Difference between revisions of "Team:IISc-Bangalore/Description"

 
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<li><a href="#introduction">Introduction<img src="https://static.igem.org/mediawiki/2017/6/68/T--IISc-Bangalore--navbar_bullet.png" /></a></li>
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<li><a href="#structure">Structure<img src="https://static.igem.org/mediawiki/2017/6/68/T--IISc-Bangalore--navbar_bullet.png" /></a></li>
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        <li><a href="#biogenesis">Biogenesis<img src="https://static.igem.org/mediawiki/2017/6/68/T--IISc-Bangalore--navbar_bullet.png" /></a></li>
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<li><a href="#bioengineering">Bioengineering<img src="https://static.igem.org/mediawiki/2017/6/68/T--IISc-Bangalore--navbar_bullet.png" /></a></li>
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<li><a href="#purification">Purification<img src="https://static.igem.org/mediawiki/2017/6/68/T--IISc-Bangalore--navbar_bullet.png" /></a></li>
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<li><a href="#references">References<img src="https://static.igem.org/mediawiki/2017/6/68/T--IISc-Bangalore--navbar_bullet.png" /></a></li>
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    </ol>
  
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<div id="contentMain">
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            <ol>
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                <li>Gas vesicles</li>
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                <li>Our analysis</li>
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                <li>Improving flotation</li>
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                <li>Assays</li>
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            </ol>
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<div id="contentMain">
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<img src="https://static.igem.org/mediawiki/2017/7/7a/T--IISc-Bangalore--Header--iFLOAT.svg" id="headerImg" />
  
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<h1>Gas vesicles</h1>
 
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Gas vesicles are hollow protein nanostructures produced by many aquatic micro-organisms like haloarchaea and cyanobacteria to provide buoyancy to the cells. In their natural environment, vertical stratification is a crucial aspect of survival: optimal access to sunlight for photosynthesis is
 
  
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The structure and arrangement is highly conserved between organisms with width being almost the only widely varying parameter. They contain gases which diffuse in during formation and are kept localised by the hydrophobicity of the inner membrane.
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<h1 id="introduction">Gas Vesicles</h1>
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Unlike true vesicles, these are made of proteins instead of phospholipids and are hence of considerable interest. Each gas vesicle is composed of two primary protein monomers, the gas vesicle forming proteins A (GvpA) and C (GvpC). The entire structure will be discussed in the following sections.
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<b>Verification of presence of Gas Vesicles</b>
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The easiest way to assay presence of gas vesicles is their disappearance under high pressure under a microscope. This was observed even during normal experiments. Fully filled micro-centrifuge tubes containing dilute gas vesicle suspensions lost their faint opalescence when the tube was closed (this did lead to a loss of samples). A more strict assay was done using DLS (See Dynamic Light Scattering) and SEM Imaging to pinpoint the exact size of the nano-particles. It was found that these gas vesicles have an effective hydrodynamic radius of around 230nm. This estimate was particularly valuable in the development of our model.
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<h1>Our analysis</h1>
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<figure>
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<img src="https://static.igem.org/mediawiki/2017/5/5a/T--IISc-Bangalore--algal-bloom-Microcystis.jpg" width="100%" height="100%">
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<br>
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<figurecaption><b>Figure 1</b>: (left) an algal bloom, (right) micrograph of Microcystis sp. showing gas vesicles
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</figurecaption>
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</figure>
  
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<h1>Improving flotation</h1>
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<p>Have you ever seen an algal bloom — a noxious mass of algae polluting eutrophic ponds and lakes? These cyanobacteria, like many other aquatic microorganisms, produce gas vesicles to help them float to the surface. Gas vesicles are hollow gas-filled organelles that make the cell buoyant enough to float in water. The synthesis and degradation of gas vesicles can be controlled by the cell to adjust its vertical position in the water column — a useful ability when competing for sunlight to photosynthesize!</p>
  
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<h1>Assays</h1>
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<h2 align="middle">Gas vesicles are ancient organelles, evolving over 3,000,000,000 years ago...</h2>
  
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<figure>
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<img src="https://static.igem.org/mediawiki/2017/e/e7/T--IISc-Bangalore--gas-vacuolated-microbes.png" width="100%" height="100%">
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<br>
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<figurecaption><b>Figure 2</b>: (clockwise from bottom-left) Haloquadratum walsbyi, Nostoc, green sulfur bacteria, Methanosarcina</figurecaption>
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</figure>
  
