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| <div class="content"> | | <div class="content"> |
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| + | <div class="jiao" > |
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| + | <div class="projtile_only"> |
| + | <h2 id="subtitleyjn1">Basic Test</h2></br> |
| + | <h2 id="subtitleyjn2">——One Connectee Binds Connector</h2> |
| + | <center><p>At first, tests should be taken to check whether TAL can bind to plasmid DNA in prokaryotic system. Here we used <i>E.coli</i>. As mentioned in the overview, in order for the protein to bind to the plasmid(the connector), we have designed two kinds of delicate fusion proteins(the connectee). One is anchored to the cell membrane, the other is free in the cytoplasm.</p></center> |
| + | </div> |
| + | |
| + | <div class="projtile" id="dingweidian2"> |
| + | <a href="#dingweidian2" title="Connectee"> |
| + | <center><h2>Connectee</h2></center></a> |
| + | </div> |
| + | |
| + | <div class="projtile"> |
| + | <a href="#connectee2:" title="Connector"> |
| + | <center> <h2>Connector</h2></center></a> |
| + | </div> |
| + | |
| + | |
| + | <div class="projtile"> |
| + | <a href="#connector:" title="Test Method"> |
| + | <center><h2>Test Method</h2></center></a> |
| + | </div> |
| + | |
| + | <div class="projtile"> |
| + | <a href="#dingweidian" title="Two Convenient Parts"> |
| + | <center><h2>Two Convenient Parts</h2></center></a> |
| + | </div> |
| + | |
| + | <div class="projtile"> |
| + | <a href="#dingweidian12" title="Reference"> |
| + | <center><h2>References</h2></center></a> |
| + | </div> |
| + | </div> |
| + | |
| + | <div style="clear:both;"></div> |
| + | |
| + | |
| <article class="post__article"> | | <article class="post__article"> |
| + | <!-- <h2 id="morethanonejewelonthecrown"><center>Basic Test</center></h2> |
| + | <h2 id="alignright" align="center">——One Connectee Binds Connector</h2> |
| + | --> |
| + | |
| + | <!-- Luo! This part has been moved up --> |
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| + | |
| + | |
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| + | |
| + | <h2 id="connectee:">Connectee:</h2> |
| + | <h3 id="schematicintroduction">Schematic Introduction</h3> |
| + | <p> <!-- Luo rewrite the part --> |
| + | |
| + | <!-- At first, tests should be taken to check whether TAL can bind to plasmid DNA in prokaryotic system. Here we used <i>E.coli</i>. As mentioned in the overview, in order for the protein to bind to the plasmid–the <strong><em>connector</em></strong>, we have designed two kinds of delicate fusion proteins–the <strong><em>connectee</em></strong>. One is anchored to the cell membrane, the other is free in the cytoplasm. --> |
| + | |
| + | The <strong><em>connectee</em></strong> consists of linkers and various sections, which are shown in the schematic diagrams below.</p> |
| + | |
| + | |
| + | |
| + | <h4>1. TAL effector: A transcription activator-like protein</h4> |
| + | <p><strong>TAL effector</strong> can bind to a target sequence on DNA. The 2012 Freiburg iGEM team has offered us a whole set of 96 TAL-protein direpeat bioparts, with which we are supposed to build functional TAL proteins. Since each TAL protein is able to identify a 14-nucleotide target sequence, the first and fourteenth nucleotide being Thymine, all the 96 parts can be used to identify more than 16 million different nucleotide sequences, which makes it very convenient for us to choose a sequence for the fusion protein to bind to.</p> |
| + | |
| + | <center><img src="https://static.igem.org/mediawiki/2014/2/2a/TAL_golden_gate.png"></img></center> |
| + | </br><center><small><strong>Figure 1.2.1 TAL Golden gate</strong></small></center></br> |
| | | |
- | <h2 id="connectee:">Connectee:</h2><br/>
| |
- | <h3 id="schematicintroduction">Schematic introduction</h3><br/>
| |
- | <p>At first, test should be taken to check whether TAL can bind plasmid DNA in prokaryotic system, here we use E.coli. As mentioned in the overview, in order for the protein to bind to the plasmid–the <strong><em>connector</em></strong>, we have designed two kinds of delicate fusion proteins–the <strong><em>connectee</em></strong>. We designed two kinds of <strong><em>connectee</em></strong>, one is anchored to the cell membrane, the other is free in the cytoplasm. Both of them consist of linkers and various sections, which are shown in the schematic diagrams below.</p>.
