Team:CSIA SouthKorea

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[[Image:CSIA_SouthKorea_team.png|right|frame|OUR FANTASTIC TEAM LOGO by Aileen Shin]]
[[Image:CSIA_SouthKorea_team.png|right|frame|OUR FANTASTIC TEAM LOGO by Aileen Shin]]
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Our team is consisted of four students who are fond of thinking creatively, sharing our knowledge with others and making contributions to the society. We hope that iGEM 2012 could be a great opportunity for us to get our feet wet in the field of synthetic biology and interact with many other students around the world who are also interested in this field!
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Our team is consisted of four students who are fond of thinking creatively, sharing our knowledge with others and making contributions to the society. We hope that iGEM 2012 could be a great opportunity for us to get our feet wet in the field of synthetic biology and interact with many other students around the world who are also interested in this field! Since our school does not have facilities for wet lab, team CSIA_SouthKorea asked professor In-Geol Choi in Korea University for guide. Professor Choi, Instructor Hyeok-Jin Ko and Hongjae Park let us to be familiar with synthetic biology, taught us how to use basic lab facilities and helped every part in our projects :)
[[Image:CSIA_SouthKorea.png|left|frame|TEAM LOGO♥]]
[[Image:CSIA_SouthKorea.png|left|frame|TEAM LOGO♥]]
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=Team=
=Team=
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[https://2012hs.igem.org/Team:CSIA_SouthKorea/ Juyeon Han ]
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: Our [[Team:CSIA_SouthKorea/team | team]] and school is introduced in this page! :)
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[https://2012hs.igem.org/Team:CSIA_SouthKorea/ Minji Kim ]
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[https://2012hs.igem.org/Team:CSIA_SouthKorea/ DaYeon Lee ]
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[https://2012hs.igem.org/Team:CSIA_SouthKorea/ Eugenie Choe ]
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=Project=
=Project=
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==Project outline, E.Coli display using repressilator==
 
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: The main mechanism that we are planning to use is the repressilator. Repressilator is a synthetic genetic regulatory network. It was reported by Michael Elowitz and Stanislas Leibler in 2000. This network is composed of 3 genes connected in a feedback loop, and each of the genes is repressed by the previous gene. This system is designed to exhibit a stable oscillation in the expression of each gene with fixed time period. In each of the wells in the 96 well plate, a colony of Escherichia Coli that expresses fluorescent protein with repressilator system will be put in. Then, by putting in different inducers in each of the wells, we are planning to control the time period and the gene expression of the E. Coli colonies and therefore express the specific shape that we are trying to express withe E. Coli display.
 
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:[[File:123.jpg]]
 
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: Using this mechanism of repressilator to make <font color="#FF9800">time difference</font> in expressing GFP, we would be able to make a certain figure on a 96 well plate. However, the periods of GFP expression in each cell will be slightly different, so we thought of applying quorum sensing to <font color="#CA19C7">synchronize</font> the periods of bacterias.
 
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: [[File:abcd.jpg]]
 
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: picture of a 96 well plate (http://avena.pw.usda.gov)
 
==Abstract==  
==Abstract==  
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: Based on the design of ''V.fischeri'', we placed luxR gene under luxpL promotor and placed luxI, Aiia, and GFP gene under luxpR promotor. In this V.fischeri quorum sensing system, LuxI synthase produces an acyl-homoserine lactone (AHL), which is a small molecule diffuses extracellularly and triggers quorum sensing. When AHL binds to LuxR, it produces LuxR–AHL complex that activates luxI promoter. This also activate GFP genes, so fluorescence can be detected. AiiA 'represses' continuing activation of luxI promotor by degrading of AHL. Therefore, fluorescence may have the cycle under right conditions.
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: Based on the design of ''V.fischeri'', we placed luxR gene under luxpL promotor and placed luxI, Aiia, and GFP gene under luxpR promotor. In this V.fischeri quorum sensing system<sup>1</sup>, LuxI synthase produces an acyl-homoserine lactone (AHL), which is a small molecule diffuses extracellularly and triggers quorum sensing. When AHL binds to LuxR, it produces LuxR–AHL complex that activates luxI promoter. This also activate GFP genes, so fluorescence can be detected. AiiA 'represses'<sup>2</sup> continuing activation of luxI promotor by degrading of AHL. Therefore, fluorescence may have the cycle under right conditions.
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In the world where people suffer from energy deficiency, we expect that this technology could be applied to many different areas. Among them, we think the most successful adaptation would be as an alternative for light bulbs such as those in night stand.
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: This network which an activator triggers its own repressor illustrates a concept of synthetic oscillator design. Further, theoretical work shows how an autoinducer leads  synchronized oscillations in a single cell and a population of cells<sup>3</sup>.  
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We got interested in synchronized oscillator while reading Team Wageningen's 2011 project. However, we modified their model a little to increase the probability of success in experiment by using only one promotor.
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: The team got interested in synchronized oscillator while reading Team Wageningen's 2011 project. However, we modified their model a little to increase the probability of success in experiment by using each luxpL and luxpR promoter for only one time.  
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=Brainstorming=
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: Our research goal is to build the circuit for synchronized oscillator and use them for display. Based on the idea that oscillatory period differs for different cell population, we plan to have a 3 x 3 array, each well consisted of different colonies. The difference in period of GFP expression will allow to display certain figure at certain time. For instance, the display could be like this:
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==Ideas from imagination==
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: [[File:gfp.jpg]]
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===To Kill a Mosquito===
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: In the world where people suffer from energy deficiency, we expect that this technology could be applied to many different areas. Among them, we think the most successful adaptation would be as an alternative for signs, secret messages and night stand.
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* From science news articles linked below, we have found some chemicals that either attracts mosquito or kills it.
 
