Brainstorm Area

Contents 1 Brainstorm Area 1.1 Interesting Links 1.2 Interesting Papers (provided by Domenico) 1.3 Decision 1.4 Top 6 Ideas 1.5 Brainstorm Ideas 1.5.1 Impulse Signal 1.5.2 Saltwater Purification 1.5.3 MicroOrganism muscle 1.5.4 Enzyme Modulation 1.5.5 Melatonin Compensation 1.5.6 Buoyant Bacteria 1.5.7 Xylitol production 1.5.8 New Antibiotic Cassettes 1.5.9 Paper Based Microfluidics with bacteria 1.5.10 Close proximity phage expression 1.5.11 Anti-Venom System 1.5.12 Birth Control Yoghurt 1.5.13 Excrement Degradation Bacteria 1.5.14 Additional Processing of Excretion for Energy 1.5.15 Targeted Delivery 1.5.16 microRNA and P53 1.5.17 Synchronization -> Flashing Colony 1.5.18 Sensor Reporter Spatially Displaced 1.5.19 Kalman Filter 1.5.20 Stopping a Cytokine storm 1.5.21 Water Soluble Vitamin Production 1.5.22 Stop Quorum Signalling -> Quenching AHL 1.5.23 Biological Isotope Separation 1.6 The early meetings 1.6.1 April 7th, 2009 1.6.2 17th April 2009

Interesting Links

• [http://openwetware.org/wiki/IGEM:Idea_exchange iGEM Idea Exchange]
• [http://openwetware.org/wiki/Tk:wanted_parts TK: Wanted Parts]
• [http://openwetware.org/wiki/IGEM:Projects_categorized iGEM:Projects categorized]
• [http://openwetware.org/wiki/Synthetic_Biology:Vectors/Wishlist Synthetic_Biology:Vectors/Wishlist]

Interesting Papers (provided by Domenico)

• [http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002815 Design and Construction of a Double Inversion Recombination Switch for Heritable Sequential Genetic Memory]
• [http://www.nature.com/nature/journal/v457/n7227/full/nature07616.html A tunable synthetic mammalian oscillator]
• [http://www.nature.com/msb/journal/v3/n1/full/msb4100187.html Programming gene expression with combinatorial promoters]
• [http://pubs.acs.org/doi/abs/10.1021/ar020219d Photoreceptor Proteins, “Star Actors of Modern Times”: A Review of the Functional Dynamics in the Structure of Representative Members of Six Different Photoreceptor Families]
• [http://www.nature.com/msb/journal/v5/n1/full/msb20091.html Environment-specific combinatorial cis-regulation in synthetic promoters]
• [http://www.ploscompbiol.org/article/info:doi%2F10.1371%2Fjournal.pcbi.1000064 Implementing Arithmetic and Other Analytic Operations By Transcriptional Regulation]
• [http://www.jbioleng.org/content/2/1/5 Engineering BioBrick vectors from BioBrick parts]
• [http://authors.library.caltech.edu/13255/ Model-guided design of ligand-regulated RNAi for programmable control of gene expression]
• [http://www.nature.com/nbt/journal/vaop/ncurrent/abs/nbt.1536.html Diversity-based, model-guided construction of synthetic gene networks with predicted functions]

Decision

We made a decision: Self destructive plasmid (a derivate of the impulse signal). Further refinement of the final idea can be seen here Team:TUDelft/Brainstorm/Plasmid.

Top 6 Ideas

1. Salt water purification (Sriram) (Orr)
2. Buoyant Bacteria (Saeed) (Orr)
3. Impulse Signal (Daniel) (Sriram)
4. Melatonin Compensation (Saeed) (Tim)
5. Microrganism Muscle (Daniel) (Tim)
6. Enzyme Modulation (Daniel) (Sriram)

The names behind the ideas indicates who is going to search background information on the subject and inspect the feasibility and application for the ideas.

Brainstorm Ideas

Impulse Signal

• one suggestion (by Federico C.) from the idea exchange was to create a self-destructing plasmid, which would express once and than destroy itself with a restriction endonuclease in its code.
  
