BerkiGEM2007 WikiPlaying2

From 2007.igem.org

(Difference between revisions)
 
(77 intermediate revisions not shown)
Line 1: Line 1:
-
<br>
 
-
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br>
 
-
 
-
 
<html>
<html>
<head>
<head>
Line 14: Line 10:
top:50px;
top:50px;
width:95%;
width:95%;
-
height:100%;
+
height:90%;
z-index:1;
z-index:1;
background-color: #FFFFFF;
background-color: #FFFFFF;
Line 22: Line 18:
background-color: #FFFFFF;
background-color: #FFFFFF;
background-repeat: repeat;
background-repeat: repeat;
 +
}
 +
a:link {
 +
color: #FF0000;
 +
}
 +
a:visited {
 +
color: #990000;
 +
}
 +
a:hover {
 +
color: #FF0000;
 +
}
 +
a:active {
 +
color: #FF0000;
 +
}
 +
h1,h2,h3,h4,h5,h6 {
 +
font-weight: bold;
 +
}
 +
h1 {
 +
font-size: xx-large;
 +
color: #000000;
 +
}
 +
h2 {
 +
font-size: x-large;
 +
color: #000000;
 +
}
 +
h3 {
 +
font-size: large;
 +
color: #000000;
}
}
-->
-->
Line 28: Line 51:
<body>
<body>
-
<div id="apDiv1">
+
<div align="center">
-
  <h1 align="center">&nbsp;</h1>
+
   <p><a href="https://2007.igem.org/Berkeley_UC">&lt;&lt;&lt; Return to UC Berkeley iGEM 2007 </a></p>
-
  <h1 align="center">Engineering Bactoblood for Oxygen Transport<br />
+
   <p><a href="https://2007.igem.org/Berkeley_Individual_Contributions">&lt;&lt;Previous Section: Individual Contributions</a> | <a href="https://2007.igem.org/BerkiGEM2007_Resources">Next Section: Team Resources&gt;&gt;</a></p>
-
  </h1>
+
</div>
-
   <p>&nbsp;</p>
+
<h1 align="center">Team Notebooks</h1>
-
  <p>The primary function of human erythrocytes is to transport oxygen to the body's tissues and remove CO2.  This is accomplished principly by high concentrations of the protein hemoglobin.  However, functional expression of hemoglobin requires the coexpression of the small molecule (heme) that specifically binds oxygen, proteins that promote the expression, folding, and addition of heme to hemoglobin, and proteins that maintain the oxidation state of hemoglobin and prevent the accumulation of toxic oxidizing species in the cell.  Bactoblood will similarly require these activities, so we designed a hierarchical genetic device that encoded this oxygen transport function.  Our design contains a heme biosynthesis devices, a hemoglobin generation device, a chaperone device, and a detoxifying device.  Additionally, we investigated alternatives to hemoglobin that may provide superior oxygen transport to Bactoblood than their human counterpart.</p>
+
<div>
-
  <h2 align="center">Heme Biosynthesis Device</h2>
+
   <h3>Team Notebooks</h3>
-
  <p align="center"><img src="https://static.igem.org/mediawiki/2007/f/fd/Berk-Figure-hemABCD.png" alt="" width="563" height="122" /></p>
+
-
  <p><br />
+
-
    Heme is a prosthetic group to hemoglobin. Generally, heme consists of  an iron atom surrounded by a porphyrin ring. Each hemoglobin molecule  is capable of binding up to four heme groups. One of the most important  functions of heme is to assist in the transportation of diatomic gases.  In red blood cells, heme and hemoglobin are the components that allow  the binding of oxygen. As a result of this interaction, red blood cells  continuously deliver oxygen to the entire body.</p>
+
-
  <h2 align="center">The Hemoglobin Generating Device</h2>
+
-
  <p align="center"><img src="https://static.igem.org/mediawiki/2007/d/d1/Berk-Figure-HbA-HbB.png" alt="" name="HemoglobinCassette" width="337" height="111" id="HemoglobinCassette" /></p>
+
-
  <p>The primary component needed for efficient oxygen transport in our  system is human hemoglobin A. (HbA) HbA is a tetramer that consists of  two different subunits, α2β2. </p>
+
-
  <p><br />
+
-
    There are several problems which we have addressed with regards to hemoglobin and oxygen transport. <br />
+
-