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: http://www.ars.usda.gov/is/pr/2010/100309.htm
 
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: http://www.scidev.net/en/news/mosquitoes-kill-their-own-in-new-dengue-control-me.html
 
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* We found that ammonia, carbon dioxide, lactic acid, octenol are the substances that attract a mosquito. (Pyriproxyfen is an insecticide which is neither lethal nor repellent to human), while geranyl acetone, citronellol, geraniol, lavonax and pyriproxifen are lethal substances to dengue mosquitoes(http://www.ncbi.nlm.nih.gov/pubmed/20127888)
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==Introduction of E.Coli display using repressilator==
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* Pyriproxifen works as a juvenile hormone analogue and prevents developmental steps of a larvae.  
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: Our [[Team:CSIA_SouthKorea/introduction | introduction]] further explains the system and goals.
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* Since a mosquito needs both its breeding area and resting place which are centered around stagnant water(toilet tanks, sinks....), it would be preferable for the lethal substance to be soluble in water.
 
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* Professor and advisors' comments:
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==Mechanism of the circuit==
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  -Although there is a way to synthesize pyriproxyfen in a chemical way, there is no known biosynthesis pathway of the substance.
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  -Many of the mosquito-attracting chemicals also appeal to different types of insects
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  -Some of the mosquito-attracting chemicals are already in sales
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===Bacterial Sunscreen===
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: This is further explanation about [[Team:CSIA_SouthKorea/mechanism | mechanism]] of the circuit and explanation about the parts we used.
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* Bacterial production of sunscreening materials has been a recent issue in microbiology.
 
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: http://www.popsci.com/science/article/2010-09/algaes-natural-bio-sunscreen-could-lead-better-skin-protection
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==Design of the circuit==
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* We found that most sunscreens could shield UV B (280-300nm) but not UV A. In addition, most sunscreens are found to have metal oxides in their components, and the effects those could lead to melanomas. We also searched about scytonemin, a pigment found in cyanobacteria which protects them from UV radiation. Such preventative mechanisms by cyanobacterias include systems that detoxify radical oxygens produced during UV stress by using enzymatic antioxidants.
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: This is the ultimate [[Team:CSIA_SouthKorea/design | design]] of the circuit and how we cloned different parts together.
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* The professor commented:
 
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: “Sunscreen that uses bacteria is one of the interesting topics. However, for iGEM project, this idea would be difficult to realize. My lab had once also considered to synthesize the bacteria, and if the process succeeds, it would be really beneficial also in industrial terms.”
 
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: And he sent us the paper (also marked in reference) that made us feel amazed.
 
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: http://www.nature.com/nrmicro/journal/v9/n11/full/nrmicro2649.html
 
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: (which is also on online)
 
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* The advisor commented:
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==Variables that determine period of the circuit==
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: The gene used in synthesizing bacteria used in sunscreen have not been completely discovered yet. Also, the pathways of the known genes are not wholly studied to use. Furthermore, about 10 to 20 operons have to be applied to synthesize bacterial sunscreen. So, it would be very difficult and complicated to make this bacteria.
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===Lactose intolerance curing E. coli===
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: Since we assumed that difference of the period in each array leads to different patterns, it is important to find the [[Team:CSIA_SouthKorea/variables | variables]] which affect the period.
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We thought about the mechanism of making E. Coli to do apoptosis when there is excessive nutrient in human colon (e.g. when a lactose intolerant individual intakes dairy product) so that it prevents excessive growth of E. Coli culture and reduces lactose-intolerant symptoms, such as diarrhea.
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* Mechanism step (hypothetical) 1. Sensing lactose
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==Simulation of the display==
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: We could not find an adaptable lactose-sensing gene. Therefore, it would be better to adopt the glucose sensing mechanism of Baker's yeast.
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# Put in lactase making gene inside the E. Coli's DNA. Put in the spliced mRNA version (of course it would be transferred into DNA by using reverse transcriptase before putting the mRNA strand into the E. Coli's gene) of Baker's yeast's glucose-sensing gene.
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# When there is lactase inside human colon, the lactase-producing gene will express and E.Coli will produce lactase.
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# Lactase will decompose lactose into galactose and glucose.
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# The glucose sensing gene will detect glucose.
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* Mechanism step 2. Apoptosis signal
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: Before doing the wet lab, in order to predict the results of our experiment, we did computer [[Team:CSIA_SouthKorea/simulation | simulation]]  of the light bulbs of the night stand that we are trying to make with colonies of ''E. coli''.
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: In ‘Baker's yeast’, the gene HXT is expressed when glucose is detected. Instead of HXT, if we put the gene that induces cell death in parts registry, we predicted that the cell will apoptosis when the glucose is detected.
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<ref>The EMBO Journal vol. 17 no. 9 pp.2566-2573, 1998 Glucose sensing and signaling by two glucose receptors in the yeast
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Saccharomyces Cerevisiae</ref>
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==Protocols==
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* Things to consider :  
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: You will see us conducting the experiments according to the [[Team:CSIA_SouthKorea/protocols | protocols]].
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: Since the cell should not die from the signal it sent to itself, the genetically modified E.Coli should be resistant to the apoptosis signal sent from itself. Further, the power of the signal should be controlled in its time and magnitude.  
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* Comments from the professor and advisors
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==Applications==
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: - I would like to know whether the fact ‘lactose-intolerant symptom is created by excessive growth of e.coli inside human intestine’ is true. It would be better if you include the related papers or references that you found during the research.
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: -Because lactose is comparatively rich carbon source, E.coli will have various lactose-sensing system.
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: -When the gene that leads the death of cell is inserted into E.coli, it is possible to recognize lactose as gene expression signal and kill the E.coli that is not manipulated. But it seems hard to kill the natural E.coli that is in the human body. Even if it is possible, the corresponding host will also be recognized as a target. In that case, before sufficient amount of substance is made, host will be killed by the initially-generated ones. We need to think of another strategy.
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===Cholesterol Degradation===
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: The page introduces possible [[Team:CSIA_SouthKorea/applications | application]] of our unique use of synthetic oscillator.
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[[File:cholesterol.png]]
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: This idea derived from an imagination to divide the macromolecular oil into several parts, as illustrated above. We primarily focused on our research of steroid compound degradation and enzyme that activates the process.
 
