Team:Tokyo-NoKoGen/Project/solubilization

From 2010.igem.org

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<img src="https://static.igem.org/mediawiki/2010/f/f8/Solubilization.JPG" style="float:right">
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<img src="https://static.igem.org/mediawiki/2010/0/09/Solubi_top.png">
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<h1>Objectives:Solubilization</h1>
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<h2>Introduction</h2>
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<p> Suppose that you would like to get a object from nature. Can you say it is always easy to pick it up?
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<img src="https://static.igem.org/mediawiki/2010/c/ce/Bitumen.png" align="right" >
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Such as a gemstone in a mine, you encounter the difficulty that a fascinating or valuable target is in a mixture.
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<div align="left">
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<p> Bitumen is hydrocarbon which adhered to oil sand. Bitumen is consisted of poisonous organic compounds such as naphthalene, phenanthrene, benzopyrene and so on. It is difficult to recycle bitumen as fuels because it is hard to extract target compound from bitumen.<br>
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Surfactants enable to solubilize organic compound, but it is difficult to biodegradate artificial surfactants which we usually use. On the other hand, biosurfactants which microbes produce by themselves are able to biodegradate. That is why, biosurfactants are very ecology.
</p>
</p>
<p>
<p>
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  We solve this problem by making rhamnolipids , which is a biosurfactant naturally secreted by Pseudomonas Aeruginosa.
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  We try to extract organic compound such as naphthalene from bitumen using solubilization device. <i>Pseudomonas aeruginosa</i> is known to produce biosurfactants called as rhamnolipids. Rhamnolipids form micelle with organic compound and micelle were able to solubilize.
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Then, we cloned RhlA and RhlB genes which are necessary to produce rhamnolopids from <i>Pseudomonas aeruginosa</i>. <i>E. coli</i> was transformed with plasmid involved RhlA or RhlB gene. We tried to construct solubilization device which <i>E. coli</i> encoded rhamnolopids genes.
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<h2>
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Objectives
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</h2>
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<p>
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We tried to construct solubilization device and evaluate it.<br>
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Due to this device, toxic organic compounds were solubilized and enabled to uptake into E. coli, so toxic organic compounds were able to eliminate from bitumen. In the case of using Eco Tanker, solubilization device makes various compounds solubilized, then, these are enabled to uptake by Eco Tanker.<br>
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That is why we hope that this device is applied to bioremediation.
</p>
</p>
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<img src="https://static.igem.org/mediawiki/2010/4/43/Gemstone1.jpg">
 
