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From 2012hs.igem.org

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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|- | Tell us more about your project. Give us background. Use this as the abstract of your project. Be descriptive but concise (1-2 paragraphs)

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Official Team Profile

Contents 1 Team 2 Brainstorming 2.1 Ideas from imagination 2.1.1 Bacterial Sunscreen 2.1.2 Lactose intolerance curing E. coli 2.1.3 Cholesterol Degradation 2.2 Ideas based on previous iGEM teams 2.2.1 Glowing Bacteria 2.2.2 Cobalt Buster 2.2.3 Plastic Degradation 2.2.4 Synthesis of Flavor using Candida Rugosa 2.2.5 Methane Sensing 3 Project 4 Diary 5 Results/Conclusions 6 Safety 7 Attributions 8 Human Practices 9 Fun! 10 References

Team

Brainstorming

Ideas from imagination

Bacterial Sunscreen

• Bacterial production of sunscreening materials has been a recent issue in microbiology.

http://www.popsci.com/science/article/2010-09/algaes-natural-bio-sunscreen-could-lead-better-skin-protection

• 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.

• The professor commented:

“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.”

And he sent us the paper (also marked in reference) that made us feel amazed.

http://www.nature.com/nrmicro/journal/v9/n11/full/nrmicro2649.html

(which is also on online)

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.

• Mechanism step (hypothetical) 1. Sensing lactose

We could not find an adaptable lactose-sensing gene. Therefore, it would be better to adopt the glucose sensing mechanism of Baker's yeast.

1. 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.
2. When there is lactase inside human colon, the lactase-producing gene will express and E.Coli will produce lactase.
3. Lactase will decompose lactose into galactose and glucose.
4. The glucose sensing gene will detect glucose.

• Mechanism step 2. Apoptosis signal

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> <ref>http://ko.wikipedia.org/wiki/칸트</ref>를 참고하면 ~

• Things to consider :

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.

• Comments from the professor and advisors

- 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

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.

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

-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.

Ideas based on previous iGEM teams

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

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!

• About the first ideas of making a bioluminescence lamp, the professor and advisors said that we might need a mechanical sensor.

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

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

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

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,

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.

Synthesis of Flavor using Candida Rugosa

• 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.

• Based on the source from openwetware(which is also a project for MIT in 2006),

http://openwetware.org/wiki/BioBuilding:_Synthetic_Biology_for_Students:_Lab_1

• 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.

• 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.

• 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.

Methane Sensing

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

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

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

Diary

• 09.20.2011

-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.

• 09.21.2011

-The professor responded very quickly. And he was really positive toward us having interest in synthetic biology and willing to participate in iGEM.

• 12.18.2011

-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.

• 12.19.2011

-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.

• 12.25.2011

-We sent our first e-mail to iGEM asking several questions about the competition.

• 12.28.2011

-We received a reply from the iGEM and she was very nice! ☺

• 01.13.2012

-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.

-More information about our ideas can be found in brainstorming section of our wiki!

• 01.13.2012

-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.

• 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!!!

Results/Conclusions

What did you achieve over the course of your semester?

Safety

Attributions

Human Practices

Fun!

References

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