Difference between revisions of "Part:BBa K2003011:Experience"

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===Results===
 
===Results===
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</br><SPAN style='font-size: 110%; font-weight: bold;'>Designing the UnaG BioBricks</SPAN>
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<p>
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<p>The IDT plasmid contains the lac-promoter so it is possible to do test expression directly with IPTG. However due to the way the final product is created, the UnaG sequence had a 50% chance to be incorporated in reverse, which was the case. Therefore different BioBrick promoters were extracted from the 2016 distribution to test for expression. Those include a medium constitutive promoter (<span style='font-size:10.0pt;font-family:Arial;mso-bidi-font-family:
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"Times New Roman";mso-bidi-theme-font:minor-bidi'><a href="https://parts.igem.org/Part:BBa_K608006"><span style='mso-bidi-font-family: "Times New Roman";color:windowtext'><u>BBa_K608006</u></span></a></span>), a strong constitutive promoter (<span style='font-size:10.0pt;
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Since summers are always too short in Sweden, there was not enough time for the Uppsala 2016 iGEM team to test this UnaG BioBrick as thoroughly as the <span
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font-family:Arial;mso-bidi-font-family:"Times New Roman";mso-bidi-theme-font:
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style='font-size:10.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";
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minor-bidi'><a href="https://parts.igem.org/Part:BBa_K880005"><span
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mso-bidi-theme-font:minor-bidi'><a
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style='mso-bidi-font-family:"Times New Roman";color:windowtext'><u>BBa_K880005</u></span></a></span>), IPTG inducible promoter (<span style='font-size:10.0pt;font-family:Arial;
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mso-bidi-font-family:"Times New Roman";mso-bidi-theme-font:minor-bidi'><a
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href="https://parts.igem.org/Part:BBa_J04500"><span style='mso-bidi-font-family:
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"Times New Roman";color:windowtext'><u>BBa_J04500</u></span></a></span>), and a T7 promoter (<span style='font-size:10.0pt;font-family:Arial;
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mso-bidi-font-family:"Times New Roman";mso-bidi-theme-font:minor-bidi'><a
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href="https://parts.igem.org/Part:BBa_K525998"><span style='mso-bidi-font-family:
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"Times New Roman";color:windowtext'><u>BBa_K525998</u></span></a></span>). All of those contain an RBS already assembled in order to speed up work. The UnaG sequence itself was excised from the IDT plasmid using EcoRI and PstI and ligated into <span style='font-size:10.0pt;font-family:Arial;mso-bidi-font-family:
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"Times New Roman";mso-bidi-theme-font:minor-bidi'><a
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href="https://parts.igem.org/Part:pSB1C3"><span style='mso-bidi-font-family:
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"Times New Roman";color:windowtext'><u>pSB1C3</u></span></a></span> backbone cut with the same set of enzymes. </p>
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<p>After successfully obtaining UnaG assembled with a variety of promoters, mutagenesis was performed in order to create several variants of the BioBrick for future use. The “stock” option contains the UnaG coding sequence. Upstream of it lies a 6xHis affinity tag, separated by an additional Serine amino acid to increase flexibility between the tag and the protein. In the registry it is annotated as <span style='font-size:10.0pt;font-family:Arial;mso-bidi-font-family:
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"Times New Roman";mso-bidi-theme-font:minor-bidi'><a
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href="https://parts.igem.org/Part:BBa_K2003010"><span style='mso-bidi-font-family:
 