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: The literature search was done on a very basic level. Pseudomonas sp. NCIB 10590 & Bacillus subtilis are able to degrade cholesterol to lower level. In human body, DHCR7 gene codes enzyme 7-dehydrocholesterol reductase. Gordonia cholesterolivorans (e.g., G. sihwensis, G. hydrophobica, G. australis, and G. neofelifaecis) has genes that codes cholesterol oxidase.
 
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* Comments from the professor and advisors
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==Results & Conclusions==
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: -Excess cholesterol may create various diseases, it is still true that cholesterol is essential in many biological processes. Breaking down cholesterol when its concentration is high would be desirable, but it is not that unique.
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: This [[Team:CSIA_SouthKorea/results | page]] shows the results and conclusions of our project so far!
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==Ideas based on previous iGEM teams==
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=Outreach & Human Practice=
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===Glowing Bacteria===
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: [[Team:CSIA_SouthKorea/outreach | Outreach]] will lead you to a page about our introducing brochure about synthetic biology, and Synbio class in Suri Nature School.  
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We came up with the idea of glowing bacteria to use it as a reading lamp. Our school always shut all the electricity on 1:00 a.m. so we have to go to sleep. So, even though when we have important homework or tests coming up, we cannot study longer than 1:00 a.m. Because of this uncomfortable system, we thought of glowing bacteria that can glow without electricity so that we can stay up late and study more(really?).  
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<font color="#38BAEB">* bioluminescence</font><br>
 
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: While searching for glowing bacteria, we found out that a Netherland company, Philips electronics, developed glowing bacteria that is fed with methane and produces luciferase to glow. However, they had a limitation: they said that their bacteria produces low-intensity light that it cannot be used as reading lamp. ☹
 
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: We found another project of making glowing bacteria. This was done by Cambridge in 2010 for iGEM. They didn’t use GFP as a glowing material. Instead, they used v. fischeri lux operon. What they did was by using long-range PCR, they extracted luxCD, luxAB, and luxE individually and assembled them into new operon. And they used Gibson Assembly to make operon consisting Lux C, D, A, B, E under the arabinose-induced promoter pBAD which can activate without the gene regulator, LuxR and AHL. Also, they had made h-ns mutants that produce much brighter light than wild type strain. This gave us a hope to make a reading lamp using glowing bacteria!
 
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* About the first ideas of making a bioluminescence lamp, the professor and advisors said that we might need a mechanical sensor. 
 
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===Cobalt Buster===
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=Brainstorming=
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Team Lyon-INSA-ENS proceeded “Cobalt Buster” project about creating a bioremediation system of using Escherichia Coli biofilm to filter radioactive cobalt from contaminated water from nuclear reactors. Reflecting the fact that the formation of E. Coli biofilm is mainly caused by the production of curli, they worked on overproduction of curli, which is a highly adhesive amyloid protein.
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* Understanding of the project
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: Please see our [[Team:CSIA_SouthKorea/brainstorming | Brainstorming]] section. We have more than ten ideas explained!
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: They used two different approaches on doing this: one is a completely <font color="#FF9800">synthetic approach</font> of creating an independent curli synthesizing pathway, and the other is <font color="#FF9800">activating the existing curli synthesis pathway</font> by cloning the superactivator ompR234 gene.
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: In addition, they tried to improve their strain by making their strain auxotrophic, which therefore prevent dispersion of the strain, and by inserting the transporter gene directly in the efflux pump gene. They used “Quick & Easy E. Coli Gene Deletion Kit” to delete a gene of amino acid biosynthesis and made their strain unable to survive without a medium that contains amino acid. Also, accounting that the transporter features are located on a plasmid that may not be stable and a Kanamycine resistant gene in rcnA gene knocks out the rcnA gene, they inserted the transporter feature in the rcnA gene.
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: They insist that they succeeded in capturing up to 85% of the radioactive cobalt, which seems to be a successful result. Nevertheless, there are also some parts that must be improved in order to be able to be used in the real-life nuclear reactors. The temperature of the primary circuit can rise up to 327 degree Celsius; however, the adequate temperature of their biofilm is between 20 degree Celsius and 45 degree Celsius. This means that there must be four to five hours of interval before opening the reactor and passing the contaminated water through the filter, which inevitably causes economic inefficiency. In addition, in primary circuits, cobalt may exist both as ions and as particle; however, the scope of this project is limited to capturing cobalt ions and could not yet reach the ability to capture cobalt particles.
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* Improvements
 
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: Being highly interested in this project, our team thought of some ways to make significant improvements on the “Cobalt Buster” project. First, in order to improve the heat resistance of the E. Coli biofilm, we thought of adding clpK gene, which is known to “render an otherwise sensitive E. Coli strain <font color="#CA19C7">resistant to lethal heat shock</font>”.
 
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: More specific plans and idea regarding the capture of cobalt particles are soon to be considered and updated.
 
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* Comments from professor and advisors
 
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: In high temperature, the protein is denatured because its three dimensional orientation is malformed. Therefore, it is not feasible to prevent denaturation by just inserting heat resisting gene in the bacteria.
 
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: The characteristics of the filter were already sufficiently discovered by the previous iGEM team. So there was not much room for improvement.
 
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: Rather, it would be interesting to study about the E.Coli's inducing mechanism against influx of Cobalt. Since bacterias are sensitive to the small intake of heavy metal compared to the influx of glucose or other essential materials, it would readily expel the ion from its internal environment. This means that present of Cobalt will greatly influence E.Coli because it is fatal. Further, it is likely that such pathways have been already discovered.
 
