project brainstorm=
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== Favourite previous iGEM projects ==
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Within the time frame of the iGEM competition, we aim at delivering a proof of concept of Dr. Coli. For the input and output of the system, i.e. sensing the disease signal and producing the appropriate amount of drug, we use a dummy system. The most important assets of our proof of concept are the different control mechanisms. Since these are very much dependent on kinetic and other constants, Dr. Coli heavily relies on proper [https://2008.igem.org/Team:KULeuven/Model/Overview modeling].
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<br>
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==Essential aspects of Dr. Coli==
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'''Maarten Breckpot'''
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===Dr. Coli delivers drugs ''in situ''===
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<div class="floatleft">[[Image:pictogram_input.png|40px]]
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[[Image:pictogram_output.png|40px]]</div>
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Dr. Coli produces a drug when it senses a certain disease signal in the human body. In our project, we replaced the input disease marker by a dummy [https://2008.igem.org/Team:KULeuven/Project/Input input] of anhydrotetracyclin. The proportional [https://2008.igem.org/Team:KULeuven/Project/Output output] drug production is mimicked with a fluorescent protein.
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'''Nathalie Busschaert'''
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===Dr. Coli self-destructs===
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* [http://parts.mit.edu/igem07/index.php/Imperial/Infector_Detector/Introduction Infector detector] - Imperial College 2007
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<div class="floatleft">[[Image:pictogram_filter.png|40px]]
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* [http://parts.mit.edu/igem07/index.php/Edinburgh Self-flavouring yoghurt] - Edinburgh 2007
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[[Image:pictogram_inverter.png|40px]]</div>
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* [http://parts.mit.edu/igem07/index.php/Paris Synthetic Multicellular Bacterium] - Paris 2007
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<div class="floatleft">[[Image:logo_reset.jpg|40px]]
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[[Image:pictogram_celldeath.png|40px]]</div>
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When the patient is cured, Dr. Coli is no longer needed and will eliminate itself from its host. Therefore, we placed an [https://2008.igem.org/Team:KULeuven/Project/Inverter inverter], a [https://2008.igem.org/Team:KULeuven/Project/Inverter molecular timer] and a [https://2008.igem.org/Team:KULeuven/Project/CellDeath cell death] mechanism in cascade. When no input signal is present, the inverter initiates the molecular timer, eventually leading to cell death. Upon renewed presence of the disease signal, the molecular timer is [https://2008.igem.org/Team:KULeuven/Project/Reset reset]. A [https://2008.igem.org/Team:KULeuven/Project/Filter filter], finally, ensures that the timer is not reset when only “noisy” disease signals  are sensed.
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'''Jonas Demeulemeester'''
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===Dr. Coli in production===
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* [http://parts.mit.edu/igem07/index.php/Ljubljana Virotrap Ljubljana 2007]
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[[Image:pictogram_memory.png|40px|left]]
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* [http://parts.mit.edu/igem07/index.php/Princeton RNAi enhanced logic circuit Princeton 2007]
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* Other nice parts/devices:
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** Caltech: Riboswitch design for targeted cell death/molecular sensor
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** Cambridge: Inducible bigger pore protein for E.coli
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** Harvard: Quorum-sensing & targeting!
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** Melbourne: Red/blue light responsive system through chimeric photoreceptors-kinases
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** Peking U: λ-based bistable switch = very powerful
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** UCSF: compartmentalization! Rewired MAPK cascade signaling through scaffolds ≅ circuit board   
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<br> 
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'''Andim Doldurucu'''
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To enable the production of Dr. Coli without it self-destructing, we included a [https://2008.igem.org/Team:KULeuven/Project/Memory memory] device. This is a stable switch that is activated by the first input signal. Only from then on, the clock can start ticking towards cell death.
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'''Jan Mertens'''
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* [http://parts.mit.edu/igem07/index.php/Ljubljana Virotrap Ljubljana 2007]
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* [http://parts.mit.edu/igem07/index.php/Berkeley_UC Bactoblood]
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'''Benjamien Moeyaert'''
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* [http://openwetware.org/wiki/IGEM:Harvard/2006/DNA_nanostructures Harvard 20006: nanostructured DNA containers]
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* [http://parts.mit.edu/igem07/index.php/Berkeley_UC Bactoblood]
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'''Stefanie Roberfroid'''
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* [http://parts.mit.edu/igem07/index.php/Cambridge Bacteria Online]
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* [http://parts.mit.edu/igem07/index.php/Berkeley_UC Bactoblood]