  

Saltwater Purification

• + heavy metal extraction
  
• diatoms
  
• Dead Sea organisms
  
• After some research, we found that this project might not be as feasible as we imagined. Searching for the keywords 'desalination' and 'bacteria' resulted in articles about the biofilm created by bacteria to deteriorate the proper function of reverse osmosis (RO) membrane
  
  

MicroOrganism muscle

• make a group of cells contract
  
  

Enzyme Modulation

• express different enzymes in different period of time and measure flux
  
• metabolic control analysis
  
  

Melatonin Compensation

• compensate the reduction in melatonin levels caused by the invention of electrical lights
  
• has been linked to lots of medical problems, see review paper [http://www.psy.ohio-state.edu/nelson/documents/JPinealRes2007.pdf The dark side of light at night: physiological, epidemiological, and ecological consequences]
  
• would need to implement melatonin detection, synthesis, and a clock
  
• work comes back to clock/delay timer
• Various papers:
• Cloning and characterization of a Chlamydomonas reinhardtii cDNA arylalkylamine N-acetyltransferase and its use in the genetic engineering of melatonin content in the Micro-Tom tomato, Masateru Okazaki, Kenji Higuchi, Yutaka Hanawa, Yoshihiro Shiraiwa and Hiroshi Ezura, http://www3.interscience.wiley.com/journal/122295358/abstract
• Non-vertebrate melatonin, Hardeland R, Poeggeler B, http://www3.interscience.wiley.com/journal/118842332/abstract?CRETRY=1&SRETRY=0
• Melatonin in the unicellular Tetrahymena pyriformis: effects of different lighting conditions, L. Köhidai, O. Vakkuri, M. Keresztesi, J. Leppäluoto, G. Csaba, http://www3.interscience.wiley.com/journal/90011815/abstract?CRETRY=1&SRETRY=0
• Melatonin and 5-methoxytryptamine in non-metazoans, http://rnd.edpsciences.org/index.php?option=article&access=standard&Itemid=129&url=/articles/rnd/abs/1999/03/RND_0926-5287_1999_39_3_ART0011/RND_0926-5287_1999_39_3_ART0011.html
• Melatonin in plants, Małgorzata M. Posmyk and Krystyna M. Janas, http://www.springerlink.com/content/r12145642683235q/
• And for project marketing:
• Melatonin present in beer contributes to increase the levels of melatonin and antioxidant capacity of the human serum, Maria D. Maldonado, Hector Morenoa and Juan R. Calvo, http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6WCM-4VPKPSC-2&_user=499885&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_acct=C000024500&_version=1&_urlVersion=0&_userid=499885&md5=93234ea24f67a57ae2f5adf2dd051889
• Putting cancer to sleep at night, David E. Blask, Robert T. Dauchy, and Leonard A. Sauer, http://www.springerlink.com/content/e7v778uj16mn8070/

Buoyant Bacteria

• for metal extraction from old mines or ocean
  
• Gvp genes of B. megaterium can be expressed functionally in Escherichia coli, rendering it buoyant by its [http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6TD0-4GHBP6F-1&_user=10&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=c7578acb7860d2a68c667623b672f5d1 gas vesicles]
  
• Might be used in mining ([http://en.wikipedia.org/wiki/In-situ_leach in-situ leach], otherwise known as solution mining, is a technique where acid is pumped into a mine, separating the ore from the soil by dissolving the ore and the ore being pumped to the surface)
  
• Several issues that need to be accounted for:
  

1. [http://www3.interscience.wiley.com/cgi-bin/fulltext/121493777/HTMLSTART Anaerobiosis inhibits gas vesicle formation in halophilic archea] – gas vesicles that allow buoyancy for bacteria require oxygen to exist. This is a major problem with mines (blackdamp – oxygen replaced by other gases)
   
2. Density of target ore – In-situ leach is often used for uranium, and can be used for copper and gold as well, all dense metals, and for buoyancy to occur, the bacteria need to have a sufficient density to allow the ore to float (copper least dense with 8.9 g/cm3, gold most dense with 19.3 g/cm3)
   
3. Amount of energy put into bacterial buoyancy – is using bacteria for the buoyancy really more efficient (energy-wise and time-wise) then normal pumping of the ore to the surface?
   

• [http://www3.interscience.wiley.com/journal/117860406/abstract Biosorption] – the 3 strains S. acidiscabies W-12, M. luteus W-20 and Bacillus sp. W-28 were found to be the most tolerating of high acidity levels and heavy metal environment. Those tolerant strains were also found to be able to absorb up to 80% aluminium and copper, and 60% uranium from mining sites
  
  

Xylitol production

• already attempted, see [http://openwetware.org/images/0/03/6-bolaji.pdf]
  
  

New Antibiotic Cassettes

• Biobricks exist for Ampicillin, Kanamycin, Chloramphenicol, and Tetracycline.
  
• New biobricks needed (see TK:Wishlist), integrate into larger project?
  
• Cationic peptides, Rhodostreptomycins [http://mit.edu/biology/sinskey/www/Kurosawa2008.pdf]
  
  

Paper Based Microfluidics with bacteria

• Improve the processing outlined in [http://www.rsc.org/delivery/_ArticleLinking/ArticleLinking.cfm?JournalCode=LC&Year=2008&ManuscriptID=b811135a&Iss=12 FLASH: A rapid method for prototyping paper-based microfluidic devices], see papers from Whitesides group at Harvard.
  
