Berkeley UC Immunity Chassis

 

 

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    <font-color: #000000>why is this still red?!?</font-color:>

  <h3 align="center">&nbsp;</h3>

  <h3 align="center">The <em>E. coli</em> outer surface</h3>

   <p align="center"><span class="style31">Oxygen Transport</span></p>

   <p align="justify" class="style36"  >To understand these modifications, we must first understand what features are present in <em>E. coli</em> strain MC1061, our starting point for Bactoblood.  Like most strains of <em>E. coli</em> used in the lab, MC1061comes from the MG1655 lineage and is a "rough"  strain. Unlike other "smooth" strains, MC1061 lacks surface-displayed  capsular polysaccharides known as K capsules and O antigens. It retains  the general 2-membrane<a href="https://2007.igem.org/Image:BerkiGEM2007-ColiOuterSurface.gif" title=""><a href="https://2007.igem.org/Image:BerkiGEM2007-ColiOuterSurface.gif" title=""><img src="https://static.igem.org/mediawiki/2007/thumb/e/e4/BerkiGEM2007-ColiOuterSurface.gif/300px-BerkiGEM2007-ColiOuterSurface.gif" alt="" width="300" height="203" align="right" longdesc="/igem07/index.php/Image:BerkiGEM2007-ColiOuterSurface.gif"></a>architecture present in gram-negative bacteria.  In between these membranes is the periplasmic space which is composed  of a gel-like carbohydrate-rich polymer called peptidoglycan.The inner  membrane is composed of a lipidbilayer and a variety of proteins. The  outer membrane similarly is a lipid bilayer, and the lipid component of  it is called lipopolysaccharide, or LPS. The structure of LPS at it's  core is a 6 fatty acid lipid called lipid X. When O antigen polymer  chains are present, they are covalently attached to the outer leaf of  LPS. K capsules are similarly embedded in the outer leaf of the outer  membrane, but they are not directly attached to LPS. Other components  of the outer membrane include a structural protein, LPP, and a variety  of other proteins. This outer surface is the critical region of the  bacterium for understanding how it interacts physically with the  outside world. When the bloodstream "looks" at E. coli, what it "sees"  is the outer membrane because everything else is stuck inside.  Modifications such as O antigens and K capsules therefore have dramatic  effects on the bacterium's interactions with the outside world. </p>

   <h3 align="center" class="style35">Capsular Polysaccharides</h3>

   <p align="justify" class="style35">The carbohydrates embedded in the outer membrane are extremely diverse within the <em>E. coli</em> species. Both K capsules and O antiens are linear carbohydrates  polymer, but at least 150 chemically-distinct O antigens exist in one <em>E. coli</em> strain or another. Similarly, at least 100 chemically-distinct K  capsules have been described. Almost all pathogenic strains of <em>E. coli</em> have some sort of capsular polysaccharide and are referred to as  "smooth" strains. The rough vs. smooth distinction refers to a visibly  discernible quality of their colonies. The particular choice of  carbohydrate present in a bacterium is essential to its ability to  survive in its living environment. For pathogenic and commensal  bacteria, specific O or K carbohydrates are appropriate for distinct  areas of the body (blood stream, urinary tract, intestines) and also  for distinct animal types (birds, pigs, humans, cows, etc.). Over 90%  of human cases of <em>E. coli</em> bacteremia (the clinical word for  having bacteria in the bloodstream) are caused by strains that have a  specific type of K capsule called K1. K1 is a long linear polymer of  sialic acid that extends about half the diameter of the bacterium  beyond its surface. Because polysialic acid is a frequent coating on  mammalian cells, the human immune system does not recognize K1 as  foreign. Bacteria with a K1 capsule are therefore resistant to both  innate and adaptive immune responses. Proper display of a K1 capsule  requires the concomitant expression of any of several O antigens. For  our studies, we have chosen O16. Genetically, the K1 capsule requires  ## genes encoded within a ##kb cassette. The O16 antigen requires ##  genes encoded within a ##kb cassette. Together, these surface  modifications allow the bacterium to avoid detection by the immune  system and are predicted to extend the serum half-life of Bactoblood to  several hours rather than the less-than-5 minutes observed with rough  strains. Both of these gene clusters have been installed into the  genome of MC1061 in the course of preparing our chassis strain. </p>

   <h3 align="center" class="style35">Lipid X and its variants</h3>

   <p align="justify" class="style35">The lipid X component of the LPS in <em>E. coli</em> contains 6 acyl  chains. Mammalian blood contains a protein called LBP that scavenges  this molecule from both live and lysed bacteria and transfers it to  toll-like receptor 4 present on mammalian cells. These events initiate  a signal transduction cascade resulting in the release of a protein  called TNFalpha. The inflammatory response to these events at low doses  helps your body fight off bacterial infections. At higher doses, it can  result in organ failure and even death. The lipid X moiety present in a  variety of other bacteria do not initiate this cascade of events.  Similarly, a pentaacylated variant of the <em>E. coli</em> lipid X is  ###x less agonistic of this response. Our bacteria synthesize this  pentaacylated variant due to the deletion of the gene responsible for  attaching the sixth acyl chain, <em>msbB</em>. </p>

   <h3 align="center" class="style35">Additional cell-surface epitopes</h3>

   <p align="justify" class="style35">Essentially any component on the surface of the bacteria has the  potential to elicit either innate or adaptive immune responses. Of  those present on MC1061's surface, type I pili and flagella are known  to elicit such responses. Each of these features is encoded within  multi-gene operons encoding protein assemblies that extend out from the  bacteral surface. Type I pili allow bacteria to adhere to the surface  of mammalian cells. Flagella are the "propellers" that allow the  bacteria to swim during chemotaxis. Bactoblood does not require either  of these activities, so these genes were deleted in the chassis strain. </p>

   <h3 align="center" class="style35">Characterization of the chassis, MC828U</h3>

   <p align="justify" class="style35">The genotype of our chassis organism is: </p>

       <pre><strong>MC828U</strong> delta(<em>araA</em>-<em>leu</em>)7697 <em>araD139</em> delta(<em>codB</em>-<em>lac</em>)=delta<em>lac74</em> <em>galK16</em>  <em>

<em>upp</em>::(Ptet-<em>wbbL</em>-<em>neuS</em>)  </pre>

   <p align="justify" class="style35">To illustrate the function of our chassis, here we show the function  of 3 critical features of chassis: its ability to survive in serum,  inability to grow in serum, and loss of chemotaxis due to the deletion  of flagella. </p>