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<div> | <div> | ||
− | <h1> Abstract</h1> | + | <h1>Abstract</h1> |
<p> The characterization of <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1319004">BBa_K1319004</a> was carried out. This part is a TEV protease (also known as Tobaco Edge Virus nuclear inclusion a endopeptidase) that was optimized for expression in E. coli. The part contains the S219V anti-self cleavage mutation. | <p> The characterization of <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1319004">BBa_K1319004</a> was carried out. This part is a TEV protease (also known as Tobaco Edge Virus nuclear inclusion a endopeptidase) that was optimized for expression in E. coli. The part contains the S219V anti-self cleavage mutation. | ||
The TEV Protease is a highly sequence specific cysteine protease from the Tobacco Edge Virus (TEV). The protease is highly sequence specific. The consensus sequence for the cut is ENLYFQ\S with \ denoting the cleaved peptide bond. This sequence can be found in the part <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1319016">BBa_K1319016</a>. Because of its stringent sequence specificity, tobacco etch virus (TEV) protease emerges as a useful reagent with wide application in the cleavage of recombinant fusion proteins. Thus, we have chosen TEV protease(sequence from BBa_K1319004) to remove the fusion protein in our another part <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2951008">BBa_K2951008</a> and be characterized. | The TEV Protease is a highly sequence specific cysteine protease from the Tobacco Edge Virus (TEV). The protease is highly sequence specific. The consensus sequence for the cut is ENLYFQ\S with \ denoting the cleaved peptide bond. This sequence can be found in the part <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1319016">BBa_K1319016</a>. Because of its stringent sequence specificity, tobacco etch virus (TEV) protease emerges as a useful reagent with wide application in the cleavage of recombinant fusion proteins. Thus, we have chosen TEV protease(sequence from BBa_K1319004) to remove the fusion protein in our another part <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2951008">BBa_K2951008</a> and be characterized. | ||
− | </p> | + | </p> |
+ | <h2>Cloning<h2> | ||
+ | <p>After obtaining TEV protease sequence in pSB1C3 from iGEM 2019 distribution kit(plate 6 21N), transforming it into DH5αand plasmid miniprep, we obtained the amount of plasmids required for construction. To characterize the TEV protease, the chosen vector for expression is pET29a which includes a 6xHis-Tag fusion on the plasmid following the insert. At both anchors at the N- and C-terminus the restriction sites for BamHI and XhoI were introduced to clone the genes into a pET29a vector by PCR with overhang primers primer1(TEV-fwd) and primer2(TEV-rev), the PCR result is observed by running a 1.8% gel and further purified(Fig.1). The modified TEV insertion were first digested with the restriction enzymes(BamH1 and Xho1)and so was the pET29a vector. (Fig.2) | ||
+ | <table style="border:1px solid #000; background-color:#A0DAAC; "> | ||
+ | <tbody><tr> | ||
+ | <th>Name</th> | ||
+ | <th>5'-3' primers sequences</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>TEV-fwd</td> | ||
+ | <td>AATTGCGGATCCGGCGAAAGCCTGTTCAAAGGTC</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>TEV-rev</td> | ||
+ | <td>AATTGCCTCGAGACCACCTTGGGAGTAGACCAGTTCATTC</td> | ||
+ | </tr> | ||
+ | </tbody></table> | ||
+ | |||
+ | </p> | ||
</div> | </div> | ||
</div> | </div> |
Revision as of 10:09, 13 October 2019
Abstract
The characterization of BBa_K1319004 was carried out. This part is a TEV protease (also known as Tobaco Edge Virus nuclear inclusion a endopeptidase) that was optimized for expression in E. coli. The part contains the S219V anti-self cleavage mutation. The TEV Protease is a highly sequence specific cysteine protease from the Tobacco Edge Virus (TEV). The protease is highly sequence specific. The consensus sequence for the cut is ENLYFQ\S with \ denoting the cleaved peptide bond. This sequence can be found in the part BBa_K1319016. Because of its stringent sequence specificity, tobacco etch virus (TEV) protease emerges as a useful reagent with wide application in the cleavage of recombinant fusion proteins. Thus, we have chosen TEV protease(sequence from BBa_K1319004) to remove the fusion protein in our another part BBa_K2951008 and be characterized.
Cloning
After obtaining TEV protease sequence in pSB1C3 from iGEM 2019 distribution kit(plate 6 21N), transforming it into DH5αand plasmid miniprep, we obtained the amount of plasmids required for construction. To characterize the TEV protease, the chosen vector for expression is pET29a which includes a 6xHis-Tag fusion on the plasmid following the insert. At both anchors at the N- and C-terminus the restriction sites for BamHI and XhoI were introduced to clone the genes into a pET29a vector by PCR with overhang primers primer1(TEV-fwd) and primer2(TEV-rev), the PCR result is observed by running a 1.8% gel and further purified(Fig.1). The modified TEV insertion were first digested with the restriction enzymes(BamH1 and Xho1)and so was the pET29a vector. (Fig.2)
Name | 5'-3' primers sequences |
---|---|
TEV-fwd | AATTGCGGATCCGGCGAAAGCCTGTTCAAAGGTC |
TEV-rev | AATTGCCTCGAGACCACCTTGGGAGTAGACCAGTTCATTC |