• use light sensor and output hydrophobic/hydrophilic chemicals that can coat the cellulose in the paper placed on an agar plate
  
  

Close proximity phage expression

• once a cell is close enough to a target, produce phages and explode
  
  

Anti-Venom System

• inject bacteria after being bitten
  
• sense which venom and produce proper anti-venom
  
  

Birth Control Yoghurt

• attacks sperm
  
• animal use
  
  

Excrement Degradation Bacteria

• dog excrements degrades quickly
  
• can be used in as excrement degrader instead of waste tank in portable toilets or [http://en.wikipedia.org/wiki/Deodorize deodorizing] excrements in chemical toilets
  
  

Additional Processing of Excretion for Energy

• Zero Growth -> convert everything to product
  
  

Targeted Delivery

• use THC for medical benefits without any psychoactive effects
  
  

microRNA and P53

• inhibit miRNA to increase activity of tumor-suppressor gene p53
  
• needs targeted delivery system
  
  

Synchronization -> Flashing Colony

• synchronized flashing
  
• covalent modification
  
  

Sensor Reporter Spatially Displaced

Kalman Filter

• integrate into larger project
  
  

Stopping a Cytokine storm

• something which could break the feedback loop in a cytokine storm
  
  

Water Soluble Vitamin Production

• produce Vitamins B and C.
  
  

Stop Quorum Signalling -> Quenching AHL

• aiiA enzyme [http://www.msk.or.kr/jsp/view_old_journalD.jsp?paperSeq=2133] already has a biobrick [http://partsregistry.org/Part:BBa_C0060 Part:BBa_C0060] but it is not secreted.
  
• halogenated furanones [http://mic.sgmjournals.org/cgi/content/full/151/11/3589?view=long&pmid=16272381] [http://mic.sgmjournals.org/cgi/content/full/148/4/1119?view=long&pmid=11932456] decrease binding activity of LuxR.
  
• [http://www.molbio.princeton.edu/index.php?option=content&task=view&id=27 Bonnie Bassler]'s research, inhibit quorum signaling with molecules structurally similar to AHL. Her recent [http://www.ted.com/index.php/talks/bonnie_bassler_on_how_bacteria_communicate.html TED Talk].
  
  

Biological Isotope Separation

• is it even possible?
  
• according to this [http://jol.liljenzin.se/BOOK.HTM book] in section 2.6.5. [http://jol.liljenzin.se/KAPITEL/CH02NY3.PDF], both chlorella algae and E.coli have isotope enrichment properties (hydrogen, deuterium). no references given.
  
• possible applications: remove radioactive isotopes from body (cancer treatments), separate heavy water from ocean water, remove carbon 14 and potassium 40 from body/food.
  
• another paper is Bioseparation of Lithium Isotopes by Using Microorganisms, Sakaguchi, Takashi | Tomita, Osamu, Resource and Environmental Biotechnology [Resour. Environ. Biotechnol.]. Vol. 3, no. 2-3, pp. 173-182. 2000. havent found a copy online.
  
  

 

The early meetings

To get acquainted with iGEM and previous projects that were done by other Universities, we all looked at different subjects.

April 7th, 2009

Orr

• Bologna 2008 - Ecoli.PROM: an Erasable and Programmable Genetic Memory with E. coli

The Bologna team created here a genetic memory for E.coli that would switch between two states as a response to a certain signal (a UVc signal would inhibit Lex A (an inhibitor of LacI), thus allowing the production of LacI, whereas an IPTG signal would inhibit LacI and allow the production of TetR).

• Cambridge 2008 - iBrain: Foundations for an Artificial Nervous System using Self-Organizing Electrical Patterning

In this project, the Cambridge team decided to do three simultaneous sub-projects: one involved the creation of an artificial neurological system for E.coli (using potassium channels to transfer the ion and thus allow for a gradient of the ions), the second involved generating Turing patterns in order to integrate two signalling systems into Bacillus subtilis, and the third part involved generating standardized tools and techniques for B. subtilis. We learned from this project an important lesson: quality is definitely more important than quantity, and there is no point of doing many simultaneous experiments if we cannot finish any one of them properly.

• KULeuven 2008 - Dr. Coli, the bacterial drug delivery system

In this project, the KU Leuven team presented the idea of using E.coli as a transporter of medication in such a way that it would be targeted at a specific location. Furthermore, the E.coli would react to the presence of foreign organisms and would express certain proteins to destroy them, followed by it's self destruction after the area would be completely cured. It is important to emphasise that the project was not intended to produce a specific medication to a specific disease, but merely as a way to show that the general concept of curing a disease in a direct manner is possible.