| |
| | | |
| | | |
- | <ol> | + | <h4>2. Membrane anchor system: ssDsbA-Lgt</h4> |
- | <li><strong>TAL effector</strong>–a transactivator-like protein. | + | <p><strong>ssDsbA</strong>, the signal sequence of DsbA, directs the fusion protein to the periplasm. <strong>Lgt</strong> is a transmembrane protein.</p> |
- | <br/><strong>TAL effector</strong> can bind DNA with target sequence. The 2012 Freiburg iGEM team has offered us a whole set of 96 TAL-protein direpeat bioparts, with which we are supposed to build functional TAL proteins. Since each TAL protein can identify a 14-nucleotide target sequence, the first and fourteenth nucleotide being Thymin, all the 96 parts can be used to identify more than 16 million different nucleotide sequences, which makes it very convenient for us to choose a sequence for the fusion protein to bind to.</li> | + | <p>The <strong> membrane anchor system</strong> has been identified by iGEM12_SJTU-BioX-Shanghai. </p> |
| | | |
- | <center><img src="https://static.igem.org/mediawiki/2014/2/2a/TAL_golden_gate.png"></img></center> | + | <h4>3. Fluorescent protein</h4> |
| + | <p><strong>CFP</strong> is a Cyan Fluorescent Protein which has an excitation peak at 439 nm and an emission peak at 476 nm.</p> |
| + | <p><strong>YFP</strong> is a Yellow Fluorescent Protein which has an excitation peak at 514 nm and an emission peak at 527 nm.</p> |
| + | <p><strong>mRFP</strong> is a Red Fluorescent Protein which has an excitation peak at 584 nm and an emission peak at 607 nm.</p> |
| | | |
| | | |
- | <li>The <strong>enzyme</strong> we want to bring together. | + | <h3 id="connectee1:">Connectee 1:</h3> |
- | <br/>The proteins we want to bring together usually are enzymes that come from a metabolic pathway.<br/>
| + | |
- | <br/>
| + | |
- | <li><strong>Membrane anchor system–ssDsbA-Lgt</strong>.
| + | |
- | <br/><strong>ssDsbA</strong> is the signal sequence of DsbA which can direct the fusion protein to the periplasm. <strong>Lgt</strong> is a transmembrane protein.</li>
| + | |
- | The <strong> membrane anchor system</strong> has been identified by iGEM12_SJTU-BioX-Shanghai.
| + | |
- | <br/><br/>
| + | |
- | <li><strong>Fluorescent protein</strong>.
| + | |
- | <br/><strong>CFP</strong> is a Cyan Fluorescent Protein which has an excitation peak at 439 nm and an emission peak at 476 nm.
| + | |
- | <br/><strong>YFP</strong> is a Yellow Fluorescent Protein which has an excitation peak at 514 nm and an emission peak at 527 nm.
| + | |
- | <br/><strong>mRFP</strong> is a Red Fluorescent Protein which has an excitation peak at 584 nm and an emission peak at 607 nm.