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* [http://parts.mit.edu/igem07/index.php/Princeton RNAi enhanced logic circuit]
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* some other nice ideas
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** [http://parts.mit.edu/igem07/index.php/Edinburgh Self-flavouring yoghurt]
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** Detection of metals: [http://parts.mit.edu/igem07/index.php/Brown Lead], [http://parts.mit.edu/igem07/index.php/Saint_Petersburg Copper]
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'''Hanne Tytgat'''
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* [http://parts.mit.edu/igem07/index.php/Berkeley_UC Bactoblood]
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* [http://parts.mit.edu/igem07/index.php/MIT Sensing & removing Hg ions - MIT 2007]
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* [http://parts.mit.edu/igem07/index.php/Imperial/Infector_Detector/Introduction Infector detector]
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'''Elke Van Assche'''
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* [http://parts.mit.edu/wiki/index.php/MIT_2006 Eau d'E.coli MIT 2006]
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* [http://parts.mit.edu/igem07/index.php/Berkeley_UC Bactoblood Berkeley UC 2007]
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* [http://parts.mit.edu/igem07/index.php/Princeton RNAi enhanced logic circuit Princeton 2007]
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'''Nick Van Damme'''
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* [http://parts.mit.edu/igem07/index.php/Davidson_Missouri_W Bacterial Computer]
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--> idea: solve a nice mathematical problem
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*  several electronical/biological components to build an entire complex combinational logic system
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** [http://parts.mit.edu/igem07/index.php/USTC Extensible Logic Circuit in Bacteria]: both components and linking
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** [http://parts.mit.edu/igem07/index.php/Valencia Comparator]
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** [http://parts.mit.edu/igem07/index.php/Bologna Schmitt trigger]
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--> idea: build an integrator to solve your own ODE's, also build a differentiator to make a PID-controller
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'''Antoine Vandermeersch'''
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*[http://parts2.mit.edu/wiki/index.php/University_of_Texas_2006 Texas 2006: Edge Detector]
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*[http://parts.mit.edu/igem07/index.php/Rice/Project_B:_Quorumtaxis Rice 2007: Quorumtaxis]
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*[http://parts.mit.edu/igem07/index.php/Berkeley_LBL Berkeley LBL 2007: Solar Bacter]
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'''Dries Vercruysse'''
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'''Sigrid De Keersmaecker'''
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* [http://parts.mit.edu/igem07/index.php/MIT Sensing & removing Hg ions - MIT 2007]
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* [http://parts.mit.edu/igem07/index.php/Edinburgh Self-flavouring yoghurt - Edinburgh 2007]
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* [http://parts.mit.edu/igem07/index.php/Missouri_Miners Biological Timer - Missouri Miners 2007]
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* [http://parts.mit.edu/igem07/index.php/Ljubljana Virotrap - Ljubljana 2007]
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* [http://parts.mit.edu/igem07/index.php/Taipei/Taipei GlucOperon - Taipei 2007]
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* [http://parts.mit.edu/igem07/index.php/Berkeley_LBL Solar Bacter - Berkeley_LBL 2007]
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* [http://parts.mit.edu/igem07/index.php/Berkeley_UC Bactoblood - Berkeley_UC 2007]
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== iGEM judging tracks ==
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* Foundational Research - basic science and engineering research
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* Information Processing - genetically encoded control, logic, and memory
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* Energy - biological fuels, feedstocks, and other energy projects
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* Environment- sensing bioremediation of environmental state
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* Health & Medicine - applied projects with the goal of directly improving the human condition
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== Other ==
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[http://openwetware.org/wiki/IGEM:Idea_exchange Idea exchange - iGEM ideas posted by other teams]
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=Ideas for our project=
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==A first idea: cancer treatment with genetically modified blood cells==
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As cancer cells need a lot of energy to replicate themselves, they should be well provided with blood. Therefore, blood cells could be the right choice for in situ treatment of cancer. First, we should immobilize these blood cells on the cancer cells. Subsequently, these blood cells should secrete specific agents that reduce the activity of the cancer cells. (These 2 steps may come in handy if we want to split up in 2 subgroups)
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===Notes===
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* Sounds like a great idea! [http://en.wikipedia.org/wiki/Avastin Anti-angiogenic therapy] is one of the big hopes for anti-tumor treatments. But let's keep in mind that angiogenesis (the formation of blood vessels) is only a late hallmark of tumors [http://www.google.be/url?sa=t&ct=res&cd=1&url=http%3A%2F%2Fwww.weizmann.ac.il%2Fhome%2Ffedomany%2FBioinfo05%2Flecture6_Hanahan.pdf&ei=-7E2SLbLGJCE1wblseHQDQ&usg=AFQjCNHirXaVMmdQNGl-72bk5jRva4106Q&sig2=L18rRG56VaP9wIB_mAENpA (more about these hallmarks of cancer -PDF)]. It is however a '''significant barrier''' to break through if the tumor has to grow past a certain (very limited) size. So this would be more like a therapy for later-stage malignancies, which would also be great because it's often the metastasis (the spreading of) of the tumor that is causing the more visible effects of the cancer. (pain, deterioration, ... and eventually, if untreated death). - [[user:Zeunas|Jonas]] 12:09, 23 May 2008 (UTC)
