"

From 2012hs.igem.org

Contents 1 Quorum sensing using V.harveyi 2 Ideas from imagination 2.1 To Kill a Mosquito 2.2 Bacterial Sunscreen 2.3 Lactose intolerance curing E. coli 2.4 Cholesterol Degradation 3 Ideas based on previous iGEM teams 3.1 Glowing Bacteria 3.2 Cobalt Buster 3.3 Plastic Degradation 3.4 Synthesis of Flavor using Candida Rugosa 3.5 Methane Sensing

Quorum sensing using V.harveyi

• During the project, we found that luxR part was inconsistent so that we had to have a backup plan. Groups of V. harveyi bacteria is known to communicate via quorum sensing, so we studied about its system. However, V. harveyi lacks a LuxI/R quorum sensing system, and instead employs a hybrid quorum-sensing circuit using AI-2.

• However, since AI-2 is both made by gram-negative and gram-positive bacteria, it was impossible for us to adopt part that is similar to luxR in V.harveyi. In our model of luxI/R quorum sensing system, luxI from V.fischeri (a gram-negative bacteria) only produces AHL, while aiia from B.subtilis (a gram-positive bacteria) only decomposes AHL.

• We concluded that the difference in composition and degradation rate of AHL drives synchronized oscillation. Since the system using AI-2 will not be able to provide accurate information about composition and degradation rate of AI-2, we decided not to use V.harveyi system.

Ideas from imagination

To Kill a Mosquito

• From science news articles linked below, we have found some chemicals that either attracts mosquito or kills it.

http://www.ars.usda.gov/is/pr/2010/100309.htm

http://www.scidev.net/en/news/mosquitoes-kill-their-own-in-new-dengue-control-me.html

• 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)

• Pyriproxifen works as a juvenile hormone analogue and prevents developmental steps of a larvae.

• 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.

• Professor and advisors' comments:

 -Although there is a way to synthesize pyriproxyfen in a chemical way, there is no known biosynthesis pathway of the substance. 
 -Many of the mosquito-attracting chemicals also appeal to different types of insects
 -Some of the mosquito-attracting chemicals are already in sales

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)

• The advisor commented:

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.

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>

• 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.

• Comments from professor and advisors

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.

The characteristics of the filter were already sufficiently discovered by the previous iGEM team. So there was not much room for improvement.

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.

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.

• Comments from professor and advisors

This type of project was widely studied by numerous previous iGEM teams. So, it would be repetitive to address the same task.

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)