<h2>Why is this device needed?</h2>
<h2>Why is this device needed?</h2>
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<div align="left">
<p>  For the first of all, we have to make the target be easily taken up from an mixture. Oil sand is a good example. It is a naturally occurring mixture of sand, clay and several minerals, and bitumen. This bitumen is a material for a usable fuels such as gasoline and diesel, though it is too viscous to produce fuels. One of  the solutions is to solubilize a object from a mixture. This is because solubilization system is the first necessary step in Eco Tanker. </p>
<p>  For the first of all, we have to make the target be easily taken up from an mixture. Oil sand is a good example. It is a naturally occurring mixture of sand, clay and several minerals, and bitumen. This bitumen is a material for a usable fuels such as gasoline and diesel, though it is too viscous to produce fuels. One of  the solutions is to solubilize a object from a mixture. This is because solubilization system is the first necessary step in Eco Tanker. </p>
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</div>
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<h2>What is this device compose inside it?</h2>
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<h2>What is this device composed inside it?</h2>
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<p>
<p>
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This device can produce two enzymes. One is transacylase, the other is rhamnosyltransferase.  
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Each protein is expressed by the gene, RhlA and RhlB respectively.  
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It is known that rhamnolipids are produced by and transacylase and rhamnosyltransferase <sup>1)2)</sup>. Transacylase is encoded RhlA gene and rhamnosyltransferase is encoded RhlB gene. Fig. 1 showed biosynthesis of rhamnolipids <sup>3)</sup>
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Fig.1 shows Rhamnolipids Biosynthetic Pathway, and you can see where two genes work for.
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It catalyzes the transfer of β-hydroxydecanoyl moieties from acyl carrier protein (ACP) to coenzyme A (CoA). With this reaction, 3-(3-hydroxyalkanoyloxy)alkanoic acids (HAAs), which Fig.2 shows, are produced.
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RhlA is required to two molecules of β-Hydrocyacyl-ACP to <br>
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3-(3-hydroxyalkanoyloxy)alkanoic acids (HAAs). <br>
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Fig. 2 showed HAA structural formula <sup>3)</sup>.
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<img src="https://static.igem.org/mediawiki/2010/a/a4/Fig2_noko.png" style="width:300px;">
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It catalyzes the reaction, which uses dTDP-L-rhamnose and an HAA as precursors, yielding mono-rhamnolipids(rhamnolipids contains both mono-rhamnolipids and di-rhamnolipid naturally(Fig.3).).  
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RhlB is rhamnolipid by the consecutive attachment of rhamnosyl groups. <br>
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Fig. 3 showed rhamnolipid structural formula<sup>3)</sup>.
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<img src="https://static.igem.org/mediawiki/2010/1/11/Fig3_noko.png" style="width:300px;" >
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<img src="https://static.igem.org/mediawiki/2010/0/06/Ramno3.jpg">
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<h2>Device component</h2>
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<h2>How does this device work in EcoTanker></h2>
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<img src="https://static.igem.org/mediawiki/2010/8/82/Genemap.jpg">
<img src="https://static.igem.org/mediawiki/2010/8/82/Genemap.jpg">
<img src="https://static.igem.org/mediawiki/2010/a/a7/Expla.jpg">
<img src="https://static.igem.org/mediawiki/2010/a/a7/Expla.jpg">
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<p>Our objective construct is as above. We ligate it to pSB1C3 and transform E. coli DH5α.
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<p>Our objective construct is as above. We ligate it to pSB1C3 and transform E. coli DH5α.</p>
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<p>P(H)const.:
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<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_J23100">
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<border="0">
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BBa_J23100
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,P(M)const.:
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<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_J23111">
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<border="0">
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BBa_J23111
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, RBS:
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<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_B0034">
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BBa_B0034
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</a>
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, Term:
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<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_B0016">
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BBa_B0016
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</a>
</p>
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<h2>progress</h2>
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<h2>Progress</h2>
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<p> Firstly, we increased the two genes, RhlA and RhlB from P. aeruginosa PAO1 strain respectively by PCR. Secondly, in order to make primers for RhlA and RhlB, we ligated former and reverse sequences which had hybridized area by PCR. Primers created ourselves cost less than that was ordered, because its length was big. Using the produced primers, we amplified the genes, promoter-RBS-RhlA-terminator and promoter-RBS-RhlB-terminator. After digested by EcoRI and PstI, we ligated the PCR products to  
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<p> At first, we cultured P. aeruginosa on agar plates including nutrient broth and we did cloning of the two genes, RhlA and RhlB from the colonies of P. aeruginosa respectively by colony PCR. Secondly, in order to add promoter, RBS and terminator to RhlA or RhlB respectively, we made mega-primer by PCR. Using the primers, we amplified the genes, promoter-RBS-RhlA-terminator and promoter-RBS-RhlB-terminator. After digested by EcoRI and PstI, we ligated the PCR products to  
<a href="http://partsregistry.org/wiki/index.php?title=Part:pSB1C3">
<a href="http://partsregistry.org/wiki/index.php?title=Part:pSB1C3">
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and transformed E coli. DH5α. We confirmed these sequences. Unfortunately, the objective constructs were not get.  
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and transformed E.coli DH5α. We confirmed these sequences. Unfortunately, the objective constructs were not got.  
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BioBricks we submitted are bellows.
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BioBricks we submitted are bellows.However, RhlA includes 1 PstI site and RhlB includes 2 PstI site.
<p>
<p>
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K317998">
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K317998">
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<h2>References</h2>
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</body>
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<p>
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1) UrsA. Ochsner <i>et al. The Journal of Biological Chemistry</i> 1994<br>
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2) Gloria soberon <i>et al. Appl Microbiol Biotechol</i> 2005<br>
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3) Kun Zhu <i>et al. Journal of Bacteriology</i> 2008<br>
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</p>
</body>
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Latest revision as of 19:44, 27 October 2010