    The first of which is the insufficiently low P50, the partial pressure  of oxygen needed for 50% saturation, of wild type human hemoglobin A.  The P50 for wild type human hemoglobin is ~3.8 at physiological  conditions. In human erythrocytes, this problem is fixed primarily by  the presence of an allosteric modifier 2,3-diphosphoglycerate (2,3-DPG)  which forces the hemoglobin conformation into the lower affinity deoxy  state, or the T-state. By pushing the hemoglobin into the T-state,  2,3-DPG is effectively pushing any bound oxygen out of the bound state  and decreasing the oxygen binding affinity of the hemoglobin. This  decreased oxygen binding affinity causes the P50 to be much higher than  if the allosteric modifier were not present. An increase of 0.4mM in  DPG concentration decreases oxygen affinity by about 1.0 torr and the  normal concentration of DPG in erythrocytes is ~5mM. <br />
+
-
    (Image of P50 curves identified for each mutant and wild type)<br />
+
-
  </p>
+
-
   <p>In our system, we have chosen to use well studied mutants of  human hemoglobin which have been engineered to be permanently in the  deoxy T-state. The mutations are named Presbyterian (beta-Asn108Lys)  and providence (beta-Lys82Asp).<br />
+
-
    The P50 is also sensitive to various ions such as Cl-, although to a much smaller amount than 2,3-DPG. The portion of hemoglobin that binds  these types of modifiers is at the N-terminal end. When hemoglobin is  expressed in E. coli, there is an extra methionine residue which is  normally cleaved off in eukaryotic cells.</p>
+
-
  <p align="center">&nbsp;</p>
+
-
  <h2 align="center">The Chaperone Device</h2>
+
-
  <p align="center"><img src="https://static.igem.org/mediawiki/2007/a/aa/Berk-Figure-AHSP.png" width="257" height="122" alt="" /></p>
+
-
  <p>The alpha subunit is more prone to precipitation because an  alpha-alpha dimer is insoluble under normal conditions. This can cause  an excess of beta subunits and decreased functional hemoglobin output. <br />
+
-
  </p>
+
-
  <p>The solution in erythrocytes is to use a chaperone named alpha  hemoglobin stabilizing protein (AHSP), which binds to single alpha  subunits and facilitates the transfer of a beta subunit to the alpha.  An alpha-beta dimer remains soluble. Our use of the bacterial chassis  as a container that mimics an erythrocyte allows us to provide the same  solution as in an erythrocyte. We include in our system the gene that  encodes for human AHSP. In addition, we have also explored a fusion of  di-alpha subunits with a glycine linker. This has been proven to give a  higher soluble output by somehow stabilizing the alpha subunits and  preventing precipitation. </p>
+
-
  <p align="center">&nbsp;</p>
+
-
  <h2 align="center">Autoxidation Control<br />
+
-
  </h2>
+
-
  <p align="center"><img src="https://static.igem.org/mediawiki/2007/7/7b/Berk-Figure-Cytochrome.png" width="531" height="113" alt="" /><img src="https://static.igem.org/mediawiki/2007/4/40/Berk-Figure-SodC-katG.png" width="282" height="105" alt="" /></p>
+
-
  <p>During the process of binding and unbinding oxygen, the four heme  groups of the hemoglobin may spontaneously undergo autoxidation,  ultimately causing the formation of damaging free radicals. This causes  two problems. The first problem is that the free radicals can cause  damage to cellular proteins, affecting their function. Another problem  is that when autoxidation occurs, an electron is transferred from the  Fe2+ iron center to the oxygen, creating a superoxide and leaving the  heme with a Fe3+ metal center. The resulting hemoglobin is known as  methemoglobin and it is unable to carry oxygen. The autoxidation occurs  to a significant amount on the order of hours. <br />
+
-
    Erythrocytes remedy the problem of free radical accumulation and damage  by containing the antioxidant enzymes catalase and superoxide  dismutase. These enzymes catalyze the breakdown of superoxide into  oxygen and H2O. Human erythrocytes have addressed the autoxidation  problem by using the NADH dependent enzyme, cytochrome b5 reductase.  The basic mechanism is shown here.<br />
+
-
  </p>
+
-
  <p>&nbsp;</p>
+
-
  <h2 align="center">Hemoglobin Alternatives</h2>
+
-
  <p>We also investigated two alternatives to the hemoglobin part in our  device: H-NOX and Myoglobin. Although the intrinsic oxygen-carrying  ability of these proteins is different from Hemoglobin, variants of  these proteins have been engineered with similar P50 values. These variants might allow Bactoblood to carry more oxygen than hemoglobin. </p>