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===Plastic Degradation===
 
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Based on the openwetware page and Team Stanford’s brainstorming ideas(http://openwetware.org/wiki/IGEM:Stanford/2009/Plastic_Degradation#Project_Summary), we researched on phenol degradation.
 
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: The idea of environment-friendly approach fascinated us despite difficulty of realizing the goals. We followed the ‘subprojects’ ideas on the page and tried to understand contents. If the goal of the project is to Engineer E. coli to metabolize phenol as a carbon source, linking it to cellular respiration,
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=Notebook=
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: Pseudomonas sp
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: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC383114/
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: or
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: Cryptanaerobacter phenolicus
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: http://ijs.sgmjournals.org/content/55/1/245
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: or
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: Rhodococcus phenolicus
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: http://www.sciencedirect.com/science/article/pii/S0723202005000986
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: should be available. However, methods of transducing bacterial plasmids into vectors that will be put in E.Coli or yeast should be investigated for further research.
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However, if we use Pseudomonas sp. as the plasmid, we would have to add Na2-succinate, which is the typical source for the strain. (The related thesis was actually about the ‘simultaneous’ Degradation of Atrazine and Phenol). Cryptanaerobacter phenolicus is known as a bacterium species that produces benzoate from phenol via 4-hydroxybenzoateRhodococcus.
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===Synthesis of Flavor using Candida Rugosa===
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: Please see our [[Team:CSIA_SouthKorea/Diary | Notebook]]. We have an extensive log from September 2011 on our experiments! :)
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* Our extensive research about lipase led to investigation of some specific function of the lipase, including flavor synthesis. The thesis linked below revealed us that C.rugosa could synthesize pentyl propanoate, isopentyl butanoate, and butyl butanoate, which are all components of apple flavor, by using lipase.
 
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* Based on the source from openwetware(which is also a project for MIT in 2006),
 
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: http://openwetware.org/wiki/BioBuilding:_Synthetic_Biology_for_Students:_Lab_1
 
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* we thought of a device generating apple flavor. In the project in openwetware, the promotor codes ATF1 enzyme, which converts isoamyl alcohol to isoamyl acetate. If we find specific lipase that makes pentyl propanoate or isopentyl butanoate , and leave it in adequate substrate(we should research more!!) we thought that the synthesis of flavor would be  feasible.
 
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* The professor first commented that it would be good to quantitatively measure the amount of fragrance(gas), but he said that gas chromatography would be unavailable and complicated. We later agreed to reserve the idea of quantitative measurement of gas.
 
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* We found out that 2006 MIT iGEM team did not either quantitatively measured the strength of fragrance. They applied relative arbitrary scale in their measurements.
 
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* Comments from professor and advisors
 
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: This type of project was widely studied by numerous previous iGEM teams. So, it would be repetitive to address the same task.
 
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===Methane Sensing===
 
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The team thought of developing methane sensing devices based on project of METU, Turkey.
 
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* Understanding of the project
 
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: They had four steps in implementing their project :
 
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: Methane sensing(in fact, MMO sensing), conversion of MMO into methanol, entrapment of methanol using enzymes, and a killswitch(cessation of replicating the sensor). The team found that the bacteria ‘Pseudomonas oleovorans’ find alkane and degrade it for carbon source.
 
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: However, according to the team,
 
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:-the synthesized methane monoxygenase(MMO) construct was such a long part. The main methane interacting region of monooxygenase could not be expressed functionally.
 
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* Improvements
 
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: We thought of improving the first step of METU’s project, by more thoroughly investigating about the activator protein AlkS. AlkS induces the transcription from PalkB promoter which initiates the expression of genes code for assimilation of alkanes.
 
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: Still, we were assured by the following informations:
 
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: -sensitive to methane presence and have mechanisms to activate transcription of related gene clusters.
 
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: -the transcription is expressed in E.coli correctly.
 
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* Comments from the professor and advisors
 
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: Since MMO is a <font color="#CA19C7">multimeric enzyme</font>, it would be difficult to be fully expressed. (This means that the problem that was in the original project could not be easily solved)
 
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=Project=
 
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=Diary=
 
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*09.20.2011
 
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: -We didn’t know any professional knowledge about mechanical biology so we decided to ask for help of experts. While searching for the professors who participated in iGEM before, we found out that a professor from Korea University has participated several times before. So, we first sent an e-mail to our instructor In-Geol Choi to ask for his help.
 
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*09.21.2011
 
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: -The professor responded very quickly. And he was really positive toward us having interest in synthetic biology and willing to participate in iGEM.
 
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*12.18.2011
 
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: -While searching through the iGEM wikis, we got interested in making bacterial sunscreen, the idea from the imperial college 2011 wiki brainstorming section. Sunscreens that we normally use can only protect UVB, which is only one part of the broad range of UV. So, we were hoping to make bacteria that can protect UVB and UVA, another kind of UV light. And according to our further research about bacterial sunscreens, scytonemin, a pigment synthesized in cyanobacteria, absorbs UVA and pityriacitrin, a pigment synthesized in malassezia yeast, absorbs a wide range of UV. Thus, we thought we could use the genes that codes for this pigments and make a bacteria that carries the genes. However, we had so much difficulty finding it that we sent the second e-mail today for help from the professor.
 
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*12.19.2011
 
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: -We received an answer from our instructor. He said our idea was interesting and worth making it, but it would be too hard for us to make it as high school students. ☹ He also sent us a review related to bacterial sunscreens from the Nature: Microbial ultraviolet sunscreens by Qunjie Gao and Ferran Garcia-Pichel.
 
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*12.25.2011
 
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: -We sent our first e-mail to iGEM asking several questions about the competition.
 