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* Anyhow, if we proceed with this idea, it will be a challenge to get everything ready and produced in the erythrocyte (red blood cell) before it loses it's nucleus and thus also the ability to initiate ''de novo'' transcription. And to keep all this machinery silent in non-docked erythrocytes. I'm liking this challenge though. Besides this has an upside as well as I feel that consequences would be less severe in this non-cell if things go awry in the system. :) - [[user:Zeunas|Jonas]] 17:30, 23 May 2008 (UTC)
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* I just thought of something that might be quite critical. If I recall correctly, there are 3 main ways in which tumors acquire blood supply.
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:# The first one is through a recapitulation of embryonic development. This is the recruitment of vascular endothelial precursors or the activation of local endothelium via factors like VEGF (angiogenic sprouting or intussusceptive growth). In this case, the 'vessels' of the tumor blood supply are lined mostly with endothelial cells which are actually NOT malignant, but are kind of working together with the tumor cells.
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:# A successful cancer metastasis (a secondary tumor, derived from the original) will co-opt blood vessels and these will thus also be lined mostly with endothelium cells.
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:# The third way to achieve blood supply is through vasculogenic mimicry, where the tumor cells actually DO line the bloodstream and mimic the normal vascular endothelium. Here tumor biomarkers should be directly displayed to the passing erythrocytes and would thus be potential targets for use in this approach.
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: OK, now for my point. In all these cases the vessels are highly abnormal, both structurally and functionally. They've got many holes, inhomogeneous bloodflow, are leaky, ... so it's very likely there will be exposed markers we can focus on but this will probably not always be the case. But anyhow, I'm really liking this idea! - [[user:zeunas|Jonas]] 14:17, 23 May 2008 (UTC)
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* There will be a lot of challenges. Some things that popped in my head: 1)What is the progenitorcell we're going to work with. When you use stemcells, you have to differentiate them in vitro into red blood cells which is pretty delicate matter. 2)finding the right markers for the recognition that are not expressed on normal cells. 3) developing an assay that proves that it works. Whith tis idea there are a lot of factors that have to be taken in account and the question is if we will get a positive result in 3 months time. There's a lot of ongoing research and a lot of things ar not yet known. But I also like the idea, next year I'm going to do my thesis about lymphangiogenesis so I'm very interested in the subject :)- [[user:Jotaro|Jan]]
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:* On the biomarker issue. There's a lot of this kind of proteomics research going on at the Biology faculty/GHB. I hope we could pick up some molecules there? Because starting our own search for these and getting results in 3 months time would be quite hopeless indeed - [[user:zeunas|Jonas]] 23:46, 23 May 2008 (UTC)
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:* I agree that working with RBC will be quite a challenge. But maybe we can use bacteria with the right proteins, just to proof the principle. If it works, another team (next year?) or research group can then try to apply this principle on RBC. Anyhow, I also really like this project. --[[User:BNathalie|BNathalie]] 16:18, 24 May 2008 (UTC)
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:* We could overcome the differentiation problem if we work with a mouse model for example. We harvest (or get them from someone) CD34+ hematopoietic progenitor cells (or other progenitors, I don't know), transfect these with our constructs + maybe an extra marker like green fluorescent protein (GFP) and then reintroduce these cells into the mouse. Differentiation should proceed normally then and we could harvest the modified and differentiated red blood cells (RBCs) by FACS analysis or something similar. The problem is that I've got no idea how long a procedure like this could take and we should be sure that our constructs work properly before trying this. We could also attempt myeloablative treatment on the mouse before reintroducing the transfected cells, this would make reharvesting a lot easier but once again, no idea how long this takes ... On the other hand, if we could harvest the modified RBCs, an ''in vitro'' assay should be quite feasible. - [[user:zeunas|Jonas]] 22:06, 24 May 2008 (UTC)
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* I've been thinking about the agent we have to deliver in order to kill the cancerous cells. We could deliver Reactive Oxygen Species (ROS) like hydroxyl radicals, superoxide, hydrogen peroxide or singlet oxygen to the tumor. This could potentially be very hazardous or lethal to the tumor cells since they often have defective DNA repair machinery and would thus be unable to cope with this amount of damaging. The erythrocyte vessel we target to the tumors is extremely '''well fitted''' to do this since it's packed with iron that could potentially catalyse the production of a lot of these ROS via the [http://www.lenntech.com/Fenton-reaction.htm Fenton reaction] etc. So all that should happen after the erythrocyte docks to the cancerous cells would be to destroy some hemoglobin, set some iron free in the red blood cell and let ROS be created locally! - [[user:zeunas|Jonas]] 09:00, 24 May 2008 (UTC)