| + | |
- | <br/>
| + | |
- | <br/>
| + | |
| | | |
| + | <p>The first kind of <strong><em>connectee</em></strong> is designed to be tested on the membrane, which consists of three major domains: membrane anchor system(ssDsbA-Lgt), mRFP and TAL effector.</p> |
| + | <p>The reasons why we chose a membrane anchor are as follows.</p> |
| | | |
- | <h3 id="connectee1:">Connectee 1:</h3> | + | <p>1. TAL could bind nucleoid which may bring some negative effect on bacteria growth.</p> |
| + | <p>2. Membrane scaffold is a natural scaffold.</p> |
| + | <p>3. Exogenous proteins often form inactive inclusion bodies when expressed in the prokaryotic system.</p> |
| | | |
- | <p>The first kind of <strong><em>connectee</em></strong> is designed to be tested on the membrane, which is consist of three major domains, membrane anchor system(ssDsbA-Lgt), mRFP and TAL effector.</p>
| |
- | <p>The reason why we choose a membrane anchor is that:</p>
| |
- | <ol>
| |
- | <li>TAL could bind nucleoid which may bring some effect on bacteria growth.</li>
| |
- | <li>Membrane scaffold is a natural scaffold.</li>
| |
- | <li>Exogenous proteins often form inactive inclusion body when expressed in the prokaryotic system</li>
| |
- | </ol>
| |
| | | |
- | <p><img src="https://static.igem.org/mediawiki/2014/7/74/MRFP-Lgt-TAL_effector.png"></img></p> | + | <p><center><img src="https://static.igem.org/mediawiki/2014/7/74/MRFP-Lgt-TAL_effector.png"></img></center></p> |
| + | </br><center><small><strong>Figure1.2.2 Connectee 1</strong></small></center></br> |
| + | <p>The membrane anchor system (ssDsbA-Lgt) comes from BBa_K771000 designed by iGEM12_SJTU-BioX-Shanghai; mRFP comes from BBa_E1010 designed by Antiquity; TAL effector comes from TAL-Protein DiRepeats (BBa_K747000 to BBa_K747095) designed by iGEM12_Freiburg. We used Helical Linker to connect mRFP and Lgt, in accord with iGEM12_SJTU-BioX-Shanghai. While in consideration of any possible stereospecific blockade when TAL binds to plasmid DNA, we chose Flexible Linker to connect Lgt and TAL effector.</p> |
| | | |
- | <p>The membrane anchor system (ssDsbA-Lgt) comes from BBa_K771000 designed by iGEM12_SJTU-BioX-Shanghai; mRFP comes from BBa_E1010 designed by Antiquity; TAL effector comes from TAL-Protein DiRepeats (Bba_K747000 to Bba_K747095) designed by iGEM12_Freiburg. We use Helical Linker to connect mRFP and Lgt, in accord with iGEM12_SJTU-BioX-Shanghai. While in consideration of any possible stereospecific blockade when TAL binds plasmid DNA, we choose Flexible Linker to connect Lgt and TAL effector.</p> | + | <h3 id="connectee2:">Connectee 2:</h3> |
| | | |
- | <h3 id="connectee2:">Connectee 2:</h3> | + | <p>The second kind of <strong><em>connectee</em></strong> is designed to be tested in the cytoplasm, which only contains TAL effector.</p> |
| | | |
- | <p>The second kind of <strong><em>connectee</em></strong> is designed to be tested in the cytoplasm, which only contain TAL effector.</p> | + | <p><center><img src="https://static.igem.org/mediawiki/2014/1/19/Random_tal_effector.png"></img></center></p> |
| + | </br><center><small><strong>Figure 1.2.3 Connectee 2</strong></small></center></br> |
| + | <h2 id="connector:">Connector:</h2> |
| | | |
