BerkiGEM2007Present1

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(The Heme Biosynthesis Device)
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<b><a href="https://2007.igem.org/Berkeley_UC"><< Back to UC Berkeley iGEM 2007</a> <font face="Arial">•</font> <a href="https://2007.igem.org/BerkiGEM2007Present4">Next: The Chassis >></a></b><br /></html>
 
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==Engineering Bactoblood for Oxygen Transport==
 
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The primary function of human erythrocytes is to transport oxygen to the body's tissues and remove CO<sub>2</sub>.  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.
 
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==The Heme Biosynthesis Device ==
 
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'''Introduction'''<br>
 
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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. 
 
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'''The Pathway'''
 
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The biosynthetic pathway for heme demonstrates that there are eight enzymes, or heme genes, involved in the production of heme.  Although bacteria already have these genes present in their genome, the amount of intermediates produced are insufficient for our purposes.  We worked solely with hemA (Delta-aminolevulinate synthase), hemB (Delta-aminolevulinic acid dehydratase), hemC (porphobilinogen deaminase), and hemD (uroporphyrinogen III synthase) because over-accumulation of heme in our cells would result in toxicity. After successful subcloning experiments, the bacterial cell pellets would become reddish-brown due to the accumulation of porphyrins. 
 
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'''Design and Construction'''
 
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[[Image:Berk-Figure-hemABCD.png]]
 
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First, we cloned hemA from R.capsulatus and hemA from CFT073, not knowing which hemA from which organism would give us the highest yield of heme. We also cloned hemB, hemC, and hemD from MG1655. Afterwards, we attached single ribosomal binding sites as well as library ribosomal binding sites. The advantage of working with library ribosomal binding sites is after transformation, one can grow single clones in a 96-well block and selectively choose the clone producing the greatest amount of heme, which correlates with the clone yielding the most red of pellets. 
 
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[[Image:BerkIGEM2007_T7AB.jpg|200px|left]]
 
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As depicted in the image to the left, because of the phenotype of porphyrins and heme, it was easy to select single colonies that combined with the strongest ribosomal binding sites of the library. These stronger clones would yield the greatest amount of heme and have the deepest red/brown color of all the clones.
 
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After the ribosomal binding sites were attached to the heme genes, the genes were subcloned onto the same plasmid in sequential order.  We placed the T7 promoter we produced at the beginning of the composite part as well as a terminator at the end.
 
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[[Image:BerkIGEM2007_chromatogram trace2.jpg|200px|right]]
 
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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.  As the graph shows, the hemA from both R.capsulatus and CFT073 combined with the hemB have significant peaks at wavelengths corresponding to the presence of heme, 412nm.
 
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===The Hemoglobin-generation device===
 
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(Image of hemoglobin cassette)
 
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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.
 
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There are several problems which we have addressed with regards to hemoglobin and oxygen transport.
 
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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. 
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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>
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(Image of P50 curves identified for each mutant and wild type)<br>
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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>
 
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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. <br>
 
