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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

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:

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

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 />

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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!
+[[Image:CSIA_SouthKorea_team.png|right|frame|OUR FANTASTIC TEAM LOGO by Aileen Shin]]
-|[[Image:CSIA_SouthKorea_logo.png|200px|right|frame]]
-|-
+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 :)
-|
-''Tell us more about your project.  Give us background.  Use this as the abstract of your project.  Be descriptive but concise (1-2 paragraphs)''
+[[Image:CSIA_SouthKorea.png|left|frame|TEAM LOGO♥]]
-|[[Image:CSIA_SouthKorea_team.png|right|frame|Your team picture]]
-|-
+[[Image:CSIA_SouthKorea2.png|left|frame|TEAM LOGO]]
-|
-|align="center"|[[Team:CSIA_SouthKorea | Team CSIA_SouthKorea]]
-|}
-<!--- Team Information Link --->
-{| style="color:#1b2c8a;background-color:#0c6;" cellpadding="3" cellspacing="1" border="1" bordercolor="#fff" width="62%" align="center"
-!align="center"|[https://igem.org/Team.cgi?year=2012&division=high_school&team_name=CSIA_SouthKorea Official Team Profile]
-|}
 =Team=
+: Our [[Team:CSIA_SouthKorea/team | team]] and school is introduced in this page! :)
-=Brainstorming=
+=Project=
-==Ideas from imagination==
-===Lactose intolerance curing E. coli===
-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.
+==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 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.
-* Mechanism step (hypothetical) 1. Sensing lactose
+: 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>.
-: We could not find an adaptable lactose-sensing gene. Therefore, it would be better to adopt the glucose sensing mechanism of Baker's yeast.
-# 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.
-# When there is lactase inside human colon, the lactase-producing gene will express and E.Coli will produce lactase.
-# Lactase will decompose lactose into galactose and glucose.
-# The glucose sensing gene will detect glucose.
-* Mechanism step 2. Apoptosis signal
+: 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.
-: 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.<ref>The EMBO Journal vol. 17 no. 9 pp.2566-2573, 1998 Glucose sensing and signaling by two glucose receptors in the yeast
-Saccharomyces Cerevisiae</ref>
-* Things to consider :
+: 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:
-: 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.
+: [[File:gfp.jpg]]
-* Comments from the professor and advisors
+: 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.
-: - 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.
-: -Because lactose is comparatively rich carbon source, E.coli will have various lactose-sensing system.
-: -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.
-===Cholesterol Degradation===
-[[File:cholesterol.png]]
-: 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.
+==Introduction of E.Coli display using repressilator==
-: 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.
-* Comments from the professor and advisors
+: Our [[Team:CSIA_SouthKorea/introduction | introduction]] further explains the system and goals.
-: -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.
+==Mechanism of the circuit==
-==Ideas based on previous iGEM teams==
+: This is further explanation about [[Team:CSIA_SouthKorea/mechanism | mechanism]] of the circuit and explanation about the parts we used.
-===Glowing Bacteria===
-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?).
-* bioluminescence
+==Design of the circuit==
-: 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. ☹
-: 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!
+: This is the ultimate [[Team:CSIA_SouthKorea/design | design]] of the circuit and how we cloned different parts together.
-===Cobalt Buster===
-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.
-* Understanding of the project
+==Variables that determine period of the circuit==
-: They used two different approaches on doing this: one is a completely synthetic approach of creating an independent curli synthesizing pathway, and the other is activating the existing curli synthesis pathway by cloning the superactivator ompR234 gene.
-: 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.
-: 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.
-* Improvements
+: 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.
-: 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 resistant to lethal heat shock”.
-: More specific plans and idea regarding the capture of cobalt particles are soon to be considered and updated.
-===Plastic Degradation===
+==Simulation of the display==
-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.
-: 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,
+: 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''.
-: Pseudomonas sp
-: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC383114/
-: or
-: Cryptanaerobacter phenolicus
-: http://ijs.sgmjournals.org/content/55/1/245
-: or
-: Rhodococcus phenolicus
-: http://www.sciencedirect.com/science/article/pii/S0723202005000986
-: 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.
-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.
-*
+==Protocols==
+: You will see us conducting the experiments according to the [[Team:CSIA_SouthKorea/protocols | protocols]].
+==Applications==
-===Methane Sensing===
+: The page introduces possible [[Team:CSIA_SouthKorea/applications | application]] of our unique use of synthetic oscillator.
-The team thought of developing methane sensing devices based on project of METU, Turkey.
-* Understanding of the project
-: They had four steps in implementing their project :
-: 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.
-: However, according to the team,
-:-the synthesized methane monoxygenase(MMO) construct was such a long part. The main methane interacting region of monooxygenase could not be expressed functionally.
-* Improvements
+==Results & Conclusions==
-: 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.
-: Still, we were assured by the following informations:
-: -sensitive to methane presence and have mechanisms to activate transcription of related gene clusters.
-: -the transcription is expressed in E.coli correctly.
-* Comments from the professor and advisors
+: This [[Team:CSIA_SouthKorea/results | page]] shows the results and conclusions of our project so far!
-: Since MMO is a multimeric enzyme, it would be difficult to be fully expressed. (This means that the problem that was in the original project could not be easily solved)
-=Project=
+=Outreach & Human Practice=
+: [[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.
-=Diary=
-* 2012.02.16: meeting with professor
-: -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:
-: -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.
-: - 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.
-: -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.
-* 2012.02.20: ideas, ideas, ideas
-: -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!!!
+=Brainstorming=
-=Results/Conclusions=
+: Please see our [[Team:CSIA_SouthKorea/brainstorming | Brainstorming]] section. We have more than ten ideas explained!
-What did you achieve over the course of your semester?
-=Safety=
-=Attributions=
+=Notebook=
+: Please see our [[Team:CSIA_SouthKorea/Diary | Notebook]]. We have an extensive log from September 2011 on our experiments! :)
-=Human Practices=
+=Safety=
-=Fun!=
+: In this page, [[Team:CSIA_SouthKorea/safety | Safety]] , you will see our team CSIA_SouthKorea's lab safety practice and learnings.
 =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 />

Team:CSIA SouthKorea