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* One last thing. The entire erythrocyte metabolism has been [http://www.tbiomed.com/content/2/1/18 simulated] ''in silico'' (including links to hemoglobin). So this project could also provide enough entertainment for the more modeling-oriented amongst us :) - [[user:zeunas|Jonas]] 23:12, 25 May 2008 (UTC)
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==A second idea: bacteria clean virusses in animals==
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This is an improvement of the idea of Ljubljana: we cannot reprogram the immune system, but we can reprogram bacteria. So, what we could do is make bacteria produce viral receptors (challenge 1) which are modified so that when a virus attaches to them, a restriction enzyme is transcribed (challenge 2). This RE degrades the viral DNA and the bacterial DNA, thus killing the bacterium. This way, the bacteria clean all virusses from the body. When this is established, we can induce a suicide signal for the bacteria (challenge 3).
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Big problems:
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* Is it possible to make a eukaryotic virus attack a prokaryote (also a fundamental question)?
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* Immunogenicity bacterium (cf. Bactoblood)! [[User:Bmoeyaert|Bmoeyaert]]
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<br>
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* I'm probably risking to get banned for nagging because of this. :P I think the tricky part would be the preferential docking of the viruses on the bacteria if you've got about 10^14 of your own cells in your body. Even if only a small subset of these would be targets for the virus, I believe the ratio of bacteria to host cells would be quite low unless you allow a very large 'bacterial infection'. This problem might be overcome if the viral receptor density is way higher on the bacteria. - [[User:zeunas|Jonas]] 17:52, 23 May 2008 (UTC)
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* Cool stuff though, the opposite system exists. A bacterial infection could be treated by administering the specific bacteriophages, here the system can also co-evolve. Problem is that you have to know the bacterial strain of the infection in order to administer the correct phage, and this can take a while. - [[User:zeunas|Jonas]] 17:52, 23 May 2008 (UTC)
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* It's an appealing concept. An eukarotic virus only has to cross the celmembrane to get its content into the human cell. When we would use E.coli, the virus would have to get  first through the outermembrane, then through the peptidoglycane and then through the innermembrane. So how does it get it in the cytoplasm? But maybe this is not necessary and is it sufficient to let the virus cross the outermembrane and come in the periplasm. If this  induces the cell to do apoptosis en kill itself, including the virus, we've got a nice system. But like Jonas said, there will always be virusparticles that infect human cells, wich are in the majority. But still, it would be quite an accomplishment to get an eukariotic virus to infect a bacterium.-[[User:Jotaro|Jan]]
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==A third idea: Bacteria that control the amount of drugs==
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=== Generic goal ===
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It's known that the effect of drugs on a human being is very dependent on the metabolism of that person. There are extensive metabolizers, people who have a normal response to a certain drug. Intermediate metabolizers show a low response and in poor metabolizers there's very little effect. Poor metabolizers need a higher amount of  drugs to respond, where ultra fast metabolizers need a lower dose. These differences in the drugmetabolism in different people is a big concern, because a wrong dose can have severe consequences. There's a need for patient-focussed therapy. If we want to know which type of metabolizer a person is, we have to know his genotype. This is a very expensive and time-consuming procedure.
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Wouldn't it be nice to develop a two bacteria-system that can replace genotyping? I' m thinking of two bacteria that live together in the intestine. The first bacteria has a sensor function. It has to monitor if the amount of the drug that is present in the intestine, is efficient. This bacteria has to signal his observations to the second bacteria. This second bacteria is the drug-producer. Like some probiotics, that are already on the market, it produces the drug at the location where it's needed. The amount of drug it produces would be controlled by the other bacteria.  In case the amount of treatment is enough, the first sensor-bacteria represses the second one. If the drug level drops again, the sensor-bacteria should re-activate the drug-producing one. In that way there's a feedback loop between the two bacterias and one can control the amount of treatment that's needed.
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--[[User:Hanne|Hanne]] 19:27, 23 May 2008 (UTC)
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* I don't think you necissarily need a 2 bacterial strain system and can also do it with one bacterial strain that has sensor- and drugproducer function. When it monitors the amount of drugs it can autoinduce itself to produce the drug and repress itself when it's enough. like the idea and think it's possible to do this in 3 months. It's also an idea in which I see our different skills can be integrated (the engineers can do modelling of pharmacokinetics for example), In the first 2 ideas that's maybe more difficult  -[[user:Jotaro|Jan]]
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=== Application to Crohn's disease ===
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* I would streamline the idea further:
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** Use a single probiotic bacteria (lots of available expertise on probiotics in the team)