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==The Chaperone Device==
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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>
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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.
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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.
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==Autoxidation==
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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.
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Human erythrocytes have addressed the autoxidation problem by using the NADH dependent enzyme, cytochrome b5 reductase. The basic mechanism is shown here.
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==Alternatives To Hemoglobin==
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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 P<sub>50</sub> values. These variants might allow Bactoblood to carry more oxygen than hemoglobin. 
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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.
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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.
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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 align="center"><a href="https://2007.igem.org/BerkiGEM2007Present4">Next Section: Chassis&gt;&gt;</a></p>
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  <h1 align="center">Engineering Bactoblood for Oxygen Transport<br />
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  </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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  <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">&nbsp;</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">&nbsp;</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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  <p align="justify">&nbsp;</p>
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  <h3 align="center">Wild Type and Mutant Hemoglobins<br />
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  </h3>
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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, and the wild type human hemoglobin. The wild type is to compare the mutant hemoglobins to. The first generation Bactoblood blood substitute will use a hemoglobin 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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  <p align="justify">The western blot to the bottom shows various mutant hemoglobins we constructed and tested for hemoglobin yield. A polyclonal antibody was used to identify the produced hemoglobin. All of these samples had heme added exogenously to the culture during growth and were allowed to grow overnight. The bands around 16kD are consistent with monomeric alpha and beta subunits of hemoglobin. Some of the constructs contain a fusion of alpha subunits with a glycine linker. More about this will be discussed in the hemoglobin solubility section. The western demonstrates that hemoglobin is being produced in our cells to a significant degree. In our potential final product, the heme will be produced in vivo and our cultures will be concentrated. This will greatly increase the concentration of hemoglobin in our cells to hopefully near physiological concentrations. </p>
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  <h3 align="center"><img src="https://static.igem.org/mediawiki/2007/b/bc/Westernpicture.jpg" alt="" width="725" height="871" border="0" align="middle"></h3>
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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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  <p align="justify">&nbsp;</p>
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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"><img src="https://static.igem.org/mediawiki/2007/b/b0/AHSPreaction.png" alt="" width="636" height="476" align="left"></h3>
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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. The mechanism for AHSP is shown in the figure to the left. </p>
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  <p><br />
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  </p>
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  <p align="justify">&nbsp;</p>
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  <p align="justify">&nbsp;</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">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><br>
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  </p>
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  <p>&nbsp;</p>
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  <h3 align="center"><img src="https://static.igem.org/mediawiki/2007/9/9b/Hemespec.jpg" alt="" width="681" height="426" align="right"></h3>
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  <h3 align="center">Verification of the Heme Device</h3>
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  <p>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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  <p>&nbsp;</p>
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  <p>&nbsp; </p>
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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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  <h3 align="center">Overview: Cytochromes and Antioxidants</h3>
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  <p align="justify">During the process of binding and unbinding oxygen, the  heme  groups of the hemoglobin may spontaneously undergo autoxidation,  ultimately causing the formation of damaging free radicals and changing the form of the iron in the heme center. To remedy these problems we created an antioxidant device and a methemoglobin reductase device. The antioxidant device contains the genes that express superoxide dismutase and catalase. The methemoglobin reductase device contains the genes that express cytochrome b5 and cytochrome b5 reductase. These two devices will keep our hemoglobin functional and our cells healthy. </p>
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  <h3 align="center">Autoxidation and Free Radical Production</h3>
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  <p align="justify">There are two problems to address in keeping our cells happy and our hemoglobin functional.  The first problem is the spontaneous autoxidation of the heme centers in hemoglobin. During the binding and unbinding of oxygen, electrons are transfered back and forth between the iron center of heme and the bound oxygen molecule. Every once in a while, an electron will transfer to the oxygen and the oxygen will then unbind from the iron center and take the electron away from the iron. This creates a superoxide and a ferric Fe3+ heme. The superoxide eventually will degrate into a hydrogen peroxide, then into a hydroxyl radical, which is very toxic to the cell. The second problem is that the ferric heme can no longer bind oxygen, leaving the hemoglobin non-functional, this form is also known as methemoglobin. This autoxidation occurs on the order of hours, therefore accumulation of non-functional methemoglobin is significant if left alone. </p>
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  <h3 align="center"><img src="https://static.igem.org/mediawiki/2007/8/87/Antioxidantreactions.jpg" alt="" width="528" height="403" align="left"></h3>
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  <h3 align="center">Antioxidants</h3>
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  <p align="justify">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. These enzymes are already used in E. Coli because free radicals are formed regularly during metabolism. However, we feel that because of our unusual accumulation of heme and hemoglobin, we must supplement the breakdown of the formed superoxides by overexpressing the catalase and superoxide dismutase genes in our system. The reaction mechanism to the left shows the pathway of superoxide degredation and the roles of superoxide dismutase and catalase. The desired breakdown of superoxide is through the two enzymes mentioned. The alternative pathway results in more non-functional ferric iron. </p>
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  <p align="justify">&nbsp;</p>
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  <p align="justify">&nbsp; </p>
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  <p align="justify">&nbsp;</p>
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  <p align="justify">&nbsp;</p>
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  <h3 align="center"><img src="https://static.igem.org/mediawiki/2007/3/3c/Cytochromereactions.jpg" alt="" width="500" height="426" align="right"></h3>
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  <h3 align="center">Cytochrome b5 / Cytochrome b5 Reductase and Methemoglobin</h3>
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  <p align="justify"> Human erythrocytes have addressed the accumulation of methemoglobin caused by the autoxidation  problem by using the NADH dependent enzyme, cytochrome b5 reductase. Working together with cytochrome b5, these two components allow for reduction of the methemoglobin heme centers back into its functional ferrous (Fe2+) form. The mechanism is shown to the right. </p>
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  <h2 align="center">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="center"><a href="https://2007.igem.org/Berkeley_UC">&lt;&lt;&lt; Return to UC Berkeley iGEM 2007 </a></div>
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Untitled Document

Next Section: Chassis>>

Engineering Bactoblood for Oxygen Transport

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.