Sriram

• Illinois - Biomolecular fluorescence biosensor system: cell bases biosensor system

• Ljubljana, Slovenia - Virotrap - A synthetic biology approach against HIV

• Ljubljana, Slovenia - Engineered Human cells - say no to sepsis

Tim Weenink

• ESBS Strasbourg - Binary generation counting in Yeast

• University of Ottowa - Puslegenerator in Yeast

• Calgary Wetware - Quorum coupled bacteriocin release (engineering a champion)

17th April 2009

Tim Vos

• MIT - Biogurt: A Sustainable and Savory Drug Delivery System

• Groningen - Conway’s Game of Life in real life

• UFreiburg - Modular Synthetic Receptor System

Daniel

• Harvard - bactricity

• ETH Zurich - Random walks towards the minimal genome

• Slovenia - Immunobricks

Saeed

• Caltech - Engenering multi-functional probiotic bacteria

The human gut houses a diverse collection of microorganisms, with important implications for the health and welfare of the host. They aimed to engineer a member of this microbial community to provide innovative medical treatments. Our work focuses on four main areas: (1) pathogen defense either by expression of pathogen-specific bacteriophage or by targeted bursts of reactive oxygen species; (2) prevention of birth defects by folate over-expression and delivery; (3) treatment of lactose intolerance by cleaving lactose to allow absorption in the large intestine; and (4) regulation of these three treatment functions to produce renewable subpopulations specialized for each function. E-Coli a non-pathogenic microbe in the guts. Nissle 1917 is a commercially available[2] non-pathogenic, probiotic strain of E. coli. Oxidative Burst.

In order to help guard against infections of the gut, we wish to engineer a strain of E. coli capable of killing bacterial pathogens. White bloodcells are able to do this, but they do migrate to large intestinal lumen where pathogens can reside. Since E-coli is adapted to this environment, it is a good candidate . Acylhomoserine lactones(AHL) are small communication molecules between bacteria. AHL is able to diffuse across membrane. This communication system contains two main components LuxI, an AHL producer and LuxR an AHL dependent transcriptional activator. Engenired E.Coli strain is able to detect AHL from pathogens. This pathogen detection activates pyruvate oxidase which uses pyruvate to produce hydrogen perioxide, to kill the pathogens. An E.Coli catalase was used to protect the cell from cells killing, so they first were able to accumulate hydrogen perioxide before oxidative burst otherwise perioxide production would lead killing of the cell only. LuxR activates several gene including LuxI. Here they used LuxR in a sensing mechanism. When LuxR binds AHL its will overexpress hydrogen perioxide coding genes. Entire system was onto a high copy plasmide in E.Coli. They showed the system is able to detect and kill pathogens by a coculture assay. They have successfully demonstrated the overproduction of hydrogen peroxide in E. coli (JI377) using the pyruvate oxidase from S. pneumoniae. This “oxidative burst” is sufficient to kill a competing strain of JI377 cells in co-culture assays

Difficulties: The most surprising finding was that the cells began expressing hydrogen peroxide a full hour before they were induced with AHL. The current hypothesis is that some component of the rich SOC media mimicked AHL and activated LuxR. However, no such autoinduction occurred in the defined minimal media M9. It would be interesting to see if LuxR spontaneously activates expression in a rich defined media

Production of hydrogen peroxide is not a normal occurrence in the large intestine, and its effects would need to be investigated before the engineered strain could be used to fight fection.

• Heidelberg - Ecolicense to kill

• Valencia - Hot Yeast

Idea: Heat is vital for life. Some organisms are able to maintain their internal temperature constant, while other not. UCP1, thermogenin is a mammalian uncoupling protein in the mitochondrial inner membrane. When protons pass through the UPC complex instead of the ATP synthase complex they produce heat instead of ATP. They tried to express the UPC1 in a S.cerevisiae strain, so this strain would be able to maintain its temperature constant in a given range.

Applications: - A cell which is able to maintain its own temperature so no external heating would be necessary, this could reduce the electrical costs in a lab. - The system could be implement in some plant species. If a plan6 would be able to maintain its own temperature it could grow in a colder temperature or survival frost.

1) In order to detect and measure temperature production they build a Liquid Cultured Calorimeter(LCC). Therefore they used commercial thermo flasks, isolation material(Armaflex), hardware and software. The isolation material Armaflex caused some problems because it reacts with water.

2) They build a UPC + strain, a UPC - strain and two mutants. Gly 76 and Gly 175 mutant(deletion in 76 or 175 Gly triplet). LCC experiments showed that the two mutants where able to heat up there own medium compared to the UPC + and UPC- strain. Growth kinetic experiments were done ass well. These showed that the UPC + and the two mutants growth much slower than the UPC – strain, due to uncoupling and thus no ATP production.

They made a proposal for an ethical code of conduct for synthetic biology based on the hot yeast project.