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<h1>About iFLOAT</h1>
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<p>Gas vesicles are found in a huge diversity of aquatic microorganisms, from green sulfur bacteria and cyanobacteria to methanogens and haloarchaea, indicating an ancient evolutionary lineage stretching back more than three billion years, when these two domains of life — bacteria and archaea — diverged from a common ocean-dwelling ancestor.</p>
  
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<p>Tell us about your project, describe what moves you and why this is something important for your team.</p>
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<figure>
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<img src="https://static.igem.org/mediawiki/2017/5/58/T--IISc-Bangalore--phylogenetic-tree.png" width="100%" height="100%">
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<figurecaption><b>Figure 3</b>: Phylogenetic tree showing evolutionary lineage of gas vesicles (green) across the domains of life — bacteria (blue), archaea (red) and eucarya (brown)</figurecaption>
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</figure>
  
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<ul>
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<h1 id="structure">Structure of Gas Vesicles</h1>
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<li> A clear and concise description of your project.</li>
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<li>A detailed explanation of why your team chose to work on this particular project.</li>
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<li>References and sources to document your research.</li>
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<li>Use illustrations and other visual resources to explain your project.</li>
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</ul>
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<p>
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<h2 align="middle">What are gas vesicles made of?</h2>
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lot of information and content on your wiki; include summaries as much as possible; consistent, accurate and unambiguous in your achievements.
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</p>
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<p>point of view of the judge evaluating you at the end of the year.</p>
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<p>Though “vesicle” conjures up images of phospholipid bilayers, gas vesicles are unique: they are made entirely of protein! Shaped like a cylinder capped by cones, these protein nanostructures consist of “ribs” made of gas vesicle protein A (GvpA), a small, hydrophobic protein that is conserved across all gas-vacuolate species. By forming linear crystalline arrays — the “ribs” — GvpA monomers become the major structural units of gas vesicles.</p>
  
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<p>record references judges can see how you thought about your project and what works inspired you.</p>
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<figure>
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  <img src="https://static.igem.org/mediawiki/2017/4/4a/T--IISc-Bangalore--Model-GVliterature.png">
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  <br>
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  <figurecaption><b>Figure 4</b>: Electron micrograph of gas vesicles isolated from Anabaena flos-aquae (left)
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and Halobacterium salinarum NRC-1 (right)</figurecaption>
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</figure>
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<p>Another gas vesicle protein, GvpC, binds to the gas vesicle surface: by interlocking the GvpA “ribs”, GvpC strengthens the gas vesicles against external pressures. However, <i>gvpC</i> is not an essential part of the gas vesicle gene cluster and several organisms produce gas vesicles without GvpC.</p>
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<h1 id="biogenesis">Biogenesis</h1>
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<h2 align="middle">How are gas vesicles made?</h2>
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<p>Current evidence suggests that gas vesicle polymerization begins from a bi-cone stage which gradually extends in length to form the cone-capped cylinder. Lengths and widths vary dramatically between species — these dimensions affect the mechanical strength of gas vesicles — but remain fairly constant between cells of the same species. Electron micrography reveals that gas-vacuolate cells contain gas vesicles at all stages of growth.</p>
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<h2 align="middle">How are they filled with gas?</h2>
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<p>Surface tension at the hydrophobic inner surface of the gas vesicle (remember, GvpA is a <i>very</i> hydrophobic protein) excludes water molecules from the interior and only allows the free diffusion of gases through the vesicle walls. As a result, no special mechanism is required to evacuate the gas vesicle; by virtue of their biochemical composition, they fill with gas even as they are synthesized!</p>
 +
 
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<h1 id="bioengineering">Bioengineering Gas Vesicles</h1>
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<h2 align="middle">How have gas vesicles been modified?</h2>
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<p>In recent years, labs around the world have bioengineered gas vesicles for diverse purposes — from ultrasonic molecular imaging to vaccine delivery — and this versatility comes from one simple structural feature of the gas vesicle: GvpC. Expressed on the gas vesicle surface, GvpC is a hydrophilic protein that accommodates insertions near its C-terminus, enabling proteins of interest to be fused to it. Antigenic peptides, fluorescent proteins and even enzymes have been successfully expressed on the surface of gas vesicles by fusion to GvpC!</p>
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<h2 align="middle">How do we do this?</h2>
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<p>The <i>in vivo</i> method — demonstrated successfully in <i>Halobacterium salinarum</i> NRC-1 — involves modifying the <i>gvpC</i> gene of gas-vacuolate microbes and purifying modified gas vesicles with the GvpC-fused protein of interest expressed on the surface.</p>
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<p>The <i>in vitro</i> method — used on gas vesicles from <i>Anabaena flos-aquae</i> — involves chemically stripping native GvpC off the surface of the gas vesicles, leaving only the GvpA shell, and refolding recombinantly-produced GvpC-fused protein of interest onto the stripped gas vesicle surface.</p>
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 +
<h1 id="purification">Purification of Gas Vesicles</h1>
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<p>Purifying gas vesicles from the microbes which synthesize them is not trivial; even getting intact gas vesicles out of the cells is a painstaking challenge. The first limitation is that gas vesicles have a critical pressure beyond which they collapse irreversibly due to mechanical failure — flattening like a crushed soda can — ruling out cell lysis by sonication or French press. This leaves enzymatic lysis, a far more expensive method that can't easily be scaled up.</p>
 +
 
