Difference between revisions of "Team:Technion Israel/Basic Part"

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<title>Team: Technion 2015</title>
  
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<p>In order to be considered for the <a href="https://2015.igem.org/Judging/Awards#SpecialPrizes">Best New Basic Part award</a>, you must fill out this page. Please give links to the Registry entries for the Basic parts you have made. Please see the Registry's <a href="http://parts.igem.org/Help:Parts#Basic_and_Composite_Parts"> Help:Parts page</a> for more information on part types.</p>
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A <b>basic part</b> is a functional unit of DNA that cannot be subdivided into smaller component parts. <a href="http://parts.igem.org/wiki/index.php/Part:BBa_R0051">BBa_R0051</a> is an example of a basic part, a promoter regulated by lambda cl.
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<p>Most genetically-encoded functions have not yet been converted to BioBrick parts. Thus, there are <b>many</b> opportunities to find new, cool, and important genetically encoded functions, and refine and convert the DNA encoding these functions into BioBrick standard biological parts. </p>
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<h1><b>Basic Part</b></h1>
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<h2><b>Introduction</b></h2>
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<p>Our basic part is the signal peptide from the <i>aprE</i> gene, which was documented in detail  and submitted as <a href="http://parts.igem.org/Part:BBa_K1674000" target="_blank">BBa_K1674000</a>. It has been successfully used in the secretion of the mCherry reporter protein by <i>B.subtilis</i>, as can be seen in our <a href="https://2015.igem.org/Team:Technion_Israel/Project/Results" target="_blank">results</a>.</p>
  
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<h2><b>The Part</b></h2>
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<p><i>Bacillus subtilis</i> is an industrially important bacterium which is commonly used as a host for the commercial production of proteins, especially enzymes such as proteases, amylases, and lipases. One of the major advantages of <i>B. subtilis</i> over many other bacteria is its ability to secrete large amounts of proteins into the growth medium, allowing efficient recovery of these protein products <sup><a href="#fn1" id="ref1">1</a></sup>.</p>
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<p><i>aprE</i> encodes for the main protease secreted by <i>B.subtilis</i> naturally, Subtilisin E. The signal peptide domain from aprE gene is part of the general secretion pathway (Sec pathway), where its role is to target pre-folded proteins to the membrane-bound protein translocation channel <sup><a href="#fn1" id="ref1">1</a>,<a href="#fn2" id="ref1">2</a></sup>.</p>
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<p>The signal peptide from <i>aprE</i> can be used for secreting various proteins by fusing it to the N or C terminal of the desired protein.  In previous research, this signal peptide has been proven to cause secretion of different proteins, such as cutinase, proinsulin, etc. <sup><a href="#fn1" id="ref1">1</a>,<a href="#fn2" id="ref1">2</a>, <a href="#fn3" id="ref1">3</a></sup></p>
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<p>Our part can be used in the future for the secretion of various proteins.  Therefore, it can help other groups, both in iGEM project and in the larger academic community, express proteins and extract them without the concern of degradation and inactivation, even when using aggressive lysis methods, such as a lysis buffer or sonication.</p>
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align="center"><img style="width: 484px; height: 361px;"
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id="Picture 17"
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src="https://static.igem.org/mediawiki/2015/c/cb/Sec_pathway.png"
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alt="Description: Description: Technion\iGEM2015\Pictures_Results\sec_pathway.jpg"></p>
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      <p class="MsoCaption" style="text-indent: 0cm;"><a href="https://static.igem.org/mediawiki/2015/c/cb/Sec_pathway.png"
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name="_Ref275820820"><span lang="EN-US">Figure
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1 </span></a><span
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<p>Schematic overview of the Sec-type signal peptide (SP) pathway in <i>B.subtilis</i>. [1]</p>
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<li> The exoprotein synthesized and directly bound to the SP and the SRP-complex (Signal Recognition
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Particle).</li>
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<li> The SecA protein interacts with the SRP-complex via the SP.</li>
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<li> The SRP-precursor-SecA complex move towards the membrane where the pore forming SecYEG
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proteins are. The SP cleaved from the precursor and the SecA-precursor complex pushed through the
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membrane. </li>
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<li> The SP is been degraded and the exoprotein is going through some last modification.</li>
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<sup id="fn1">1. Kolkman, M. A. B.; Van der Ploeg, R.; Bertels, M.; Van Dijk, M.; Van der Laan, J.; Van Dijl, J. M.; Ferrari, E.: The Twin-Arginine Signal Peptide of Bacillus subtilis YwbN Can Direct either Tat- or Sec-Dependent Secretion of Different Cargo Proteins: Secretion of Active Subtilisin via the B. subtilis Tat Pathway. Applied and Environmental Microbiology. 2008, 74, 24, 7507-7513.</sup></br>
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<sup id="fn2">2. Brockmeier, U.:New strategies to optimize the secretion capacity for heterologous proteins in Bacillus subtilis.PhD thesis, Biowissenschaften der Ruhr-Universita ̈t, Bochum, 2006.
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</sup></br>
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<sup id="fn3">3.Westers, L.; Westers, H.; Quax, W. J.: Bacillus subtilis as cell factory for pharmaceutical proteins: a biotechnological approach to optimize the host organism. Biochimica et Biophysica Acta. 2004, 1694, 299-310.
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</sup></br>
 