 

The Hemoglobin Generating Device

Overview: Human Hemoglobin A and Methionine Aminopeptidase

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.

Oxygen Binding Affinity and P50

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.

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.

 

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.

 

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.

 

Wild Type and Mutant Hemoglobins

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, and the wild type human hemoglobin. The wild type is to compare the mutant hemoglobins to. The first generation Bactoblood blood substitute will use a hemoglobin 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.

The western blot to the bottom shows various mutant hemoglobins we constructed and tested for hemoglobin yield. A polyclonal antibody was used to identify the produced hemoglobin. All of these samples had heme added exogenously to the culture during growth and were allowed to grow overnight. The bands around 16kD are consistent with monomeric alpha and beta subunits of hemoglobin. Some of the constructs contain a fusion of alpha subunits with a glycine linker. More about this will be discussed in the hemoglobin solubility section. The western demonstrates that hemoglobin is being produced in our cells to a significant degree. In our potential final product, the heme will be produced in vivo and our cultures will be concentrated. This will greatly increase the concentration of hemoglobin in our cells to hopefully near physiological concentrations.

Methionine Aminopeptidase

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.

 

The Chaperone Device

Overview: Alpha Hemoglobin Stabilizing Protein

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.

Hemoglobin Solubility

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. The mechanism for AHSP is shown in the figure to the left.


 

 

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.


Heme Biosynthesis Device

Overview: Heme

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.

Heme Biosynthesis Pathway

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 precursors 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.

Construction of the Heme Device

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.


 

Verification of the Heme Device

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.

 

 

 

 

 

 

 

 

 

Autoxidation Control

Overview: Cytochromes and Antioxidants

During the process of binding and unbinding oxygen, the heme groups of the hemoglobin may spontaneously undergo autoxidation, ultimately causing the formation of damaging free radicals and changing the form of the iron in the heme center. To remedy these problems we created an antioxidant device and a methemoglobin reductase device. The antioxidant device contains the genes that express superoxide dismutase and catalase. The methemoglobin reductase device contains the genes that express cytochrome b5 and cytochrome b5 reductase. These two devices will keep our hemoglobin functional and our cells healthy.

Autoxidation and Free Radical Production

There are two problems to address in keeping our cells happy and our hemoglobin functional. The first problem is the spontaneous autoxidation of the heme centers in hemoglobin. During the binding and unbinding of oxygen, electrons are transfered back and forth between the iron center of heme and the bound oxygen molecule. Every once in a while, an electron will transfer to the oxygen and the oxygen will then unbind from the iron center and take the electron away from the iron. This creates a superoxide and a ferric Fe3+ heme. The superoxide eventually will degrate into a hydrogen peroxide, then into a hydroxyl radical, which is very toxic to the cell. The second problem is that the ferric heme can no longer bind oxygen, leaving the hemoglobin non-functional, this form is also known as methemoglobin. This autoxidation occurs on the order of hours, therefore accumulation of non-functional methemoglobin is significant if left alone.

Antioxidants

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. These enzymes are already used in E. Coli because free radicals are formed regularly during metabolism. However, we feel that because of our unusual accumulation of heme and hemoglobin, we must supplement the breakdown of the formed superoxides by overexpressing the catalase and superoxide dismutase genes in our system. The reaction mechanism to the left shows the pathway of superoxide degredation and the roles of superoxide dismutase and catalase. The desired breakdown of superoxide is through the two enzymes mentioned. The alternative pathway results in more non-functional ferric iron.

 

 

 

 

Cytochrome b5 / Cytochrome b5 Reductase and Methemoglobin

Human erythrocytes have addressed the accumulation of methemoglobin caused by the autoxidation problem by using the NADH dependent enzyme, cytochrome b5 reductase. Working together with cytochrome b5, these two components allow for reduction of the methemoglobin heme centers back into its functional ferrous (Fe2+) form. The mechanism is shown to the right.

 

 

 

 

 

 

 

 


 

Hemoglobin Alternatives

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.

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.

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.

Next Section: Chassis>>