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*12.28.2011
 
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: -We received a reply from the iGEM and she was very nice! ☺
 
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*01.13.2012
 
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: -Today, we sent another e-mail to our instructor with several ideas. All of these ideas were from previous team wikis. From open wet ware, we found the rough outline of Stanford 2009 iGEM project. This team tried to degrade plastic, specifically phenol and formaldehyde, into carbon source of e.coli. However, we couldn’t find any sign that shows the team actually conducted the experimentation. Other one was making an e.coli that can sense methane. This idea was based on METU 2011 project. This team, had several projects-sensing methane, conversion, entrapment, killswitch- but they didn’t accomplish their goal. So we thought we could try part of their project, methane sensing. The last idea we sent was making e.coli that produces an apple smell. We found a similar project, MIT 2006 project, in biobuilder homepage that was to make a banana odor generator with e.coli.
 
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: -More information about our ideas can be found in brainstorming section of our wiki!
 
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*01.13.2012
 
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: -We received some negative comments on our ideas. He told us that since methane oxygenase is multimeric enzyme, we would have hard time sensing methane and also plastic degrading would be a tough task for us. But he said maybe using other kind of ester to produce odor could be possible. And he also suggested us to improve Korea University 2010 project.
 
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*02.16.2012: meeting with professor
 
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: -Our team had a meeting with professor In-Geol Choi in order to discuss our topic for the igem project. We had some creative ideas of our own, but we weren’t sure if they were appropriate topics for this project. As our discussion went on, professor Choi gave us some advices in selecting the topic:
 
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: -First, the project should have some kind of purpose – something like ‘saving environment’. We need to synthesize and create biological machine of certain function that can fulfill the fundamental purpose of our own.
 
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: - Second, considering that we don’t have much time nor the professional knowledge for the research and experiments, as high school students, it might be more appropriate to work with relatively easy project. He recommended us to do some research on websites such as biobuilders, and read about some labs that students can conduct.
 
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: -Third, note the parts registry that we can use. In parts registry site ( http://partsregistry.org/Catalog) we can find out which devices and functions we are able to use, and that will help us with selecting our topic. We decided to consider these points and brainstorm more about the topics. We are to determine our topic by the end of this February.
 
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*02.20.2012: ideas, ideas, ideas
 
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: -We first start to have an idea of making glowing bacteria to use it as a reading lamp. And having done some research, we found out that most of the glowing products were containing a protein called GFP (green fluorescent protein). To make a new challenge, we thought of making a new color of light other than green light. However, we soon found out that other great scientist have already made numerous mutation of GFP and produced many other colors. So, now… we are stuck!!!
 
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*02.17.2012
 
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: -After some discussions and research, we came up with some ideas: Lacotse intolerance curing ecoli, cholesterol degradation, and synthesis of scent. We referred to many of the previous igem projects and the research materials to determine necessary steps for each of the project.
 
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: -Lactose intolerance curing e.coli : mechanism of making E.Coli to do apoptosis when ther is excessive nutrient in human colon
 
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: It prevents excessive growth of E.Coli culture and reduces lactose-intolerant sypmtoms
 
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: 1) Sensing lactose
 
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: 2) Apoptosis signal
 
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:-because cell can’t die due to its own signal, genetically engineered E.Coli should be made resistant to apoptosis signal
 
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:-To prevent complete eradication of E.coli or too minimal deaths of E.Coli, the strength or period of signal should be controlled
 
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: -Cholesterol degradation
 
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: Bacteria called Pseudomonas sp. NCIB 10590 and Bcillus subtillis have the gene to degrade cholesterol. In human bodies, DHCR7 gene codes for enzyme 7-dehydrocholesterol reductase.
 
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: -Synthesis of scent using Candida rugose
 
-
: (*openwetware)
 
-
 
-
* 03.05.2012
 
-
: -The idea of synthesis of scent
 
-
: When we looked over the banana scent-synthesing project that MIT had conducted before, we found out that they applied arbitrary standard for the quantity of scent. But other than that, we couldn’t find a method of quantitatively measuring specific gas. We are still looking for mechanical sensors..
 
-
 
-
* 03.07.2012
 
-
: We thought of additional ideas: killing mosquitoes and toggle switch.
 
-
: -Killing mosquitoes
 
-
: Mosquitoes have a preference for dextorotatory ocetenol molecules that are emitted y mammals.
 
-
During the research, we found out how to lure the mosquitoes and chemicals that can kill them. Engineering E.Coli that can synthesize both chemicals would be the goal.
 
-
 
-
: -Toggle switch
 
-
: Two positive feeback loop that represses each other
 
-
: Idea 1) make an input that can sensor pH, and distribute in in the soil so that weak base can be released when needed
 
-
: Idea 2) because toggle switch has similar mechanism with neuron, it might be able to model a simple neuron circuit.
 
-
 
-
* 03.12.2012
 
-
: We received some negative feedbacks for our “cobalt buster” proposal. We made this proposal based on previous igem projects. However, he told us that it doesn’t seem to be able to be significantly improved, nor be more developed, since previous igem team had already completed much of the work. Instead, what we should focus on is developing the projects that prvious igem team had failed. What he advised was that because there is not enough time for highschool-division to go over all the errors and fix them, it would be better to plan a project with interesting ideas. He also suggested that in Cobalt Buster project, we can modify the operon so that we can create a circuit that has cobalt as the inducer.
 
-
 
-
* 03.13.2012
 
-
: Professor Choi also sent us some feedbacks on our ideas. He first commented on our “mosquito-kiling” that it is a great idea, but we should consider more about the feasibility, and how long it would take to complete the project.
 
-
: Also, he told us to do some more research, comparing toggle switch and repressilator. (http://en.wikipedia.org/wiki/Repressilator)
 
-
: Also, we decided to make lists of our ideas and decide one among those by the next week.
 
-
 
-
* 03.14.2012
 
-
: Another feedback arrived:
 
-
: 1. Pyriproxyfen, the substance that kills mosquito work as juvenile hormone analogue to prevent larvae’s development. However, we need a way to synthesize this substance other than in chemical way.
 
-
: 2. Toggle switch seems to be a similar system with the repressilator. It would be interesting to create microorganism with circadian rhythm or biological clock. We will discuss more about how to use it.
 
