|
|
| Line 32: |
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| | <strong>[CHECK these steps with: TO / AD / LB / DT]</strong> | | <strong>[CHECK these steps with: TO / AD / LB / DT]</strong> |
| | <ol> | | <ol> |
| - | <li><a href="/Team:UCL/experiments#Genes">Genes, Plasmids, and BioBricks</a> (Genes and Parts that we're working with)</li>
| |
| | <li><a href="/Team:UCL/experiments#Expt00">Expt 00: Identifying and Confirming Necessary BioBricks from iGEM Parts Registry / Distribution</a> (ID, transform, miniprep (MP), digest, gel)</li> | | <li><a href="/Team:UCL/experiments#Expt00">Expt 00: Identifying and Confirming Necessary BioBricks from iGEM Parts Registry / Distribution</a> (ID, transform, miniprep (MP), digest, gel)</li> |
| | <li><a href="/Team:UCL/experiments#Expt01">Expt 01: Isolating and Purifying Genes from Lisbon Plasmids</a> (Transform, MP)</li> | | <li><a href="/Team:UCL/experiments#Expt01">Expt 01: Isolating and Purifying Genes from Lisbon Plasmids</a> (Transform, MP)</li> |
| Line 41: |
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| | <li><a href="/Team:UCL/experiments#Expt06">Expt 06: Characterisation- Azoreductase Assays</a> (See <a href="https://docs.google.com/document/d/1sU1CWSoi1Anydh2AkGb7ZnfdKoWgiAIJ7hat0gvctS0/edit?usp=sharing">Standard Operating Procedure</a> (SOP))</li> | | <li><a href="/Team:UCL/experiments#Expt06">Expt 06: Characterisation- Azoreductase Assays</a> (See <a href="https://docs.google.com/document/d/1sU1CWSoi1Anydh2AkGb7ZnfdKoWgiAIJ7hat0gvctS0/edit?usp=sharing">Standard Operating Procedure</a> (SOP))</li> |
| | </ol></p> | | </ol></p> |
| - | <h2><a name="Genes">Genes, Plasmids, and BioBricks</a></h2>
| |
| - | <strong>[CHECK: column width adjustment!]
| |
| - | <br>[Internal hyperlinks to different sections?]
| |
| - | <br></strong>
| |
| - | <table class="table table-striped table-bordered">
| |
| - | <!--HELP REQUIRED WITH COL WIDTH ADJUSTMENT
| |
| - | <col width="5%">
| |
| - | <col width="10%">
| |
| - | <col width="10%">
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| - | <col width="10%">
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| - | <col width="10%">
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| - | <col width="10%">
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| - | <col width="15%">
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| - | <col width="30%">
| |
| - | -->
| |
| - | <thead>
| |
| - | <tr>
| |
| - | <th> No. </th>
| |
| - | <th> ID </th>
| |
| - | <th> Name / Function </th>
| |
| - | <th> Source </th>
| |
| - | <th> State / Concentration / Date Made </th>
| |
| - | <th> Gene Size / Sequence </th>
| |
| - | <th> Initial Plasmid / Vector </th>
| |
| - | <th> Comments </th>
| |
| - | </tr>
| |
| - | </thead>
| |
| - | <tbody>
| |
| - | <!--Lisbon plasmids-->
| |
| - | <tr> <!--(1) pAzoR / BBa_K1336000 REQUIRES OVERVIEW-->
| |
| - | <td> 1 </td>
| |
| - | <td> pAzoR / <a href="/Team:UCL/biobricks#BBa_K1336000">BBa_K1336000</a> </td>
| |
| - | <td> FMN-dependent NADH-azoreductase 1 </td>
| |
| - | <td> <em>Pseudomonas putida</em> </td>
| |
| - | <td> Miniprep,
| |
| - | <br>48 ng/uL,
| |
| - | <br>15/07/17, TO. </td>
| |
| - | <td> 597 bp <strong>[Check! Not 612 bp?]</strong>,
| |
