BerkiGEM2007 WikiPlaying2

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   <div align="center"><a href="https://2007.igem.org/Berkeley_UC">&lt;&lt;&lt; Return to UC Berkeley iGEM 2007 </a></div>
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   <p><a href="https://2007.igem.org/Berkeley_UC">&lt;&lt;&lt; Return to UC Berkeley iGEM 2007 </a></p>
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   <p align="center"><a href="https://2007.igem.org/BerkiGEM2007Present4">Next &gt;&gt;</a></p>
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   <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>
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  <h1 align="center">Engineering Bactoblood for Oxygen Transport<br />
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<h1 align="center">Team Notebooks</h1>
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  <p align="justify">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>
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  <p align="justify">&nbsp;</p>
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   <h3>Team Notebooks</h3>
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  <h2 align="center">The Hemoglobin Generating Device</h2>
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  <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" /><img src="https://static.igem.org/mediawiki/2007/9/9e/Berk-Figure-Map.png" width="251" height="115" alt=""></p>
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  <h3 align="center">Overview: Human Hemoglobin A and Methionine Aminopeptidase</h3>
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  <p align="justify">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. We constructed a device that expresses both the alpha (HbA) and beta (HbB) subunits of human hemoglobin A, under the control of a T7 promoter. We also created a similar device that will express mutant versions of human hemoglobin. To cleave the extra methionine residue that is present when expressed in prokaryotic cells, we have also constructed a device which expresses the Map gene, which encodes for methionine aminopeptidase (MetAP). By expressing both of these devices in our system, we achieve the expression of unmodified, fully functional adult human hemoglobin. </p>
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  <h3 align="center">Oxygen Binding Affinity and P50</h3>
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  <p align="justify">The first problem to be addressed is the insufficiently low P50 of wild type human hemoglobin. The P50 is the partial pressure  of oxygen needed for 50% saturation. A low P50 means that the oxygen affinity is too high, which  inhibits the ability of the hemoglobin to deliver oxygen to the needed tissues.  The P50 for wild type human hemoglobin is ~3.8 torr under normal physiological  conditions, however this varies with temperature and pH.</p>
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  <p align="justify"><img src="https://static.igem.org/mediawiki/2007/e/e6/P50Comparison.gif" alt="" width="644" height="439" align="left"></p>
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  <p align="justify">Demonstrated by George J. Brewer's image to the left, an increase in P50 is the same as shifting the oxygen binding curve to the right. A shift to the right is shown to increase the oxygen transport, thus a higher P50 is desirable for efficient oxygen transport. </p>
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  <p align="justify">In human erythrocytes, the  oxygen binding affinity is decreased by  the presence of an allosteric modifiers, primarily 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 from the heme center. This effectively lowers the oxygen binding affinity of the hemoglobin and increases the P50. An increase of 0.4mM in  DPG concentration decreases oxygen affinity by about 1.0 torr. Since the  normal concentration of DPG in erythrocytes is ~5mM, this raises the P50 to ~16.3 torr. </p>
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  <p align="justify">DPG isn't the only allosteric modifier of hemoglobin, to a lesser extent, various ions bind to a region near the N-terminal end of the hemoglobin protein and act to increase the P50 as well. In recombinant hemoglobin expressed in E. coli, there is an extra methionine residue at the N-terminal region of expressed proteins. For the case of hemoglobin, that extra methionine acts to inhibit the allosteric effects of certain ions. </p>
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  <h3 align="justify">Mutant Hemoglobin<br />
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  <p align="justify">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. First generation Bactoblood will use a mutant which has two mutations applied to the beta subunit. The mutations are named Presbyterian (beta-Asn108Lys)  and providence (beta-Lys82Asp). It has been reported that human hemoglobin with these two point mutations  has a P50 in the range of ~44 torr, an acceptable P50 value for a good blood substitute.</p>
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  <h3 align="center">Methionine Aminopeptidase</h3>
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  <p align="justify">In prokaryotic cells, the N-terminal end of proteins retain the initial methionine residue from translation. In order to create human hemoglobin in the same form as it exists in erythrocytes, this extra methionine needs to be cleaved. The enzyme that does this occurs natrually in prokaryotes and is called methionine aminopeptidase (MetAP), encoded for by the Map gene. However, recombinant hemoglobin largely retains this extra methionine residue. By overexpressing metAP, it has been shown that a large percentage of the produced hemoglobin can be free of this extra methionine residue. It has also been shown that removal of the extra methionine residue increases the P50 slightly. </p>