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** Use a peptide or protein drug - probably a lot easier than synthetisizing a small molecule
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** As probiotic bacteria live in the gut, peptide or protein drug could probably enter the blood stream
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** Make a sensor for a signal that indicates the reaction to the treatment 
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** I was thinking about Crohn's disease (because it happens in the gut). Produce a peptide such as vasoactive intestinal peptide (potential treatment for Crohn). Sense the inflammatory response present in the gut. If high inflammation, increase drug production - up to some maximum dosage to avoid overdosing. Do not respond too fast to avoid overdosing.
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** Crohn's expertise available in Leuven (P. Rutgeerts, S. Vermeiren) --[[user:moreau|Yves]]
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* I also like this idea. However, you can't really control how long the bacteria stay inside your gut. So I think this is a system that is best used for chronic conditions (like Crohn's disease) that require continuous treatment. I like the example of Crohn's disease, because you can build in a good control devise to avoid overdosing. In this system, we need a bacteria that produces a drug only when: a) there is a low concentration of the drug inside the gut, and b) when the gut is inflamed. Practically, this means a receptor that will stop the production of the drug upon binding to the drug. I don't know how to sense inflammation though. --[[User:BNathalie|BNathalie]] 16:35, 24 May 2008 (UTC)
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* I was indeed thinking of applications for chronic disease. --Y
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* In the case of Crohn's, I think it would be quite doable to sense inflammation as there are many molecules typical of this process. We would have to look up which ones though. --Y
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* An additional advantage is that Crohn's disease is characterized by local inflammation, so that the system would probably produce more drug exactly where it is needed most. --[[user:moreau|Yves]]
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* Genetically modified ''Lactococcus lactis'' bacteria constitutively expressing Interleukin-10 (anti-inflammatory) are [http://www.ncbi.nlm.nih.gov/pubmed/16716759?ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum already in stage I clinical trials] for the treatment of Crohn's disease. The dosage regulation by sensing could be a very nice addition to a system like this. I don't know however to what degree Crohn's disease is temporally variable and thus might benefit from such a system. - [[user:zeunas|Jonas]] 23:28, 25 May 2008 (UTC)
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* People in Leuven (Gastroenterology lab) are collaborating on the studies on IL-10-producing lactobacillus. This product is being developed by a spin-off of the Flanders Institute for Biotechnology (VIB) called Actogenix (www.actogenix.com) --Y
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* I also like this idea. Like Jan says it's a project were we are going to need all our different skills. Maybe this is more interesting for the engineers among us because it needs a lot of modeling. --[[user:Stefanie|stefanie]]
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=== Control ===
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* As an engineer this also seems the most challenging proposal. To regulate the proportion of drugs it seems that we are going to need a PID-controller (proportional with the amount already available, integrating to keep the level steady and differentiating to avoid responding too fast). It would be very challenging if we could also achieve this kind of controlling system in the bacteria. --[[user:nickvd|Nick]]
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* I think you see it somewhat wrong. It's about sensing what amount of drugs a certain patient needs. So there is a variable, not only in one patient, but also between different patients! It's about sensing how much every person needs. I think the idea of implementing this system for the treatment of Crohn's disease is really interesting. It's true that Lactobacillus lactis already delivers the drugs at the right spot in the body, but the control mechanism isn't there yet. So I think we could make an important improvement to this approach. This system can than serve as a model for treating other diseases. --[[User:Hanne|Hanne]] 09:13, 26 May 2008 (UTC)
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* The reference signal for the PID would simply be the local inflammation, but does this mean the bacteria would extract no information about the specific metabolism of the host (person)? If it would, the PID-controller could be optimised for every person by means of self-tuning (retrieving optimal parameters that characterise the PID-controller behaviour and determines it stability). Anyway, I like this idea a lot. EDIT: just saw Hanne's post, this is indeed what I ment --[[User:Avdmeers|Avdmeers]]
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* Ideally, the dosage would be patient specific and variable in space and time. In space, because the disease is not uniform across the intestine (some regions are in a better condition while others are worse). But I guess that because the mixing and diffusion that happens in a fluid environment, it is hard to control for production at a very specific spot on the intestine, except if the bacteria would stick to the intestine wall itself (might be possible with the right bacteria). However, patient specific dosage and time variability would be a very interesting advantage as not all patients have the same severity of the disease and as the disease progresses with up-and-downs ("The disease is characterized by active periods, known as flare-ups, followed by periods of remission, during which symptoms diminish or disappear altogether. Its cause is not known." - ehealthmd.com). Basically, the drug would be self-dosing. --Y