Introduction

Bitumen is hydrocarbon which adhered to oil sand. Bitumen is consisted of poisonous organic compounds such as naphthalene, phenanthrene, benzopyrene and so on. It is difficult to recycle bitumen as fuels because it is hard to extract target compound from bitumen.
Surfactants enable to solubilize organic compound, but it is difficult to biodegradate artificial surfactants which we usually use. On the other hand, biosurfactants which microbes produce by themselves are able to biodegradate. That is why, biosurfactants are very ecology.

We try to extract organic compound such as naphthalene from bitumen using solubilization device. Pseudomonas aeruginosa is known to produce biosurfactants called as rhamnolipids. Rhamnolipids form micelle with organic compound and micelle were able to solubilize. Then, we cloned RhlA and RhlB genes which are necessary to produce rhamnolopids from Pseudomonas aeruginosa. E. coli was transformed with plasmid involved RhlA or RhlB gene. We tried to construct solubilization device which E. coli encoded rhamnolopids genes.

Objectives

We tried to construct solubilization device and evaluate it.
Due to this device, toxic organic compounds were solubilized and enabled to uptake into E. coli, so toxic organic compounds were able to eliminate from bitumen. In the case of using Eco Tanker, solubilization device makes various compounds solubilized, then, these are enabled to uptake by Eco Tanker.
That is why we hope that this device is applied to bioremediation.

Why is this device needed?

For the first of all, we have to make the target be easily taken up from an mixture. Oil sand is a good example. It is a naturally occurring mixture of sand, clay and several minerals, and bitumen. This bitumen is a material for a usable fuels such as gasoline and diesel, though it is too viscous to produce fuels. One of the solutions is to solubilize a object from a mixture. This is because solubilization system is the first necessary step in Eco Tanker.

What is this device composed inside it?

It is known that rhamnolipids are produced by and transacylase and rhamnosyltransferase 1)2). Transacylase is encoded RhlA gene and rhamnosyltransferase is encoded RhlB gene. Fig. 1 showed biosynthesis of rhamnolipids 3)


Transacylase:

RhlA is required to two molecules of β-Hydrocyacyl-ACP to
3-(3-hydroxyalkanoyloxy)alkanoic acids (HAAs).
Fig. 2 showed HAA structural formula 3).


Rhamnosyltransferase:

RhlB is rhamnolipid by the consecutive attachment of rhamnosyl groups.
Fig. 3 showed rhamnolipid structural formula3).

Device component

Our objective construct is as above. We ligate it to pSB1C3 and transform E. coli DH5α.

P(H)const.: BBa_J23100 ,P(M)const.: BBa_J23111 , RBS: BBa_B0034 , Term: BBa_B0016

Progress

At first, we cultured P. aeruginosa on agar plates including nutrient broth and we did cloning of the two genes, RhlA and RhlB from the colonies of P. aeruginosa respectively by colony PCR. Secondly, in order to add promoter, RBS and terminator to RhlA or RhlB respectively, we made mega-primer by PCR. Using the primers, we amplified the genes, promoter-RBS-RhlA-terminator and promoter-RBS-RhlB-terminator. After digested by EcoRI and PstI, we ligated the PCR products to pSB1C3 and transformed E.coli DH5α. We confirmed these sequences. Unfortunately, the objective constructs were not got. BioBricks we submitted are bellows.However, RhlA includes 1 PstI site and RhlB includes 2 PstI site.

BBa_K317998 : RhlA

BBa_K317999 : RhlB

References

1) UrsA. Ochsner et al. The Journal of Biological Chemistry 1994
2) Gloria soberon et al. Appl Microbiol Biotechol 2005
3) Kun Zhu et al. Journal of Bacteriology 2008

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