href="https://parts.igem.org/Part:BBa_K2003010"><span style='mso-bidi-font-family:
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"Times New Roman";color:windowtext'><u>BBa_K2003010</u></span></a></span>. Note that this part is designed with </span><span style='font-size:10.0pt;
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"Times New Roman";color:windowtext'><u>BBa_K2003010</u></span></a></span> was tested. However, BBa_K2003011 was made by PCR mutagenesis with the <span
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font-family:Arial;mso-bidi-font-family:"Times New Roman";mso-bidi-theme-font:
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minor-bidi'><a href="https://parts.igem.org/Help:Assembly_standard_25"><span
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style='mso-bidi-font-family:"Times New Roman";color:windowtext'><u>RFC25 (Freiburg
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Standard)</u></span></a></span><span style='font-size:10.0pt;font-family:Arial;
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mso-bidi-font-family:"Times New Roman"'> prefix and suffix in mind, hence it contains additional restriction sites, that do not affect normal 3A assembly but enable in-frame protein fusion without creating stop codons. Downstream of the part lies a short Glycine-rich flexible linker to minimize the effects of possible protein fusion. However the base part still contains the double TAA codons before this flexible linker.</p>
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<p>The stop signal has been removed with PCR in <span style='font-size:10.0pt;
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font-family:Arial;mso-bidi-font-family:"Times New Roman";mso-bidi-theme-font:
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minor-bidi'><a href="https://parts.igem.org/Part:BBa_K2003011"><span
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style='mso-bidi-font-family:"Times New Roman";color:windowtext'><u>BBa_K2003011</u></span></a></span>. This is part retains all the features, including the RFC25 prefix and suffix, but now the flexible linker is properly expressed and in case of fusing to another CDS would not cause premature termination.</p>
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<p>Finally, to avoid potential interference from the 6xHistidine affinity tag, <span
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style='font-size:10.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";
 
style='font-size:10.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";
 
mso-bidi-theme-font:minor-bidi'><a
 
mso-bidi-theme-font:minor-bidi'><a
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href="https://parts.igem.org/Part:BBa_K2003012"><span style='mso-bidi-font-family:
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href="https://parts.igem.org/Part:BBa_K2003010"><span style='mso-bidi-font-family:
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"Times New Roman";color:windowtext'><u>BBa_K2003012</u></span></a></span> was created based on <span style='font-size:10.0pt;
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"Times New Roman";color:windowtext'><u>BBa_K2003010</u></span></a></span> BioBrick as template. BBa_K2003011 is therefore expected  to function similarly to BB_K2003010 during expression. </p>
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font-family:Arial;mso-bidi-font-family:"Times New Roman";mso-bidi-theme-font:
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minor-bidi'><a href="https://parts.igem.org/Part:BBa_K2003011"><span
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style='mso-bidi-font-family:"Times New Roman";color:windowtext'><u>BBa_K2003011</u></span></a></span>. In this part, the six amino acids were removed (again through PCR), as well as the Serine linker in between. This part has been designed for studying the properties of the protein in vivo, since the high positive charge of the affinity tag could interfere with its function or localization. At each step of the experiments, sequencing was performed using the iGEM standard VF2 and VR verification primers to ensure CDS integrity and especially to avoid introduced frameshift mutations.</p>
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</br><SPAN style='font-size: 110%; font-weight: bold;'>HEADING</SPAN>
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<p>PARAGRAPH</p>
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</br><SPAN style='font-size: 110%; font-weight: bold;'>HEADING</SPAN>
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<p>PARAGRAPH</p>
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</br><SPAN style='font-size: 110%; font-weight: bold;'>HEADING</SPAN>
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<p>PARAGRAPH</p>
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</br><SPAN style='font-size: 110%; font-weight: bold;'>HEADING</SPAN>
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Latest revision as of 14:25, 19 October 2016


This experience page is provided so that any user may enter their experience using this part.
Please enter how you used this part and how it worked out.

Results

Since summers are always too short in Sweden, there was not enough time for the Uppsala 2016 iGEM team to test this UnaG BioBrick as thoroughly as the BBa_K2003010 was tested. However, BBa_K2003011 was made by PCR mutagenesis with the BBa_K2003010 BioBrick as template. BBa_K2003011 is therefore expected to function similarly to BB_K2003010 during expression.

User Reviews

UNIQ64d5dc786740e95c-partinfo-00000001-QINU UNIQ64d5dc786740e95c-partinfo-00000002-QINU