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* The project does indeed bring together the interests of many people: a realistic medical application, work on bacteria that looks doable, and a tractable modeling problem based on well-known engineering principles (control engineering). --Y
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* As a security mechanism, I guess that the bacteria are simply sensitive to antibiotics and thus a course of antibiotics is sufficient to stop the treatment altogether. --Y
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* Possible extension for later: sensing adverse reaction. Some patients might respond badly to the treatment. We could have a system that senses physiological distress from the patient and then shuts down the drug production. This could be faster than giving antibiotics. Something like that might also work for patients who metabolize the drug differently (slower and faster) and for whom baseline level (i.e., for the same degree of inflammation) of the drug need to be lower or higher. --[[user:moreau|Yves]]
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* By having a system with signaling molecule - nuclear receptor - response element - reporter/drug (such as PGE2-NR4A2 - NBRE - luciferase), we implement a proportional P controller. We can set the the constant K of the proportional controller by setting the strength of the promoter (for example, with multiple copies of the response element (3xNBRE in the colon cancer construct). --Y
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* To get an integration I controller, we would need a construct that controls a protein that will accumulate in the bacteria and that then controls the reporter (signal - nuclear receptor -  response element - intermediate transcription factor - accumulation - response element - reporter). --Y
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* I have no idea about how to implement a differentiation D controller. --Y
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* Depending on characteristics of the reference signal (local inflammation) and how it responds to the output signal (drugs concentration released) --- so the reference signal is in itself not independant of the output signal, there is an implicit coupling --- certain blocks could be omitted for simplification. The differentation term is ment for minimizing overshoots during rather '''abrupt transitions''' of the reference signal, so we could need information on time delays. --[[User:Avdmeers|Antoine]]
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** remark: mathematically speaking is the reference signal the setpoint of the system that must be reached, so i think it's better not to call the local inflammation the reference signal. The local inflammation is the output of our "proces" (a CV: controlled variable) which has to be compared to the reference signal (setpoint) that i assume you take equal to zero (we want to reach zero inflammation). the difference between these signals can then be considered as error-variable that is used as input to the controller. In that way the inflammation is indeed equal to the signal that is given to the controller ... --[[User:nickvd|nick]]
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* The [http://parts.mit.edu/igem07/index.php/Valencia/Controller#Sensibility_of_the_system_to_the_different_parameters| Valencia team] for example simplified their PID-controller to a regular P-controller. [[User:Avdmeers|Antoine]]
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** That means we have found our challenge: first test their proportional controller (maybe try to improve it) and then add the integral (and maybe differential) controller. --[[User:nickvd|nick]]
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* for the integral/differential components, couldn't it be a good idea to implement the electronic equivalences and make somekind of 'simulink-diagram' that interconnects them?
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=== Sensing ===
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* You could sense several cytokines, such as TNF-alpha, interleukin-1, interleukin-6 as a measure of disease status. I guess for a cytokine, you could use a corresponding cytokine receptor as sensing device. --Y
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* An important issue is whether  those disease-marker proteins are measurable in the gut. I would assume so since they are probably produced at the inflammation site (but not sure about that). --Y
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* Other important markers are fecal calprotectin (so most probably present in the gut) and C-reactive protein. --Y
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* Like Yves, I was also thinking about what we could sense while remaining on the luminal side of the gut. The localisation of the cytokines might not be a problem since the surface of the gut lumen is severely damaged in the patient and even blood can be exposed. Since the inverse works; the IL-10 can get in, the others should be able to get out I presume. I don't know how long these proteins last in the gut though (because f degradation by other bacteria/our own proteases) and thus how long they are able to constitute a valid signal. But once again the Interleukin-10 in clinical trials seems to last long enough, so this might not be a problem either. - [[user:zeunas|Jonas]] 16:09, 26 May 2008 (UTC)
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* If we go through with this project, I feel like we should keep modularity of the system in mind. This could make the introduction of other sensors or other output molecules a lot easier if knowledge about the disease progresses. So we would basically be creating a platform for Crohn's or other inflammatory bowel diseases on which anyone could further build, switching inputs, outputs and stuff. And if we've got spare time during the summer we could implement extra sensors or switching output molecules ourselves. - [[user:zeunas|Jonas]] 16:09, 26 May 2008 (UTC)