- | <p><img src="https://static.igem.org/mediawiki/2014/1/19/Random_tal_effector.png"></img></p> | + | <p>As iGEM12_Freiburg designed, we can choose a TAL protein to recognize a 14-nucleotide-long sequence, TXXXXXXXXXXXXT, on the plasmid DNA. |
| + | The plasmid here is called <strong><em>connector</em></strong>. </p> |
| | | |
- | <h2 id="connector:">Connector:</h2>
| |
| | | |
- | <p>As iGEM12_Freiburg designed, we can choose a 14-nucleotide-long Transactivator-like (TAL) protein TXXXXXXXXXXXXT to recognize the same sequence on the plasmid DNA.<br/> | + | <p>The principle for choosing a TAL recognizing sequence: </p> |
- | The plasmid here we called <strong><em>connector</em></strong>. </p>
| + | |
| | | |
- | <blockquote> | + | <p>I. It does not exist in expression vector.</p> |
- | <p>The principle for choose a TAL recognize sequence: </p> | + | <p>II. It does not exist in the sequence of <strong><em>connectee</em></strong>.</p> |
| | | |
- | <ol>
| |
- | <li>Not exist in expression vector;</li>
| |
- | <li>Not exist in the sequence of <strong><em>connectee</em></strong>;</li>
| |
- | </ol>
| |
- | </blockquote>
| |
| | | |
- | <h2 id="testmethod:">Test method:</h2>
| |
| | | |
- | <p>In consideration of our multiple-enzyme system may be applied in the following experiment, we choose pRSFDuet–1(<em>NOVAGEN</em>) as the expression vector. </p> | + | <h2 id="testmethod:">Test Method:</h2> |
| | | |
- | <p><img src="https://static.igem.org/mediawiki/2014/d/d3/Prsf.png"></img></p> | + | <p>1. pBluescript II KS(+) is chosen as the <strong><em>connector</em></strong> in our test for several reasons: </p> |
| | | |
- | <blockquote>
| |
- | <p>pBluescript II KS(+) is chose as the <strong><em>connector</em></strong> for test for several reasons: </p>
| |
| | | |
- | <ol> | + | <p>I. High copy number;</p> |
- | <li>High copy number;</li>
| + | <p>II.Medium length—2961bp;</p> |
- | <li>Medium length—2961bp</li> | + | <p>III. Easy to detect whether binding a TAL may affect gene expression — through lacI & blue-white spot screening.</p> |
- | <li>Easy to test whether binding a TAL may have effect on gene expression—lacI & blue-white spot screening</li> | + | |
- | </ol>
| + | |
- | </blockquote>
| + | |
| | | |
- | <p><img src="https://static.igem.org/mediawiki/2014/8/81/Pks_ii_original.png"></img></p>
| |
| | | |
- | <p>Similar with the cross-linked ChIP, we use formaldehyde to cross-link <strong><em>connectee</em></strong> and <strong><em>connector</em></strong></p> | + | <p><center><img src="https://static.igem.org/mediawiki/2014/8/81/Pks_ii_original.png"></img></center></p> |
- | <p>After that we do immunoprecipitation to get the protein-plasmid complex and digest protein</p> | + | </br><center><small><strong>Figure 1.2.4 pBluescript II KS(+)</strong></small></center></br> |
- | <p>Finally, doing a PCR to check is there any plasmid DNA exist.</p> | + | <p>2. After checking the sequence of pBluescript II KS(+), we chose TTCGATATCAAGCT as the recognition sequence for test and designed TAL1.</p> |
| | | |
- | <h2 id="aconvenientpart:">A convenient part:</h2> | + | <p>Two kinds of connectee with TAL1 are shown below:</p> |
| + | <p><center><img src="https://static.igem.org/mediawiki/2014/4/41/Membrane_TAL1.png"></img></center></p> |
