User:Wolfgang Pernice

Contact Info



 * Wolfgang Pernice
 * Imperial College London
 * 18 Courtfield Gardens
 * SW5 0PD
 * London, UK
 * wolfgang.pernice08@imperial.ac.uk

I work in the IGEM:IMPERIAL/2010 at Imperial College London. I learned about OpenWetWare from my uni, and I've joined because of my participation in the Imperial College iGEM 2010 Team.

Education

 * 2nd, BS, Imperial College London, life sciences

iGEM ideas
I think the major directions we could take the project into are the following:
 * 1) Using a micriobial organism as a bio-detector and/or bio-filter. Several ideas were already discussed during the first two brain-storming sessions, with focus on public water-systems or dialysis. The combination of detection and filtering approaches would allow to build uppon the results of previous iGEM teams, such as the camebridge 2009 project. For example a water-filter approach could include a recombinant, bio-film-forming bacterial species which endocytoses, or otherwise sequesters contaminants in water-circuits. The accumulation of target molecules in/on the cell could be used as trigger to induce loss of bio-film attachment so that the "loaded" cell can be filtered out by a secondary, mechanical filter. Additionally, the receptor could be linked to a signalling pathway leading to the synthesis of a visual signal. Key questions here would be: are there contaminants that would suit such a system; can we find a suitable receptor and express it in the target organism; can we ensure the safety of the system (no recombinant organisms introduced into the open circuit). Linked to this area are other variations of filter mechanisms such as for oil or for salts. I think we have a relatively high chance to realize a project in this area; something of potential use could already be created with just modifying one function: detection/binding of extracellular molecules. The more complex desired response pathways are, the more difficult will the realization be.
 * 2) Second, a microbial organism could be used for bio-synthesis of a desired substance. In the workshops we discussed a H2 sythesising bacterium. Another idea would be the synthesis of graphene (e.g. Gain et al. 2007), a new material with extraordinary properties, e.g. and intrinsic breaking strength up to 200x stronger than steel (Lee et al. 2008)and as transistors exceeding the on-off frequence of silicon by approximately 10x (100 GHz) (Bourzac 2010); also Xia et al. 2009for photonic rather than electronic properties. Graphene consists of a one atom thick monolayer of carbon atoms forming a honeycomb grid. In a 3D complex multiple layers form graphite. The production of graphene has been extremely expensive and has thus limited it's commercial value. While several advanced methods now exist which allow for less costly growing of graphene, a bio-synthetical approach in my opinion has potential to significantly increase the value-cost ratio. We would need to define a suitable entry point in the carbon metabolism cycles in a target organism which would most likely be located in the anabolism of polysaccharides (e.g. cellulose, starch) especially of membrane-targeted glycans. Figure 1 suggests a basic strategy for biological graphene synthesis [[Image: SchemeGrapheneproduction.bmp|thumb|right|400px|x300px| Figure 1]].As this will require the design of new, or the substantial editing of existing anabolic pathways I consider the bio-synthesis area as significantly more ambitious and possibly not realizable in the limited time of our project.
 * 3) A third area, that was already mentioned in the workshops is the manipulation of human cutaneous commensal bacteria to enhance their existing protective features: the continuous production and secretion of drugs for the long-term treatment of dermatological problems, to support wound healing, of scent-neutralizing or -producing agents or with transpiration-suppressing function are only a few examples. For the medical or pharmacological character of these ideas our work and progress here would most likely limited by substantial ethical and legal barriers.
 * 4) Cell-cell communication: e.g. a recombinant cell which reacts to signals from its surrounding cellular environment. As such the recombinant cell could buffer intracellular signalling by secreting antagonists to an undesired signalling pathway active in the normal cell environment. Could be useful in stabilizing a target cell population in a specific state. This would require systems in which multiple signals are integrated in order to induce a specific cellular response.
 * 5) Virus build battery

A more techniqual approach would include the following as topics for our project:
 * 1) Robust cell-cell communication pathways. Could be illustrated by e.g. stable and efficient communication between cells or directional cell migration in an environment with increased chemical noise.
 * 2) Switches, oscillators or logic gates with increased robustness to noises. A great example of how the processivity and functionality of such operators can be illustrated is shown by Tabor et al. 2009: several genetic circuits were linked to an optical sensoring module in e.coli which caused the aggregation of bacterial colonies along light exposed areas. Thus the bacterial lawn reprinted a given projected pattern.
 * 3) Stabilizing genetic information to ensure safety of recombinant organisms.

Fv-fragment guided input module
To design detection-input mechanism with modular character, as opposed to the use of possibly existing two-component signalling cascades with specificity to e.g. parasite larval stages, we could exploit the specificity of antibodies to their substrate. Several teams in iGEM have used Ab's, attempting to alter the specificity of cell-surface receptors towards a desired target (e.g. Illinois 2008). To circumvent problems with direct manipulation of the receptor molecule, while incorporating the wide spectrum of potential targets, that could be recognized in an anitbody based approach, the Fv fragment of a given antibody is fused to the ligand of a suitable native two-component system. Figure 2 illustrates the principle of the Fv-fragment guided detection system.


 * 1) . Expression of the Fv-ligand-fusion protein (detector protein). Since the production of Fv fragments in prokaryotes has not been successful so far, a eukaryotic system is preferred (e.g. Yeast).
 * 2) . The detector protein is secreted into the extracellular environment.
 * 3) . Due to the high specificity of the Fv-fragment to its substrate, the detector protein binds to a given pathogen.
 * 4) . Spatial co-localization of detector proteins on the pathogens surface is mirrored by clustering of receptors for the ligand-fragment, in the detector cell. This leads to the induction of a downstream signalling cascade to trigger a response module.

The system has several features which render it interesting:


 * An extremely broad array of potential targets that can be detected, exploiting the power of molecular recognition via Antibody fragments.
 * A highly modular character. Exchangebility of specificity and potential to link the system to virtually any given response system
 * Very close to real world applications (parasites, toxins etc).

Two central issues come along with this system:


 * A suitable secretion system has to be found to allow efficient trafficing of the detector protein into the extracellular space.
 * Production of false positives has to be prevented. A requirement for clustering and crosslinking of several receptors for signal generation could solve this problem.

Schistosoma sensoring via Cercarial proteases
The cercarial stage of the schistosoma life-cycle represents the infective form for humans. Living in open or closed water systems, upon detection of skin-lipids the excretion of various proteases from primary secretory glands is triggered by thermal and chemical signals associated with skin lipids. Two major classes of proteases have been identified: a chemotrypsin like protease with specifity for large hydrophobic side chains and a trypsin like with preference for positively charged side chains ((Salter et al 2000). A highly potent option is cercarial elastase (reference).

These proteases could be used to cleave membrane associated Fret-pairs, or release other response mediators from a membrane-scaffhold. This would e.g. produce a color response in the sample.

Three key problems exist here:


 * Artificial triggering of protease secretion of potential circaria in the sample. This can be achieved by using linoleic acid residues which can mimic skin lipids.
 * Targeting of the response carrier protein to the extracellular membrane.
 * Releasing a sufficiently strong signal to be detected by simple means. As e.g. spores can be stored easily and used in desired quantities, an additional way exists in which we can increase total concentration of e.g. color-response molecules to be released, in addition to high-level expression mechanisms.

Useful links

 * Introductory tutorial
 * OpenWetWare help pages
 * iGEM 2010 Imperial College London
 * heidelberg license to kill