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* PS: maybe Eicosanoids or other non-protein inflammatory molecules could also be used to detect the inflammation. These wouldn't be bothered by proteases and can travel through cell membranes so they should definitely be present in Liver Receptor Homologue-1the gut (if they are produced in Crohn's) and they can be easily picked up by nuclear hormone receptors. - [[user:zeunas|Jonas]] 16:14, 26 May 2008 (UTC)
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* Both measuring cytokines and eicosanoids seem viable options (see http://gut.bmj.com/cgi/content/full/46/4/487 with TNF-alpha and IL1-beta (and IFN-gamma) as cytokines, and Thromboxane B2 and prostaglandin E2 as eicosanoids). So it would be a question of what is easier for the experimental work. My impression though is that many eicosanoids are detected by transmembrane receptors rather than nuclear receptors. --Y
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* We still have to face the problem of what organism to use and how it will sense what we want it to sense. For example ''L. lactis'' is gram-positive with a big layer of peptidoglycan shielding off it's membrane surface. So how will the signaling molecule be sensed? The TNF-alpa trimer is quite big. If we use a gram-negative like ''E. coli'' we will still have to have our signal transduced through the periplasm. This might prove to be quite tricky - [[user:zeunas|Jonas]] 17:19, 26 May 2008 (UTC)
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* Most eicosanoids do indeed bind to GPCR transmembrane receptors but some can also bind to PPAR subfamily nuclear receptors, these form heterodimers with the retinoic X receptors and can immediately induce gene expression. With a GPCR we'd have to construct a longer signal transduction cascade in the bug (which could allow additional regulation). Nuclear or membrane, it shouldn't make a big difference for eicosanoids since they can get through the membrane anyhow. For the cytokines we only have transmembrane receptors which won't work/be accessible in bacteria I think. - [[user:zeunas|Jonas]] 22:46, 26 May 2008 (UTC)
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* Sensing is indeed a problem. For example, expression of GPCRs is toxic for the bacterial membrane (see http://linkinghub.elsevier.com/retrieve/pii/S0167779904003348). A nuclear receptor (NR) would probably be more appropriate (plus direct activation of transcription indeed). The question then is whether we can find an appropriate eicosanoid/NR combination suitable for Crohn's or whether we can engineer/evolve an NR for an eicosanoid of our choice (e.g., Thromboxane B2) (probably a lot more challenging). --Y
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* Prostaglandin E2 seems to target also nuclear receptors: Liver Receptor Homologue-1 LRH-1 (HUGO: NR5A2), which itself activates transcription of aromatase (http://cancerres.aacrjournals.org/cgi/content/abstract/65/2/657) + NR4A2 (NURR1), which binds to the NBRE response element - a construct expressing a reporter through PGE2->NR4A2->3xNBRE->reporter has been demonstrated in a colon cancer line (http://www.jbc.org/cgi/content/full/281/5/2676).
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* There are still other possibilities though. The PhoP - PhoQ histidine kinase system of ''S. typhimurium'' should be able to detect antimicrobial peptides produced by the immune system and direct gene expression changes. Also, the OprF outer membrane protein of ''P. aeruginosa'' can detect IFN-gamma (for these 2 cases we do need a gram-negative bacterium). Another even more exotic option could be to engineer a bacterial AHL receptor to recognize our eicosanoids. It should be feasible since AHL's and eicosanoids are similar lipid-derived molecules. Only thing is, that this is probably not the 3-month-time kind of feasible, especially if we don't want cross-reactions. (though it would be extremely fancy) :) - [[user:zeunas|Jonas]] 19:56, 27 May 2008 (UTC)
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=== Proof of concept ===
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* For a proof of concept, we would need to be able to set up such a system in vitro. The drug production could be "simulated" by replacing it by the production of GFP. We would then need a system for controling the level of the signaling protein (e.g., TNF-alpha cytokine). We can easily increase the level of the signaling protein by spiking in a fixed amount of it. But how do we decrease it? We cannot really have a model of the response of the gut to the drug. Maybe we could precipitate the signaling protein with an antibody? And how do we measure the concentration? By fluorescently labeling the signaling protein? --Y
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* Another setup (probably much easier) could be to
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# Grow the bacteria on a dish
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# Remove them from the original medium
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# Put them in a medium with a high concentration of signaling protein (i.e., TNF-alpha)
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# Observe that production of GFP SLOWLY ramps up (maybe up to some maximum threshold to avoid overdosing)
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# Put the bacteria back into a medium with a low or zero concentration of signaling protein
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# Observe that GFP production is turned off slowly --Y
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* As an easier, intermediate goal, we could simply have a sensing system that directly turns on the reporter (that would be like a proportional controller).
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=Our project=
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Abstract
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= Our project details=
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== Part 2 ==
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== The Experiments ==
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== Part 3 ==
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= Results =
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Latest revision as of 21:33, 3 October 2008