| + | </br><center><small><strong>Figure 1.2.5 ssDsbA-mRFP-Lgt-TAL1-His Tag</strong></small></center></br> |
| + | <p><center><img src="https://static.igem.org/mediawiki/2014/e/e6/Free_TAL1.png"></img></center></p> |
| + | </br><center><small><strong>Figure 1.2.6 TAL1-His Tag</strong></small></center></br> |
| | | |
- | <p>In order to be used cooperatively with TAL-Protein DiRepeats (Bba_K747000 to Bba_K747095), we also design a part named ssDsbA-mRFP-Lgt-TAL adapter-His Tag (BBa_K1453000). </p> | + | <p>3. Considering our multiple-enzyme system might be applied in the following experiment, we chose pRSFDuet–1(<em>NOVAGEN</em>) as the expression vector.</p> |
| | | |
- | <p><img src="https://static.igem.org/mediawiki/2014/9/9b/Part%EF%BC%9ABBa_K1453000.png"></img></p> | + | <p id="dingweidian"><center><img src="https://static.igem.org/mediawiki/2014/d/d3/Prsf.png"></img></center></p> |
| + | </br><center><small><strong>Figure 1.2.7 Expression</strong></small></center></br> |
| + | <p>4. Similar to the cross-linked ChIP, we used formaldehyde to cross-link <strong><em>connectee</em></strong> and <strong><em>connector</em></strong></p> |
| + | <p>5. After that we did immunoprecipitation to obtain the protein-plasmid complex and digestd protein.</p> |
| + | <p>6. Finally, we used PCR to check whether there was any existing plasmid DNA.</p> |
| + | |
| | | |
- | <p>This part is consists of T1 sequence, T14 sequence and two sites for type II restriction enzyme BsmBI. When digested with BsmBI, this part can produce two sticky-ends. One can complement with the first TAL-Protein DiRepeat (Bba_K747000 to Bba_K747015) at 5’ of the sequence, while the other can complement with the sixth TAL-Protein DiRepeat (Bba_K747080 to Bba_K747095) at 3’. </p> | + | <h2 id="aconvenientpart:">Two Convenient Parts:</h2> |
| + | <p>In order to integrate TAL-Protein DiRepeats (BBa_K747000 to BBa_K747095) properly into our system, we also designed two corresponding parts; one is ssDsbA-mRFP-Lgt-TAL USB-His Tag (BBa_K1453000), the other is Lgt-TAL USB-His Tag (BBa_K1453006). </p> |
| | | |
- | <p>With the rest of TAL-Protein DiRepeats (Bba_K747016 to Bba_K747079), users can synthesize a 14-nucleotide-long Transactivator-like (TAL) protein to recogize their own <strong><em>connector</em></strong> and design their own polymerization. (Golden Gate Cloning) </p> | + | <p><center><img src="https://static.igem.org/mediawiki/2014/9/9b/Part%EF%BC%9ABBa_K1453000.png"></img></center></p></br> |
| + | </br><center><small><strong>Figure 1.2.8 ssDsbA-mRFP-Lgt-TAL USB-His Tag</strong></small></center></br> |
| + | <p><center><img src="https://static.igem.org/mediawiki/2014/0/0d/FL-TAL_USB-His_Tag.png"></img></center></p> |
| + | </br><center><small><strong>Figure 1.2.9 TAL USB-His Tag</strong></small></center></br> |
| + | <p>The TAL USB of these parts all consist of T1 sequence, T14 sequence and two sites for type II restriction enzyme BsmBI. When digested with BsmBI, these parts produce two sticky-ends. One can complement with the first TAL-Protein DiRepeat (BBa_K747000 to BBa_K747015) at 5’of the sequence, while the other can complement with the sixth TAL-Protein DiRepeat (BBa_K747080 to BBa_K747095) at 3’. </p> |
| | | |