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<h2 align="middle">There's a more fundamental problem...</h2>
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<p>Once the cells are lysed and the gas vesicles are released into suspension, another hurdle awaits us — separating the gas vesicles from the remaining cell fractions is not as easy as it sounds. Intuitively, the solution seems obvious: simply allowing the suspension to stand for enough time should make the gas vesicles float to the surface; after all, the gas vesicles have an incredibly low density.</p>
 +
 
 +
<h2 align="middle">But that does not work.</h2>
 +
 
 +
<p>In fact, the gas vesicle suspension must be centrifuged in a swinging-bucket rotor at very low speeds of ~60 g overnight (to avoid hydrostatic pressure induced collapse) before gas vesicles rise to the surface as a milky-white suspension that must be carefully skimmed off. To ensure sample purity, this tedious process must be serially repeated by diluting the suspension, centrifuging, and skimming off the surface over and over again.</p>
 +
 
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<h2 align="middle">Gas vesicles <i>don't</i> float well <i>in vitro</i>.</h2>
 +
 
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<p>For some reason, isolated gas vesicles don't float well at all, unlike gas-vacuolate cells. No bioengineered gas vesicle — despite all its remarkable acoustic, mechanical and surface characteristics — can ever be exploited for its most fundamental property: buoyancy.<p>
 +
 
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<h2 align="middle">And so, for iGEM 2017, we want to make gas vesicles float again — iFLOAT!</h2>
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<h1 id="references">References</h1>
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[1]https://microbewiki.kenyon.edu/index.php/File:CyanobacteriaMicroscope.jpeg <br>
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[2]http://www.amyhremleyfoundation.org/images/conservation/html/GasVesicles-07.html <br>
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[3]http://www.noaa.gov/what-is-harmful-algal-bloom <br>
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[4]http://news.mit.edu/2012/nanotubes-energy-transfer-0706 <br>
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[5]Walsby AE. Gas vesicles. Microbiological Reviews. 1994;58(1):94-144. <br>
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[6]Methanosarcina acetivorans C2A courtesy of Whitehead Institute Center of Genome Research. <br>
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[7]http://www.nature.com/news/2004/041011/full/news041011-3.html <br>
  
 
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Latest revision as of 02:56, 2 November 2017

  1. Introduction
  2. Structure
  3. Biogenesis
  4. Bioengineering
  5. Purification
  6. References

Gas Vesicles


Figure 1: (left) an algal bloom, (right) micrograph of Microcystis sp. showing gas vesicles

Have you ever seen an algal bloom — a noxious mass of algae polluting eutrophic ponds and lakes? These cyanobacteria, like many other aquatic microorganisms, produce gas vesicles to help them float to the surface. Gas vesicles are hollow gas-filled organelles that make the cell buoyant enough to float in water. The synthesis and degradation of gas vesicles can be controlled by the cell to adjust its vertical position in the water column — a useful ability when competing for sunlight to photosynthesize!

Gas vesicles are ancient organelles, evolving over 3,000,000,000 years ago...


Figure 2: (clockwise from bottom-left) Haloquadratum walsbyi, Nostoc, green sulfur bacteria, Methanosarcina

Gas vesicles are found in a huge diversity of aquatic microorganisms, from green sulfur bacteria and cyanobacteria to methanogens and haloarchaea, indicating an ancient evolutionary lineage stretching back more than three billion years, when these two domains of life — bacteria and archaea — diverged from a common ocean-dwelling ancestor.

Figure 3: Phylogenetic tree showing evolutionary lineage of gas vesicles (green) across the domains of life — bacteria (blue), archaea (red) and eucarya (brown)

Structure of Gas Vesicles

What are gas vesicles made of?

Though “vesicle” conjures up images of phospholipid bilayers, gas vesicles are unique: they are made entirely of protein! Shaped like a cylinder capped by cones, these protein nanostructures consist of “ribs” made of gas vesicle protein A (GvpA), a small, hydrophobic protein that is conserved across all gas-vacuolate species. By forming linear crystalline arrays — the “ribs” — GvpA monomers become the major structural units of gas vesicles.


