Team:UiOslo Norway/Project

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<h1>Project Overview</h1>
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<h1 style="font-family:Syncopate; color: blue;">Project Details</h1>
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<h2>Our Project - The microOrganizer</h2>
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<p>The aim of our project was to build a system for physically connecting different bacteria in a predetermined manner. We have tried to design surface markers that enable bacteria to bind to each other and to respond to the binding by a mechanism that would allow us to select for bound bacteria. A succesful system would allow us to organize different types of bacteria in a culture and we have therefore named our project «The microOrganizer».</p>
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<h3>Organ</h3>
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<h2>The Parts</h2>
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<p>The cell is the basic structural, functional, and biological unit of all known living organisms. Because the cells have all equipment and expertise necessary to carry out the functions of the life, they are considered the smallest unit of life that can replicate independently.</p>
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<h3>Split Enzyme Principle</h3>
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<p>The unicellular organisms are made up with just one cell and their main groups are: bacteria, archaea, protozoa, unicellular algae and unicellular fungi.</p>
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<p>A split enzyme is an enzyme split into two or more non-functional parts with intact tertiary structure. This means that the enzyme can be active if the different parts come together and assemble into the original enzyme. The enzyme parts have a natural affinity for each other and can assemble spontanously. We wanted to expose two different parts of a split enzyme on the surface of different bacteria.</p>
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<p>On the other hand the multicellular organisms are more complex and consist of multiple cells. The animals, including human beings, are multi-cellular. An adult human body is composed of about 100,000,000,000,000 cells!</p>
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<p>Each cell has basic requirements to sustain it, and the body's organ systems are largely built around providing the many trillions of cells with those basic needs (such as oxygen, food, and waste removal).</p>
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<p>A tissue, such as muscle, nervous and connective tissues, is an organized system composed with different kinds of specialized cells.</p>
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<p>Similarly, an organ is an organized and differentiated structure of various tissues performing independently a specific function, such as the stomach, the skin and the brain.</p>
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<p>The organs carry out functions as:</p>
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<li>Communication between cells</li>
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<li>Supplying the cells with nutrients</li>
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<li>Controlling exchanges with the environment</li>
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<p>Source: <a href="http://www.nature.com/scitable/ebooks/essentials-of-cell-biology-14749010/15625600 8/09/2011">http://www.nature.com/scitable/ebooks/essentials-of-cell-biology-14749010/15625600 8/09/2011</a></p>
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<h3>Microorganism</h3>
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<h3>Autotransporters</h3>
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<p>A microorganism or microbe is a diverse unicellular or multicellular mostly microscopic organism that includes all the bacteria and archaea and almost all the protozoa. They have also some members of the fungi, algae, and animals.</p>
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<p>Wild type <em>E.coli</em> can express proteins on their surface by using protein channeling systems called autotransporters. These proteins assemble themselves to a channel in the bacterial membrane and any proteins connected to them will be threaded through this channel and exposed to the outside of the bacteria. The channel itself functions as a membrane anchor. We aimed to connect our split enzyme parts to the membrane part of the autotransporter and present them on the bacterial surface.</p>
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<p>They are considered the oldest form of life on Earth and wandered it hundreds of millions of years before the dinosaurs.</p>
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<p>Microorganisms can be found and are able to live in every part of the biosphere including terrestrial, aquatic and extraterrestrial habitats, extreme environments and in other organisms, such as our digestive systems. Similarly, they are tolerant to many different conditions such as limited water accessibility, high salt content and low oxygen levels. Although they are well adapted, not every microorganism can survive in all habitats, as each type of it has developed to live within a narrow range of conditions.</p>
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<p>These organisms are essential components of every ecosystem. Indeed, they are essential to nutrient recycling and other compounds throughout the environment, by acting as decomposers and oxygen generators.</p>
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<p>Furthermore, they are very exploited in the biotechnology field, such as in food and beverage preparation, in water treatment, in energy production, in manufacturing of chemicals, enzymes and other bioactive molecules and in science. A small percentage of microorganisms are pathogenic and cause disease or even death in plants and animals, having, consequently, an important role in health.</p>
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<p>Sources:</p>
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<li><a href="http://www.sciencedaily.com/articles/m/microorganism.htm">http://www.sciencedaily.com/articles/m/microorganism.htm</a></li>
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<li>Madigan M, Martinko J (editors) (2006). Brock Biology of Microorganisms (13th ed.). Pearson Education. p. 1096. ISBN 0-321-73551-X.</li>
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<h3>Our Project</h3>
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<h3>Selection Mechanism</h3>
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<p>The mOrgan or micro-Organizer is a layered system for organizing E. coli in a predetermined and organ-like way.</p>
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<p>The enzyme activity is an obvious marker of a succesful surface interaction between the bacteria. We therefore wanted an enzyme which substrate could induce a particular response that the product could not. The enzyme activity could protect the interacting bacteria and do the job as a selection mechanism.</p>
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<p>This system can be applied in several fields of science and needs. In this context, the mOrgan is built up with different types of bacteria that form an organized structure and communicate to each other to perform specific functions, like organs.</p>
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<p>Thus, whatever is done in mOrgan occurs in a multi-step process within the different layers of the system.</p>
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<p>The E.coli will have a surface identity given by regulation of downstream genes and the mOrgan will be formed in an organized way. This property helps to avoid random bacteria linking to each other and creating uncontrolled growing colonies, keeping the correct configuration.</p>
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<h3>Applications</h3>
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<p>For instance, mOrgan can be useful in industrial manufacturing, industrial degradation, remediation processes, drug delivery systems, drug metabolism studies, catalytically amplified sensors, regulatory networks, biocomputers and so forth.</p>
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<p>We chose β-galactosidase as our enzyme and β-galactosyl glycerol as our substrate. β-galactosyl glycerol is a small β-galatoside that can cross the <em>E.coli</em> membranes and enter the cytoplasm through a constitutively expressed galactose permease. β-galactosyl glycerol can also bind to the Lac operon repressor in the same manner as allolactose and inhibit its repressor activity – thus function as an inducer of the Lac operon. β-galactosyl glycerol also makes a potential substrate for β-galactosidase which splits β-galactosyl glycerol into glycerol and galactose<sup>1</sup>.</p>
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<p>β-galactosidase is expressed in wild type <em>E.coli</em> from the LacZ gene which is a part of the Lac operon. β-galactosidase can be split into two different parts with an intact tertiary structure and is therefore a good alternative for our split enzyme. The two parts are coded by what has been called LacZ α and LacZ Ω.</p>
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<p>The final version of the microorganizer system would be as follows: two different strains of bacteria would express a fusion protein consisting of the membrane part of an autotransporter and one of either LacZ α or LacZ Ω. Both strains would also have a toxic gene under the control of the Lac promoter. The two strains would be mixed in a medium containing β-galactosyl glycerol. Bacteria with no partners would have β-galactosyl glycerol flowing into cytoplasma and induce transcription from the Lac promoter and kill them. Bacteria that are bound to each other through the β-galactosidase parts will cleave galactosyl glycerol into galactose and glycerol and make sure the bacteria can survive.</p>
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<p>To avoid a short cut in the mechanism we would have to use bacteria were the Lac operon is deleted so there is no cytoplasmic β-galactosyl activity. We identified a strain, <em>E. coli</em> NCM17, which meet this criteria.</p>
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<h4>Sources</h4>
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<p>1) Egel, R. (1988) The "lac" operon: an irrelevant paradox? Trends in Genetics 4:31.</p>
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Latest revision as of 15:39, 17 October 2014