- | <p>More details about membrane anchor, please view <a href="https://2012.igem.org/Team:SJTU-BioX-Shanghai">this page</a>.</p> | + | <p id="dingweidian12">With the rest of TAL-Protein DiRepeats (BBa_K747016 to BBa_K747079), users can synthesize a specific TAL protein to recognize their own <strong><em>connector</em></strong> and design their own polymerization. (Golden Gate Cloning) </p> |
| | | |
- | <p>More details about TAL and Golden Gate Cloning, please view <a href="https://2012.igem.org/Team:Freiburg">this page</a>.</p> | + | <p > For more details about membrane anchor, please view <a href="https://2012.igem.org/Team:SJTU-BioX-Shanghai">this page</a>.</p> |
| | | |
- | <h2 id="reference">Reference:</h2> | + | <p>For more details about TAL and Golden Gate Cloning, please view <a href="https://2012.igem.org/Team:Freiburg">this page</a>.</p> |
| | | |
- | <ol> | + | |
- | <li>GONTERO, Brigitte, María Luz CÁRDENAS, and Jacques RICARD. “A functional five‐enzyme complex of chloroplasts involved in the Calvin cycle.” European journal of biochemistry 173.2 (1988): 437–443.</li> | + | <h2 id="reference">References:</h2> |
- | <li>Bogdanove, Adam J., and Daniel F. Voytas. “TAL effector: customizable proteins for DNA targeting.” Science 333.6051 (2011): 1843–1846.</li> | + | <ol style="font-style: italic;"> |
- | <li>Deng, Dong, et al. “Structural basis for sequence-specific recognition of DNA by TAL effector.” Science 335.6069 (2012): 720–723.</li> | + | <li>Bogdanove, Adam J., and Daniel F. Voytas. “TAL effector: customizable proteins for DNA targeting.” Science 333.6051 (2011): 1843–1846.</li> |
- | <li>Pailler, J., W. Aucher, et al. (2012). “Phosphatidylglycerol: prolipoprotein diacylglyceryl transferase (Lgt) of Escherichia coli has seven transmembrane segments, and its essential residues are embedded in the membrane.” Journal of bacteriology 194(9): 2142–2151.</li> | + | <li>Deng, Dong, et al. “Structural basis for sequence-specific recognition of DNA by TAL effector.” Science 335.6069 (2012): 720–723.</li> |
- | <li>Schierle, C. F., M. Berkmen, et al. (2003). “The DsbA signal sequence directs efficient, cotranslational export of passenger proteins to the Escherichia coli periplasm via the signal recognition particle pathway.” Journal of bacteriology 185(19): 5706–5713.</li> | + | <li>Pailler, J., W. Aucher, et al. (2012). “Phosphatidylglycerol: prolipoprotein diacylglyceryl transferase (Lgt) of Escherichia coli has seven transmembrane segments, and its essential residues are embedded in the membrane.” Journal of bacteriology 194(9): 2142–2151.</li> |
- | <li>Scholze, H. & Boch, J. TAL effector are remote controls for gene activation. ‘‘Current Opinion in Microbiology’’ 14, 47–53 (2011).
</li> | + | <li>Schierle, C. F., M. Berkmen, et al. (2003). “The DsbA signal sequence directs efficient, cotranslational export of passenger proteins to the Escherichia coli periplasm via the signal recognition particle pathway.” Journal of bacteriology 185(19): 5706–5713.</li> |
- | <li>Moscou, M. J. & Bogdanove, A. J. A Simple Cipher Governs DNA Recognition by TAL Effector. ‘‘Science’’ 326, 1501–1501 (2009).</li> | + | <li>Scholze, H. & Boch, J. TAL effector are remote controls for gene activation. ‘‘Current Opinion in Microbiology’’ 14, 47–53 (2011).