Figure 4: Electron micrograph of gas vesicles isolated from Anabaena flos-aquae (left) and Halobacterium salinarum NRC-1 (right)

Another gas vesicle protein, GvpC, binds to the gas vesicle surface: by interlocking the GvpA “ribs”, GvpC strengthens the gas vesicles against external pressures. However, gvpC is not an essential part of the gas vesicle gene cluster and several organisms produce gas vesicles without GvpC.

Biogenesis

How are gas vesicles made?

Current evidence suggests that gas vesicle polymerization begins from a bi-cone stage which gradually extends in length to form the cone-capped cylinder. Lengths and widths vary dramatically between species — these dimensions affect the mechanical strength of gas vesicles — but remain fairly constant between cells of the same species. Electron micrography reveals that gas-vacuolate cells contain gas vesicles at all stages of growth.

How are they filled with gas?

Surface tension at the hydrophobic inner surface of the gas vesicle (remember, GvpA is a very hydrophobic protein) excludes water molecules from the interior and only allows the free diffusion of gases through the vesicle walls. As a result, no special mechanism is required to evacuate the gas vesicle; by virtue of their biochemical composition, they fill with gas even as they are synthesized!

Bioengineering Gas Vesicles

How have gas vesicles been modified?

In recent years, labs around the world have bioengineered gas vesicles for diverse purposes — from ultrasonic molecular imaging to vaccine delivery — and this versatility comes from one simple structural feature of the gas vesicle: GvpC. Expressed on the gas vesicle surface, GvpC is a hydrophilic protein that accommodates insertions near its C-terminus, enabling proteins of interest to be fused to it. Antigenic peptides, fluorescent proteins and even enzymes have been successfully expressed on the surface of gas vesicles by fusion to GvpC!

How do we do this?

The in vivo method — demonstrated successfully in Halobacterium salinarum NRC-1 — involves modifying the gvpC gene of gas-vacuolate microbes and purifying modified gas vesicles with the GvpC-fused protein of interest expressed on the surface.

The in vitro method — used on gas vesicles from Anabaena flos-aquae — involves chemically stripping native GvpC off the surface of the gas vesicles, leaving only the GvpA shell, and refolding recombinantly-produced GvpC-fused protein of interest onto the stripped gas vesicle surface.

Purification of Gas Vesicles

Purifying gas vesicles from the microbes which synthesize them is not trivial; even getting intact gas vesicles out of the cells is a painstaking challenge. The first limitation is that gas vesicles have a critical pressure beyond which they collapse irreversibly due to mechanical failure — flattening like a crushed soda can — ruling out cell lysis by sonication or French press. This leaves enzymatic lysis, a far more expensive method that can't easily be scaled up.

There's a more fundamental problem...

Once the cells are lysed and the gas vesicles are released into suspension, another hurdle awaits us — separating the gas vesicles from the remaining cell fractions is not as easy as it sounds. Intuitively, the solution seems obvious: simply allowing the suspension to stand for enough time should make the gas vesicles float to the surface; after all, the gas vesicles have an incredibly low density.

But that does not work.

In fact, the gas vesicle suspension must be centrifuged in a swinging-bucket rotor at very low speeds of ~60 g overnight (to avoid hydrostatic pressure induced collapse) before gas vesicles rise to the surface as a milky-white suspension that must be carefully skimmed off. To ensure sample purity, this tedious process must be serially repeated by diluting the suspension, centrifuging, and skimming off the surface over and over again.

Gas vesicles don't float well in vitro.

For some reason, isolated gas vesicles don't float well at all, unlike gas-vacuolate cells. No bioengineered gas vesicle — despite all its remarkable acoustic, mechanical and surface characteristics — can ever be exploited for its most fundamental property: buoyancy.

And so, for iGEM 2017, we want to make gas vesicles float again — iFLOAT!

References

[1]https://microbewiki.kenyon.edu/index.php/File:CyanobacteriaMicroscope.jpeg
[2]http://www.amyhremleyfoundation.org/images/conservation/html/GasVesicles-07.html
[3]http://www.noaa.gov/what-is-harmful-algal-bloom
[4]http://news.mit.edu/2012/nanotubes-energy-transfer-0706
[5]Walsby AE. Gas vesicles. Microbiological Reviews. 1994;58(1):94-144.
[6]Methanosarcina acetivorans C2A courtesy of Whitehead Institute Center of Genome Research.
[7]http://www.nature.com/news/2004/041011/full/news041011-3.html