-
: 3. Cobalt buster seems to be unfeasible only with a few heat-resistant genes.
 
-
 
-
* 03.16.2012
 
-
: We did some research about what our professor wanted us to do about toggle switch. Toggle switch is a similar system with repressilator, and the only difference is that toggle switch has two loops repressing each other and repressilator has three. And we thought circadian rhythm idea was really great.
 
-
: Also, we developed our previous idea of making glowing bacteria using mechanical sensors. We thought of making a grid with 10*10 or more spaces, place gfp, and let each space have signal of 0 or 1, controlling the coloring of the bacteria in each space. It would be sort of like making the bacteria-neon sign.
 
-
Then we got reply from the professor. He told us that mechanical sensor would be found if we search previous iGEMs. For the grid idea, it would be possible with e.coli with toggle switch and turn it on or off.
 
-
 
-
* 03.17.2012
 
-
: (comments from professor & advisors)
 
-
: 1. MIT team in 2010 iGEM made a system with mammalian cell that responds to the pressure. It is uncertain whether we can apply eukaryotic promoter to the prokaryote or not. Also, it wouldn’t be so easy to find a system in which microorganism responds to the physical stimulus. Instead, it might be possible to use ‘heat-sensing’ property, shaking it to raise the temperature and making it respond.
 
-
https://2010.igem.org/files/poster/MIT.pdf (MIT 2012 iGEM poster)
 
-
 
-
: 2. To develop more about the idea of coding the grid system with toggle switch, we can use the fluorescent-protein as a reporter for the switch. When switch is off, it will have value of 0 and 1 when it is on. We can use filter paper and put only on some of them the inducer that turns the switch on, and place it on e.coli. Then, it will have fluorescent-protein appearing on only some of filter papers, and when we send it, receiver can use the wave that excites the protein to get the message.
 
-
 
-
: We will be meeting with the professor next Wednesday (3/21) and make final decision on our topic for the iGEM project ☺
 
-
 
-
* 03.18. 2012
 
-
: Professor Choi commented that Electronic display using bacteria seems to be the best project for us to experiment.
 
-
: He said that “The example of the code below is the “on only” experiment which doesn’t need toggle switch to turn it on and off.
 
-
: The other possible suggestion is that first, make the circuit that works with toggle switch, and in the 96 well plate, control on-off of the each well and make the fluorescence oscillate (on and off). Then it would seem like electronic display.
 
-
: I will discuss it with assistant and tell you whether it is possible or not. You can talk with him on Wednesday and make final decision.”
 
-
 
-
* 03.19.2012
 
-
: We organized our brainstormed ideas before we have a meeting on Wednesday:
 
-
 
-
: Sunblock utilizing bacteria
 
-
: Resisting mosquitoes
 
-
: Electronic display using bacteria (and toggle switch)
 
-
: cobalt detection (modifying the operon in order to create a circuit that has cobalt as the inducer)
 
-
: glowing bacteria with heat sensor
 
-
: lactose intolerance
 
-
: cholesterol degradation
 
-
: glowing bacteria
 
-
: plastic degradation
 
-
: synthesis of flavor
 
-
: methane sensing
 
-
 
-
: We are also organizing wiki with the ideas that came up till now.
 
-
 
-
* 03.23.2012
 
-
: First, we made decision on our research project! We are planning to work on bacteria-display.
 
-
: We also uploaded wiki with what we discussed with the assistant on Wednesday.
 
-
: Professor Choi commented that-
 
-
: Substance that allures mosquitoes applies not only to mosquitoes but to all the insects, so it won’t work specifically to the mosquitoes.
 
-
: Biosynthetic pathway isn’t well known, and the substance itself is already commercialized in the market, so it doesn’t seem to have much meaning.
 
-
: Also, 1. Try making team logo 2. Upload team introduction to wiki.
 
-
 
-
* 03.25.2012
 
-
: We asked several questions.
 
-
 
-
: 1. 96 well plate has a lot of wells each, and we are curious how gfp in separate wells uses quorum sensing to synchronize their oscillation periods.
 
-
: 2. You told us that the period that fluorescence appears and disappears is the same, so that we should use a video; we are curious how long it goes!
 
-
 
-
* 03.27.2012
 
-
: Instructor told us that our logo seems to be simple and nice^^ and added that it would also be nice to make a logo that represents the project. He also told us that for iGEMHS, the part registration is not required; igem homepage says that special awards are given to teams who register parts with “new, cool and important function”
 
-
 
-
: And here are the answers:
 
-
: 1. Synchronization of gfp expression in one well is explained in the paper linked below.
 
-
: In Fig1(a) diagram, luxI promoter that is originally used in quorum sensing can be modified to function as repressilator.
 
-
: http://www.nature.com/nature/journal/v463/n7279/full/nature08753.html
 
-
: 2. According to the paper, repressilator has about 150 minutes long period, and quorum sensing about 70 minutes. But it can differ according to the laboratory environment, so we will have to measure it ourselves during the experiment.
 
-
 
-
: We should first search the parts related to repressilator or toggle switch, and see if we can utilize it.
 
-
 
-
* 03.28.2012
 
-
: We didn’t really understand why quorum sensing has 70 minutes of period, so we asked the instructor. 
 
-
: In fact, reading the paper was an excruciating process but the result was worth it.
 
-
 
-
: 1. If we use E.coli with shorter period then we can reduce the time; does it mean that the period can be shorter than 70 minutes?
 
-
: 2. We found a picture for repressilator, but it says that c gene at the end represses gfp, so we aren’t really sure what’s right…
 
-
: 3.  We are looking up parts registry site to find gene that goes into repressilator, but we are not sure what gene is needed☹ Are we using the same genes that Elowitz and Leibler used?
 
-
 
-
* 03.29.2012
 
-
: Instructor sent us two papers, the one with Elowitz and Leibler & the one that is linked above.
 