</li> |
- | <li>Conrado, R. J., G. C. Wu, et al. (2012). “DNA-guided assembly of biosynthetic pathways promotes improved catalytic efficiency.” Nucleic acids research 40(4): 1879–1889.</li> | + | <li>Moscou, M. J. & Bogdanove, A. J. A Simple Cipher Governs DNA Recognition by TAL Effector. ‘‘Science’’ 326, 1501–1501 (2009).</li> |
- | <li>Yang, Zhong, et al. “Highly efficient production of soluble proteins from insoluble inclusion bodies by a two-step-denaturing and refolding method.” PloS one 6.7 (2011): e22981.</li> | + | <li>Conrado, R. J., G. C. Wu, et al. (2012). “DNA-guided assembly of biosynthetic pathways promotes improved catalytic efficiency.” Nucleic acids research 40(4): 1879–1889.</li> |
- | </ol> | + | <li>Yang, Zhong, et al. “Highly efficient production of soluble proteins from insoluble inclusion bodies by a two-step-denaturing and refolding method.” PloS one 6.7 (2011): e22981.</li> |
| + | </ol> |
| </article> | | </article> |
| </div></html> | | </div></html> |
| {{Team:SJTU-BioX-Shanghai/footer}} | | {{Team:SJTU-BioX-Shanghai/footer}} |
Basic Test
——One Connectee Binds Connector
At first, tests should be taken to check whether TAL can bind to plasmid DNA in prokaryotic system. Here we used E.coli. As mentioned in the overview, in order for the protein to bind to the plasmid(the connector), we have designed two kinds of delicate fusion proteins(the connectee). One is anchored to the cell membrane, the other is free in the cytoplasm.
Connectee:
Schematic Introduction
The connectee consists of linkers and various sections, which are shown in the schematic diagrams below.
1. TAL effector: A transcription activator-like protein
TAL effector can bind to a target sequence on DNA. The 2012 Freiburg iGEM team has offered us a whole set of 96 TAL-protein direpeat bioparts, with which we are supposed to build functional TAL proteins. Since each TAL protein is able to identify a 14-nucleotide target sequence, the first and fourteenth nucleotide being Thymine, all the 96 parts can be used to identify more than 16 million different nucleotide sequences, which makes it very convenient for us to choose a sequence for the fusion protein to bind to.
Figure 1.2.1 TAL Golden gate
2. Membrane anchor system: ssDsbA-Lgt
ssDsbA, the signal sequence of DsbA, directs the fusion protein to the periplasm. Lgt is a transmembrane protein.
The membrane anchor system has been identified by iGEM12_SJTU-BioX-Shanghai.
3. Fluorescent protein
CFP is a Cyan Fluorescent Protein which has an excitation peak at 439 nm and an emission peak at 476 nm.
YFP is a Yellow Fluorescent Protein which has an excitation peak at 514 nm and an emission peak at 527 nm.
mRFP is a Red Fluorescent Protein which has an excitation peak at 584 nm and an emission peak at 607 nm.
Connectee 1:
The first kind of connectee is designed to be tested on the membrane, which consists of three major domains: membrane anchor system(ssDsbA-Lgt), mRFP and TAL effector.
The reasons why we chose a membrane anchor are as follows.
1. TAL could bind nucleoid which may bring some negative effect on bacteria growth.
2. Membrane scaffold is a natural scaffold.
3. Exogenous proteins often form inactive inclusion bodies when expressed in the prokaryotic system.
Figure1.2.2 Connectee 1
The membrane anchor system (ssDsbA-Lgt) comes from BBa_K771000 designed by iGEM12_SJTU-BioX-Shanghai; mRFP comes from BBa_E1010 designed by Antiquity; TAL effector comes from TAL-Protein DiRepeats (BBa_K747000 to BBa_K747095) designed by iGEM12_Freiburg. We used Helical Linker to connect mRFP and Lgt, in accord with iGEM12_SJTU-BioX-Shanghai. While in consideration of any possible stereospecific blockade when TAL binds to plasmid DNA, we chose Flexible Linker to connect Lgt and TAL effector.
Connectee 2:
The second kind of connectee is designed to be tested in the cytoplasm, which only contains TAL effector.
Figure 1.2.3 Connectee 2
Connector:
As iGEM12_Freiburg designed, we can choose a TAL protein to recognize a 14-nucleotide-long sequence, TXXXXXXXXXXXXT, on the plasmid DNA.
The plasmid here is called connector.