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  <h2 align="center">&nbsp;</h2>
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  <h2 align="center">The Chaperone Device</h2>
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  <p align="center"><img src="https://static.igem.org/mediawiki/2007/a/aa/Berk-Figure-AHSP.png" width="257" height="122" alt="" /></p>
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  <h3 align="center">Overview: Alpha Hemoglobin Stabilizing Protein</h3>
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  <p>Alpha hemoglobin stabilizing protein (AHSP) is a chaperone protein normally found in erythrocytes. Because AHSP is important in the proper folding of hemoglobin, we created a device under a T7 promoter which will express AHSP in Bactoblood. </p>
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  <h3 align="center">Hemoglobin Solubility</h3>
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  <p>The alpha subunit of hemoglobin 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 output of functional tetrameric hemoglobin. In order to prevent alpha subunits from binding to themselves and precipitating out of solution, human erythrocytes contain an alpha hemoglobin stabilizing protein (AHSP), which acts as a chaperone. AHSP has the ability to bind to the alpha subunit of hemoglobin while keeping it soluble. The AHSP bound alpha subunit readily gives up its AHSP for a beta subunit which then goes on to form functional tetrameric hemoglobin. By expressing AHSP in Bactoblood, we expect to increase the yield of functional, soluble tetrameric hemoglobin. <br />
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  </p>
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  <p align="justify">In addition, we have also explored a fusion of  di-alpha subunits with a glycine linker. This has been shown to give a  higher soluble output by  stabilizing the alpha subunits and  preventing precipitation. </p>
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  <p align="justify"><br />
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  </p>
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  <h2 align="center">Heme Biosynthesis Device</h2>
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  <p align="center"><img src="https://static.igem.org/mediawiki/2007/f/fd/Berk-Figure-hemABCD.png" alt="" width="563" height="122" /></p>
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  <h3 align="center"><br />
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    Overview: Heme</h3>
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  <p align="justify">Heme is a prosthetic group to hemoglobin. Heme consists of  an iron atom surrounded by a porphyrin ring. Each hemoglobin tetramer  is capable of binding up to four heme groups. One of the most important  functions of heme, and the fuction we are interested in, is to assist in the transportation of diatomic gases, namely oxygen. The heme biosynthesis pathway is already present in E. coli, however, to achieve the high concentrations of functional hemoglobin needed for Bactoblood, we need lots of heme. We have constructed a device containing thefour genes hemA, hemB, hemC, and hemD. These genes are the primary bottlenecks in the heme biosynthesis pathway. We hope to greatly increase the production of heme by overexpressing these four genes under a T7 promoter. </p>
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  <h3 align="center"><strong>Heme Biosynthesis Pathway</strong> </h3>
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  <p>The biosynthetic pathway contains primarily eight enzymes.  We  included  hemA (Delta-aminolevulinate synthase), hemB  (Delta-aminolevulinic acid dehydratase), hemC (porphobilinogen  deaminase), and hemD (uroporphyrinogen III synthase) in our system. These genes overproduce <em>precursors</em> to heme in our cells because over-accumulation of heme itself  would result in toxicity. After  successful subcloning experiments, the bacterial cell pellets would  become reddish-brown due to the accumulation of porphyrins and heme.</p>
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  <p><img src="https://static.igem.org/mediawiki/2007/0/01/96wellplateheme.jpg" alt="" width="498" height="308" align="left"></p>
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  <h3 align="center">Construction of the Heme Device</h3>
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  <p>The hemA gene was cloned from both R.capsulatus and  CFT073. Both were cloned to figure out which versin would give a greater yield of heme precursors. We also cloned hemB, hemC, and hemD from MG1655. We attached single ribosomal binding sites to the genes, and we also attached a library of ribosomal  binding sites to the genes. We used a library of ribosome binding sites so that we could grow up many clones in a 96 well plate and determine which ribosome binding sites were the strongest. The image of such a 96 well plate is shown to the left. Because  colors change is a phenotype of  porphyrins and heme, it was easy to select single colonies that  corresponded to the stronger ribosomal binding sites in library.  These stronger clones would yield the greatest amount of heme and heme precursors and have  the deepest red/brown color of all the clones. </p>
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  <p>&nbsp;</p>