-
 
-
: He also told us that reducing the oscillation period shorter should be considered after we check that the system works properly. He said: “About gfp repression, you will know better if you read the paper. Based on the picture that you sent me, TetR gene is repressing the gfp expression, and gfp is expressed when λ cI gene is expressed. Also, I think that it would be too hard to create a new promoter and make oscillation system. It would be both easier and much certain to use the same method as Elowitz and Leibler’s. If you search ‘igem repressilator’ on google, you’ll see that there were many previous igem teams who used the same circuit. Laboratory schedule isn’t decided specifically. I’ll tell you as soon as I can after I discuss it with the professor.”
 
-
 
-
* 03.30.2012
 
-
: We asked several questions again!
 
-
: 1. Both two papers are about gfp oscillation occurring in one colony; I’m curious whether we have to connect every single cells in 96 well plate into one colony
 
-
: 2. When doing the lab, is the trapping chamber necessary for making critical cell density as shown in 2010 paper? If we do so in 96 well plate, then how can we control flow rate?
 
-
: 3. In 2000 paper is the IPTG inducer that has to be inserted from the external environment? And moving the cell from place with IPTG to without IPTG resulted in the occurrence of gfp fluorescence oscillation?
 
-
On the other hand, AHL is substance synthesized by luxI, so amount of AHL also oscillated in the 2010 paper?
 
-
Then I’m curious that if we have to use autoinducer similar to AHL.
 
-
: 4. I also want to ask if substance that is used to do cell-to-cell coupling wasn’t used in 2000 paper. We haven’t been able to read closely the previous igem projects yet. Always thank you for kind reply!
 
-
 
-
* 04.02.2012
 
-
: We kept reading a thesis by Elowitz & Leibler.
 
-
: We were curious about why the oscillation slows down suddenly at the post-septation phase. (Then, doesn’t the oscillation happen in the E. coli after about 600 minutes, as shown in the graph?) Further, it was hard to understand why only about 40% of the cells exhibits oscillation behavior. In the thesis, it is stated that transient pulse of IPTG might be capable of synchronizing, because IPTG hinders the repression of LacI. We do not understand why. We asked these questions to professor Choi.
 
-
 
-
* 04.03.2012
 
-
: This is the reply from professor Choi☺
 
-
: According to the thesis, when the cell (E. coli) enters the stationary phase, the oscillation stops. (p.335 first paragraph, last sentence). The reason for only 40% of the cells showing the oscillatory characteristic may vary. Fundamentally, the oscillation is the result of complex cellular metabolism, not simple chemical reactions, so the oscillation may be highly affected by various external circumstances, such as the size of the cell, metabolism rate, diffusion rate, etc. So, as the time that the cell is exposed to IPTG becomes longer, synchronization will be hindered more, due to the same reason. I think making the circuit is not very difficult, but obtaining preferred results will not be a easy thing. We’ll make up the schedule to proceed experiments for building the circuits with your instructor.
 
-
 
-
* 04.05.2012
 
-
: We thought about make oscillation of GFP expression in one colony in a 96-well-plate and size of the colony.  Instructor told us that the thesis about the repressilator is about oscillation in a single E. coli cell, and the other thesis is about synchronization of oscillations in a cell colony, not a single cell. In our experiment, we will use the 96-well-plate to raise the cells in different wells as independent colonies. We will not make the whole plate as a single colony.
 
-
 
-
: We were also curious about use of ‘trapping chamber’ in order to create the critical cell density. Our experiment will be conducted inside a 96-well-plate; we wonder how we could control the flow rate inside the plate, if we use the same mechanism as the ‘trapping chamber’ in order to obtain the critical cell density. We concluded that it will be quite difficult to use a trapping chamber to make a continuous culture. Instead, we will use batch culture system to raise the cells.
 
-
 
-
: We asked our instructor whether we understood the theses correctly: is IPTG an inducer that must be injected from external source? And because AHL is a substance that luxI synthesizes, the amount of AHL oscillates together, right? Then, if we use quorum sensing in the experiment, do we need to use autoinducer, such as AHL?
 
-
He replied that IPTG is a chemically synthesized substance, and it is to be injected from external source. AHL is auto-synthesized. Therefore, the concentration of AHL also shows oscillation, and we do not need to put in AHL.
 
-
 
-
: We also discussed that due to the difference in the period of oscillations among the cells, preferred results may not come out in our experiment. But, because repressilator parts are already registered in the parts registry by previous iGEM teams, the experiment is relatively easy to conduct right now. Instructor said that we will start preparing for the experiment and order the parts from the part registry, so we should design a theoretical model of our experiment to see if our experiment is actually plausible.
 
-
 
-
 
-
 
-
 
-
 
-
 
-
 
-
 
-
=Results/Conclusions=
 
-
What did you achieve over the course of your semester?
 
=Safety=
=Safety=
-
 
-
 
-
 
-
=Attributions=
 
-
 
-
 
-
 
-
=Outreach & contribution=
 
-
 
-
 
-
==Creating a introducing brochure about synthetic biology==
 
-
 
-
: Although synthetic biology is a fascinating field, it is not well known to the public. Since we hoped to share our understanding of the synthetic biology work, we made a small pamphlet that explains DNA, synthetic biology, and iGEM. We wish it would be a helpful material to all those who wish to introduce synthetic biology to everyone!
 
-
: We also introduced iGEM at the end of the brochure, too.
 
-
: The files we made is now distributed to several institutions, not only including schools but also institutions and teacher's associations.
 
-
 
-
[https://2012hs.igem.org/Team:CSIA_SouthKorea/brochureigem1]
 
-
[https://2012hs.igem.org/Team:CSIA_SouthKorea/brochureigem2]
 
-
 
-
: We'll upload the English version soon:)
 
-
 
-
==Synthetic Biology class in Suri Nature School ==
 
-
: Suri Nature School located in Gunpo, Korea, is a NGO that runs educational programs for teens. CSIA_SouthKorea participated as mentoring students who discussed students with subjects related to biology and genetic technology. 
 