  dock/undock dropdown  

Dr. Coli, the bacterial drug delivery system

Our team’s project is Dr. Coli, an E. coli bacterium that produces a drug when and where it is needed in the human body. It does this in an intelligent way, such that the drug production meets the individual patient’s needs. And when the patient is cured, Dr. Coli eliminates itself from the body. To achieve this, a molecular timer registers the time since the last disease signal sensed. Then after a certain time, Dr. Coli self-destructs. However, when the disease flares up again – above a certain noise level - the timer is reset and new drug is produced. Finally, the timer will not start counting during the production of Dr. Coli, thanks to its disease-memory.

Dr. Coli thus has several advantages over classical drugs, and, if proven successful, could have many medical applications. One example could be the delivery of a vasoactive intestinal peptide as a potential treatment for Crohn's disease.

Within the time frame of the iGEM competition, we aim at delivering a proof of concept of Dr. Coli. For the input and output of the system, i.e. sensing the disease signal and producing the appropriate amount of drug, we use a dummy system. The most important assets of our proof of concept are the different control mechanisms. Since these are very much dependent on kinetic and other constants, Dr. Coli heavily relies on proper modeling.

Essential aspects of Dr. Coli

Dr. Coli delivers drugs in situ

Pictogram input.png Pictogram output.png

Dr. Coli produces a drug when it senses a certain disease signal in the human body. In our project, we replaced the input disease marker by a dummy input of anhydrotetracyclin. The proportional output drug production is mimicked with a fluorescent protein.

Dr. Coli self-destructs

Pictogram filter.png Pictogram inverter.png
Logo reset.jpg Pictogram celldeath.png

When the patient is cured, Dr. Coli is no longer needed and will eliminate itself from its host. Therefore, we placed an inverter, a molecular timer and a cell death mechanism in cascade. When no input signal is present, the inverter initiates the molecular timer, eventually leading to cell death. Upon renewed presence of the disease signal, the molecular timer is reset. A filter, finally, ensures that the timer is not reset when only “noisy” disease signals are sensed.

Dr. Coli in production

Pictogram memory.png

To enable the production of Dr. Coli without it self-destructing, we included a memory device. This is a stable switch that is activated by the first input signal. Only from then on, the clock can start ticking towards cell death.

Retrieved from "http://2008.igem.org/Team:KULeuven/Project"