The principle for choosing a TAL recognizing sequence:
I. It does not exist in expression vector.
II. It does not exist in the sequence of connectee.
Test Method:
1. pBluescript II KS(+) is chosen as the connector in our test for several reasons:
I. High copy number;
II.Medium length—2961bp;
III. Easy to detect whether binding a TAL may affect gene expression — through lacI & blue-white spot screening.
Figure 1.2.4 pBluescript II KS(+)
2. After checking the sequence of pBluescript II KS(+), we chose TTCGATATCAAGCT as the recognition sequence for test and designed TAL1.
Two kinds of connectee with TAL1 are shown below:
Figure 1.2.5 ssDsbA-mRFP-Lgt-TAL1-His Tag
Figure 1.2.6 TAL1-His Tag
3. Considering our multiple-enzyme system might be applied in the following experiment, we chose pRSFDuet–1(NOVAGEN) as the expression vector.
Figure 1.2.7 Expression
4. Similar to the cross-linked ChIP, we used formaldehyde to cross-link connectee and connector
5. After that we did immunoprecipitation to obtain the protein-plasmid complex and digestd protein.
6. Finally, we used PCR to check whether there was any existing plasmid DNA.
Two Convenient Parts:
In order to integrate TAL-Protein DiRepeats (BBa_K747000 to BBa_K747095) properly into our system, we also designed two corresponding parts; one is ssDsbA-mRFP-Lgt-TAL USB-His Tag (BBa_K1453000), the other is Lgt-TAL USB-His Tag (BBa_K1453006).
Figure 1.2.8 ssDsbA-mRFP-Lgt-TAL USB-His Tag
Figure 1.2.9 TAL USB-His Tag
The TAL USB of these parts all consist of T1 sequence, T14 sequence and two sites for type II restriction enzyme BsmBI. When digested with BsmBI, these parts produce two sticky-ends. One can complement with the first TAL-Protein DiRepeat (BBa_K747000 to BBa_K747015) at 5’of the sequence, while the other can complement with the sixth TAL-Protein DiRepeat (BBa_K747080 to BBa_K747095) at 3’.
With the rest of TAL-Protein DiRepeats (BBa_K747016 to BBa_K747079), users can synthesize a specific TAL protein to recognize their own connector and design their own polymerization. (Golden Gate Cloning)
For more details about membrane anchor, please view this page.
For more details about TAL and Golden Gate Cloning, please view this page.
References:
- Bogdanove, Adam J., and Daniel F. Voytas. “TAL effector: customizable proteins for DNA targeting.” Science 333.6051 (2011): 1843–1846.
- Deng, Dong, et al. “Structural basis for sequence-specific recognition of DNA by TAL effector.” Science 335.6069 (2012): 720–723.
- Pailler, J., W. Aucher, et al. (2012). “Phosphatidylglycerol: prolipoprotein diacylglyceryl transferase (Lgt) of Escherichia coli has seven transmembrane segments, and its essential residues are embedded in the membrane.” Journal of bacteriology 194(9): 2142–2151.
- Schierle, C. F., M. Berkmen, et al. (2003). “The DsbA signal sequence directs efficient, cotranslational export of passenger proteins to the Escherichia coli periplasm via the signal recognition particle pathway.” Journal of bacteriology 185(19): 5706–5713.
- Scholze, H. & Boch, J. TAL effector are remote controls for gene activation. ‘‘Current Opinion in Microbiology’’ 14, 47–53 (2011).
- Moscou, M. J. & Bogdanove, A. J. A Simple Cipher Governs DNA Recognition by TAL Effector. ‘‘Science’’ 326, 1501–1501 (2009).
- Conrado, R. J., G. C. Wu, et al. (2012). “DNA-guided assembly of biosynthetic pathways promotes improved catalytic efficiency.” Nucleic acids research 40(4): 1879–1889.
- Yang, Zhong, et al. “Highly efficient production of soluble proteins from insoluble inclusion bodies by a two-step-denaturing and refolding method.” PloS one 6.7 (2011): e22981.