UiOslo IGEM 2014

Project Details

Our Project - The microOrganizer

The aim of our project was to build a system for physically connecting different bacteria in a predetermined manner. We have tried to design surface markers that enable bacteria to bind to each other and to respond to the binding by a mechanism that would allow us to select for bound bacteria. A succesful system would allow us to organize different types of bacteria in a culture and we have therefore named our project «The microOrganizer».

The Parts

Split Enzyme Principle

A split enzyme is an enzyme split into two or more non-functional parts with intact tertiary structure. This means that the enzyme can be active if the different parts come together and assemble into the original enzyme. The enzyme parts have a natural affinity for each other and can assemble spontanously. We wanted to expose two different parts of a split enzyme on the surface of different bacteria.

microOrganizer

Autotransporters

Wild type E.coli can express proteins on their surface by using protein channeling systems called autotransporters. These proteins assemble themselves to a channel in the bacterial membrane and any proteins connected to them will be threaded through this channel and exposed to the outside of the bacteria. The channel itself functions as a membrane anchor. We aimed to connect our split enzyme parts to the membrane part of the autotransporter and present them on the bacterial surface.

Selection Mechanism

The enzyme activity is an obvious marker of a succesful surface interaction between the bacteria. We therefore wanted an enzyme which substrate could induce a particular response that the product could not. The enzyme activity could protect the interacting bacteria and do the job as a selection mechanism.

The Details

We chose β-galactosidase as our enzyme and β-galactosyl glycerol as our substrate. β-galactosyl glycerol is a small β-galatoside that can cross the E.coli membranes and enter the cytoplasm through a constitutively expressed galactose permease. β-galactosyl glycerol can also bind to the Lac operon repressor in the same manner as allolactose and inhibit its repressor activity – thus function as an inducer of the Lac operon. β-galactosyl glycerol also makes a potential substrate for β-galactosidase which splits β-galactosyl glycerol into glycerol and galactose1.

β-galactosidase is expressed in wild type E.coli from the LacZ gene which is a part of the Lac operon. β-galactosidase can be split into two different parts with an intact tertiary structure and is therefore a good alternative for our split enzyme. The two parts are coded by what has been called LacZ α and LacZ Ω.

The final version of the microorganizer system would be as follows: two different strains of bacteria would express a fusion protein consisting of the membrane part of an autotransporter and one of either LacZ α or LacZ Ω. Both strains would also have a toxic gene under the control of the Lac promoter. The two strains would be mixed in a medium containing β-galactosyl glycerol. Bacteria with no partners would have β-galactosyl glycerol flowing into cytoplasma and induce transcription from the Lac promoter and kill them. Bacteria that are bound to each other through the β-galactosidase parts will cleave galactosyl glycerol into galactose and glycerol and make sure the bacteria can survive.

To avoid a short cut in the mechanism we would have to use bacteria were the Lac operon is deleted so there is no cytoplasmic β-galactosyl activity. We identified a strain, E. coli NCM17, which meet this criteria.

Sources

1) Egel, R. (1988) The "lac" operon: an irrelevant paradox? Trends in Genetics 4:31.

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