-
: A student in Suri nature school wanted to learn about GMO, so this is the worksheet designed to solve any basic questions regarding GMO.
 
-
: We made this quiz to grasp some basic notions about GMO & to let people know that there are some questions in genetic engineering that cannot be clearly answered "Yes" or "No"
 
-
 
-
: [[File:gmoquizengg.pdf|400px|thumb|left|FUN INTERACTIVE GMO QUIZZES for elementary school kids]] [[File:gmoanswer.pdf|400px|thumb|right|ANSWERS for FUN INTERACTIVE GMO QUIZZES for elementary school kids]]
 
-
 
-
: We made several students in Suri Nature school to solve the quizzes, and some of them were surprised by the fact that DNA is so long and it is digested in the intestines after taken by the body.
 
-
 
-
=Fun!=
 
 +
: In this page, [[Team:CSIA_SouthKorea/safety | Safety]] , you will see our team CSIA_SouthKorea's lab safety practice and learnings.
-
==References==
 
-
<references />
 
 +
=References=
 +
# Danino et al, A synchronized quorum of genetic clocks”, Nature vol. 463, 326-330 (2010)
 +
# Liu, D. et al. Mechanism of the quorum-quenching lactonase (AiiA) from Bacillus thuringiensis. 1. Product-bound structures. Biochemistry 47, 7706–7714 (2008).
 +
# Garcia-Ojalvo, J., Elowitz, M. & Strogatz, S. Modeling a synthetic multicellular clock: repressilators coupled by quorum sensing. Proc. Natl Acad. Sci. USA 101, 10955–10960 (2004).
 +
# http://avena.pw.usda.gov
<forum_subtle />
<forum_subtle />

Latest revision as of 11:12, 17 June 2012

OUR FANTASTIC TEAM LOGO by Aileen Shin

Our team is consisted of four students who are fond of thinking creatively, sharing our knowledge with others and making contributions to the society. We hope that iGEM 2012 could be a great opportunity for us to get our feet wet in the field of synthetic biology and interact with many other students around the world who are also interested in this field! Since our school does not have facilities for wet lab, team CSIA_SouthKorea asked professor In-Geol Choi in Korea University for guide. Professor Choi, Instructor Hyeok-Jin Ko and Hongjae Park let us to be familiar with synthetic biology, taught us how to use basic lab facilities and helped every part in our projects :)

TEAM LOGO♥
TEAM LOGO











Contents

Team

Our team and school is introduced in this page! :)

Project

Abstract

Based on the design of V.fischeri, we placed luxR gene under luxpL promotor and placed luxI, Aiia, and GFP gene under luxpR promotor. In this V.fischeri quorum sensing system1, LuxI synthase produces an acyl-homoserine lactone (AHL), which is a small molecule diffuses extracellularly and triggers quorum sensing. When AHL binds to LuxR, it produces LuxR–AHL complex that activates luxI promoter. This also activate GFP genes, so fluorescence can be detected. AiiA 'represses'2 continuing activation of luxI promotor by degrading of AHL. Therefore, fluorescence may have the cycle under right conditions.
This network which an activator triggers its own repressor illustrates a concept of synthetic oscillator design. Further, theoretical work shows how an autoinducer leads synchronized oscillations in a single cell and a population of cells3.
The team got interested in synchronized oscillator while reading Team Wageningen's 2011 project. However, we modified their model a little to increase the probability of success in experiment by using each luxpL and luxpR promoter for only one time.
Our research goal is to build the circuit for synchronized oscillator and use them for display. Based on the idea that oscillatory period differs for different cell population, we plan to have a 3 x 3 array, each well consisted of different colonies. The difference in period of GFP expression will allow to display certain figure at certain time. For instance, the display could be like this:
Gfp.jpg
In the world where people suffer from energy deficiency, we expect that this technology could be applied to many different areas. Among them, we think the most successful adaptation would be as an alternative for signs, secret messages and night stand.


Introduction of E.Coli display using repressilator

Our introduction further explains the system and goals.


Mechanism of the circuit

This is further explanation about mechanism of the circuit and explanation about the parts we used.


Design of the circuit

This is the ultimate design of the circuit and how we cloned different parts together.


Variables that determine period of the circuit

Since we assumed that difference of the period in each array leads to different patterns, it is important to find the variables which affect the period.


Simulation of the display

Before doing the wet lab, in order to predict the results of our experiment, we did computer simulation of the light bulbs of the night stand that we are trying to make with colonies of E. coli.

Protocols

You will see us conducting the experiments according to the protocols.


Applications

The page introduces possible application of our unique use of synthetic oscillator.


Results & Conclusions

This page shows the results and conclusions of our project so far!


Outreach & Human Practice

Outreach will lead you to a page about our introducing brochure about synthetic biology, and Synbio class in Suri Nature School.



Brainstorming

Please see our Brainstorming section. We have more than ten ideas explained!



Notebook

Please see our Notebook. We have an extensive log from September 2011 on our experiments! :)



Safety

In this page, Safety , you will see our team CSIA_SouthKorea's lab safety practice and learnings.


References

  1. Danino et al, A synchronized quorum of genetic clocks”, Nature vol. 463, 326-330 (2010)
  2. Liu, D. et al. Mechanism of the quorum-quenching lactonase (AiiA) from Bacillus thuringiensis. 1. Product-bound structures. Biochemistry 47, 7706–7714 (2008).
  3. Garcia-Ojalvo, J., Elowitz, M. & Strogatz, S. Modeling a synthetic multicellular clock: repressilators coupled by quorum sensing. Proc. Natl Acad. Sci. USA 101, 10955–10960 (2004).
  4. http://avena.pw.usda.gov


<forum_subtle />

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