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  <p><img src="https://static.igem.org/mediawiki/2007/9/9b/Hemespec.jpg" alt="" width="832" height="537" align="right">With our construct complete, we employed UV-Vis to confirm that the  red product we are seeing is, in fact, heme. The maximum absorbance for  heme occurs at 412 nm. The graph to the right verifies that by alleviating the bottlenecks in the heme biosynthesis pathway, we have increased the concentration of heme in our system. </p>
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  <h2 align="justify">&nbsp;</h2>
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  <h2 align="center">Autoxidation Control<br />
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  </h2>
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  <p align="center"><img src="https://static.igem.org/mediawiki/2007/7/7b/Berk-Figure-Cytochrome.png" width="582" height="112" alt="" /><img src="https://static.igem.org/mediawiki/2007/4/40/Berk-Figure-SodC-katG.png" width="294" height="105" alt="" /></p>
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  <p align="justify">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 />
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    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 />
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  <p align="justify">&nbsp;</p>
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  <h2 align="justify">Hemoglobin Alternatives</h2>
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  <p align="justify">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>
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  <p align="justify">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>
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  <p align="justify">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>
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  <div align="justify"><a href="https://2007.igem.org/Berkeley_UC">&lt;&lt;&lt; Return to UC Berkeley iGEM 2007 </a></div>
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   <p align="justify"><a href="https://2007.igem.org/BerkiGEM2007Present4">Next &gt;&gt;</a></p>
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  <p align="justify">&nbsp;</p>
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<hr>
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<p><br>
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    <a href="https://2007.igem.org/John_Dueber_Notebook" title="John Dueber Notebook"> John Dueber's Notebook</a><br>
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    <a href="https://2007.igem.org/Christopher_Anderson_Notebook" title="Christopher Anderson Notebook"> Christopher Anderson's Notebook</a><br>
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    <a href="https://2007.igem.org/Farnaz_Nowroozi_Notebook" title="Farnaz Nowroozi Notebook"> Farnaz Nowroozi's Notebook</a><br>
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    <a href="https://2007.igem.org/Amin_Hajimorad_Notebook" title="Amin Hajimorad Notebook"> Amin Hajimorad's Notebook</a><br>
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    <a href="https://2007.igem.org/Rickey_Bonds_Notebook" title="Rickey Bonds Notebook"> Rickey Bonds' Notebook</a><br>
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</p>
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<p><br>
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    <em>Keep your wiki notebooks,  sequencing/construction logs, and the registry updated!</em> </p>
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<p><br>
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    <a href="https://2007.igem.org/Arthur_Yu_Notebook" title="Arthur Yu Notebook"> Arthur Yu's 1337 Notebook</a><br>
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    <a href="https://2007.igem.org/Austin_Day_Notebook" title="Austin Day Notebook"> Austin Day's Notebook</a><br>
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    <a href="https://2007.igem.org/David_Tulga_Notebook" title="David Tulga Notebook"> David Tulga's Notebook</a><br>
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    <a href="https://2007.igem.org/Kristin_Doan_Notebook" title="Kristin Doan Notebook"> Kristin Doan's Notebook</a><br>
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    <a href="https://2007.igem.org/Samantha_Liang_Notebook" title="Samantha Liang Notebook"> Samantha's Notebook (June - July 19, 2007</a><br>
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    <a href="https://2007.igem.org/Samantha_Liang_Notebook2" title="Samantha Liang Notebook2"> Samantha's Notebook (July 20, 2007 - present)</a><br>
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    <a href="https://2007.igem.org/Vaibhavi_Umesh_Notebook" title="Vaibhavi Umesh Notebook"> Vaibhavi Umesh's Notebook</a><br>
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    <a href="https://2007.igem.org/Kristin_Fuller_Notebook" title="Kristin Fuller Notebook"> Kristin Fuller's Notebook</a><br>
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<p><br>
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    <a href="https://2007.igem.org/Vincent_Parker_Notebook" title="Vincent Parker Notebook"> Vincent Parker's Notebook</a><br>
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    <a href="https://2007.igem.org/Nhu_Nguyen_Notebook" title="Nhu Nguyen Notebook"> Nhu Nguyen's Notebook</a><br>
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  <a href="https://2007.igem.org/Hannah_Cole_Notebook" title="Hannah Cole Notebook"> Hannah Cole's Notebook</a></p>
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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

 

 

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