+
-
   <p>H-NOX is a heme-based sensor that is found in bacteria. H-NOX is  able to bind Oxygen using a distal pocket tyrosine. For this gene I  added the T7 promoter we created for this project, an RBS site, and  lastly a Bca1092 terminator. When we assayed this part the results were  inconclusive. The part was assembled correctly but the assay didn't  show strong signs of expression. </p>
+
-
  <p>The second gene we explored was Sperm Whale Myoglobin.  Myoglobin is a monomeric protein that behaves as an intracellular  oxygen storage site. Sperm whale myoglobin in particular is easily  found in large amounts in the whale's muscle tissue. The construction  of this part was very similar to that of the H-NOX composite part. It  used the same promoter, terminator, and RBS. The assay for Myoglobin  showed a bit more promise but couldn't conclusively show that Myoglobin  was binding to oxygen. </p>
+
-
  <p align="center">&nbsp;</p>
+
-
  <p>&nbsp;</p>
+
</div>
</div>
 +
<hr>
 +
<p><br>
 +
    <a href="https://2007.igem.org/John_Dueber_Notebook" title="John Dueber Notebook"> John Dueber's Notebook</a><br>
 +
    <a href="https://2007.igem.org/Christopher_Anderson_Notebook" title="Christopher Anderson Notebook"> Christopher Anderson's Notebook</a><br>
 +
    <a href="https://2007.igem.org/Farnaz_Nowroozi_Notebook" title="Farnaz Nowroozi Notebook"> Farnaz Nowroozi's Notebook</a><br>
 +
    <a href="https://2007.igem.org/Amin_Hajimorad_Notebook" title="Amin Hajimorad Notebook"> Amin Hajimorad's Notebook</a><br>
 +
    <a href="https://2007.igem.org/Rickey_Bonds_Notebook" title="Rickey Bonds Notebook"> Rickey Bonds' Notebook</a><br>
 +
</p>
 +
<p><br>
 +
    <em>Keep your wiki notebooks,  sequencing/construction logs, and the registry updated!</em> </p>
 +
<p><br>
 +
    <a href="https://2007.igem.org/Arthur_Yu_Notebook" title="Arthur Yu Notebook"> Arthur Yu's 1337 Notebook</a><br>
 +
    <a href="https://2007.igem.org/Austin_Day_Notebook" title="Austin Day Notebook"> Austin Day's Notebook</a><br>
 +
    <a href="https://2007.igem.org/David_Tulga_Notebook" title="David Tulga Notebook"> David Tulga's Notebook</a><br>
 +
    <a href="https://2007.igem.org/Kristin_Doan_Notebook" title="Kristin Doan Notebook"> Kristin Doan's Notebook</a><br>
 +
    <a href="https://2007.igem.org/Samantha_Liang_Notebook" title="Samantha Liang Notebook"> Samantha's Notebook (June - July 19, 2007</a><br>
 +
    <a href="https://2007.igem.org/Samantha_Liang_Notebook2" title="Samantha Liang Notebook2"> Samantha's Notebook (July 20, 2007 - present)</a><br>
 +
    <a href="https://2007.igem.org/Vaibhavi_Umesh_Notebook" title="Vaibhavi Umesh Notebook"> Vaibhavi Umesh's Notebook</a><br>
 +
    <a href="https://2007.igem.org/Kristin_Fuller_Notebook" title="Kristin Fuller Notebook"> Kristin Fuller's Notebook</a><br>
 +
</p>
 +
<p><br>
 +
    <a href="https://2007.igem.org/Vincent_Parker_Notebook" title="Vincent Parker Notebook"> Vincent Parker's Notebook</a><br>
 +
    <a href="https://2007.igem.org/Nhu_Nguyen_Notebook" title="Nhu Nguyen Notebook"> Nhu Nguyen's Notebook</a><br>
 +
  <a href="https://2007.igem.org/Hannah_Cole_Notebook" title="Hannah Cole Notebook"> Hannah Cole's Notebook</a></p>
 +
<p>&nbsp;</p>
 +
<p align="justify">&nbsp;</p>
</body>
</body>
</html>
</html>

Latest revision as of 23:07, 26 October 2007

Untitled Document

<<< Return to UC Berkeley iGEM 2007

<<Previous Section: Individual Contributions | Next Section: Team Resources>>

Team Notebooks

Team Notebooks


John Dueber's Notebook
Christopher Anderson's Notebook
Farnaz Nowroozi's Notebook
Amin Hajimorad's Notebook
Rickey Bonds' Notebook


Keep your wiki notebooks, sequencing/construction logs, and the registry updated!


Arthur Yu's 1337 Notebook
Austin Day's Notebook
David Tulga's Notebook
Kristin Doan's Notebook
Samantha's Notebook (June - July 19, 2007
Samantha's Notebook (July 20, 2007 - present)
Vaibhavi Umesh's Notebook
Kristin Fuller's Notebook


Vincent Parker's Notebook
Nhu Nguyen's Notebook
Hannah Cole's Notebook

 

 

Retrieved from "http://2007.igem.org/wiki/index.php/BerkiGEM2007_WikiPlaying2"