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Latest revision as of 21:54, 18 September 2015

Team: Technion 2015

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Basic Part

Introduction

Our basic part is the signal peptide from the aprE gene, which was documented in detail and submitted as BBa_K1674000. It has been successfully used in the secretion of the mCherry reporter protein by B.subtilis, as can be seen in our results.

The Part

Bacillus subtilis is an industrially important bacterium which is commonly used as a host for the commercial production of proteins, especially enzymes such as proteases, amylases, and lipases. One of the major advantages of B. subtilis over many other bacteria is its ability to secrete large amounts of proteins into the growth medium, allowing efficient recovery of these protein products 1.

aprE encodes for the main protease secreted by B.subtilis naturally, Subtilisin E. The signal peptide domain from aprE gene is part of the general secretion pathway (Sec pathway), where its role is to target pre-folded proteins to the membrane-bound protein translocation channel 1,2.

The signal peptide from aprE can be used for secreting various proteins by fusing it to the N or C terminal of the desired protein. In previous research, this signal peptide has been proven to cause secretion of different proteins, such as cutinase, proinsulin, etc. 1,2, 3

Our part can be used in the future for the secretion of various proteins. Therefore, it can help other groups, both in iGEM project and in the larger academic community, express proteins and extract them without the concern of degradation and inactivation, even when using aggressive lysis methods, such as a lysis buffer or sonication.

Description: Description: Technion\iGEM2015\Pictures_Results\sec_pathway.jpg

Figure 1

Schematic overview of the Sec-type signal peptide (SP) pathway in B.subtilis. [1]

  1. The exoprotein synthesized and directly bound to the SP and the SRP-complex (Signal Recognition Particle).
  2. The SecA protein interacts with the SRP-complex via the SP.
  3. The SRP-precursor-SecA complex move towards the membrane where the pore forming SecYEG proteins are. The SP cleaved from the precursor and the SecA-precursor complex pushed through the membrane.
  4. The SP is been degraded and the exoprotein is going through some last modification.

1. Kolkman, M. A. B.; Van der Ploeg, R.; Bertels, M.; Van Dijk, M.; Van der Laan, J.; Van Dijl, J. M.; Ferrari, E.: The Twin-Arginine Signal Peptide of Bacillus subtilis YwbN Can Direct either Tat- or Sec-Dependent Secretion of Different Cargo Proteins: Secretion of Active Subtilisin via the B. subtilis Tat Pathway. Applied and Environmental Microbiology. 2008, 74, 24, 7507-7513.
2. Brockmeier, U.:New strategies to optimize the secretion capacity for heterologous proteins in Bacillus subtilis.PhD thesis, Biowissenschaften der Ruhr-Universita ̈t, Bochum, 2006.
3.Westers, L.; Westers, H.; Quax, W. J.: Bacillus subtilis as cell factory for pharmaceutical proteins: a biotechnological approach to optimize the host organism. Biochimica et Biophysica Acta. 2004, 1694, 299-310.

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