| - | <br><a href=" http://www.ncbi.nlm.nih.gov/nuccore/26986745?report=fasta&from=3267527&to=3268138">Genomic Sequence</a> </td>
| |
| - | <td> Expression vector <a href="http://www.addgene.org/browse/sequence_vdb/2549/ ">pET-21a (+) <a href=" http://biochem.web.utah.edu/hill/links/pET21.pdf">(ampicillin resistant (ampR))</a>, initially cloned between <em>NdeI</em> and <em>BamHI</em>. </td>
| |
| - | <td> Plasmid provided by Lisbon; with <a href="http://www.ncbi.nlm.nih.gov/pubmed/21655981">Paper</a>. </td>
| |
| - | </tr>
| |
| - |
| |
| - | <tr> <!--(2) p1B6 / BBa_K1336001-->
| |
| - | <td> 2 </td>
| |
| - | <td> p1B6 (AzoR 1B6) / <a href="/Team:UCL/biobricks#BBa_K1336001">BBa_K1336001</a> </td>
| |
| - | <td> Mutant: Heat-stable; FMN-dependent NADH-azoreductase 1 </td>
| |
| - | <td> <em>Pseudomonas putida</em> </td>
| |
| - | <td> Miniprep,
| |
| - | <br>68 ng/uL,
| |
| - | <br>15/07/14, TO.</td>
| |
| - | <td> 597 bp <strong>[Check! Not 612 bp?]</strong>,
| |
| - | <br><a href="">Genomic Sequence</a><strong>[Add sequence! Sent from Lisbon, see DT]</strong> </td>
| |
| - | <td> Expression vector <a href="http://www.addgene.org/browse/sequence_vdb/2549/ ">pET-21a (+) <a href=" http://biochem.web.utah.edu/hill/links/pET21.pdf">(ampR)</a>, initially cloned between <em>NdeI</em> and <em>BamHI</em>. </td>
| |
| - | <td> Plasmid provided by Lisbon; with <a href=" http://www.ncbi.nlm.nih.gov/pubmed/24475252">Paper</a>. </td>
| |
| - | </tr>
| |
| - | <tr> <!--(3) pCotA / BBa_K1336002-->
| |
| - | <td> 3 </td>
| |
| - | <td> pCotA / <a href="/Team:UCL/biobricks#BBa_K1336002">BBa_K1336002</a> </td>
| |
| - | <td> Spore Coat Protein Laccase </td>
| |
| - | <td> <em>Bacillus subtilis</em> </td>
| |
| - | <td> Miniprep,
| |
| - | <br>103 ng/uL,
| |
| - | <br>15/07/14, TO. </td>
| |
| - | <td> 1733 bp <strong>[Check! Not 1539 bp?]</strong>
| |
| - | <br><a href="">Genomic Sequence</a><strong>[Add sequence! Sent from Lisbon, see DT]</strong> </td>
| |
| - | <td> Expression vector <a href="http://www.addgene.org/browse/sequence_vdb/2549/ ">pET-21a (+) <a href=" http://biochem.web.utah.edu/hill/links/pET21.pdf">(ampR)</a>, initially cloned between <strong><em>NheI</em></strong> and <em>BamHI</em>. </td>
| |
| - | <td> Plasmid provided by Lisbon; with <a href=" http://www.itqb.unl.pt/martins/index_files/JBC2002.pdf">Paper</a>. </td>
| |
| - | </tr>
| |
| - | <tr> <!--(4) pLiP / BBa_K1336003-->
| |
| - | <td> 4 </td>
| |
| - | <td> LiP / <a href="/Team:UCL/biobricks#BBa_K1336003">BBa_K1336003</a> </td>
| |
| - | <td> Lignin Peroxidase </td>
| |
| - | <td> <em>Phanerochaete chrysosporium</em> (White-Rot Fungi) </td>
| |
| - | <td> [Being synthesised by <a href="">Gene Oracle</a>],
| |
| - | <br>X ng/uL,
| |
| - | <br>dd/mm/yy, Gene Oracle. </td>
| |
| - | <td> X bp,
| |
| - | <br><a href="">Genomic Sequence</a> </td>
| |
| - | <td> [Cloned directly into expression vector, <a href="">pSB1C3</a>, between <em>EcoRI</em> and <em>PstI</em>.] </td>
| |
| - | <td> Synthesised (for free) by <a href="">Gene Oracle</a>. Sequence from <a href="">Paper</a>. </td>
| |
| - | </tr>
| |
| - | <tr> <!--(5) pBsDyP / BBa_K1336004-->
| |
| - | <td> 5 </td>
| |
| - | <td> pBsDyP / <a href="/Team:UCL/biobricks#BBa_K1336004">BBa_K1336004</a> </td>
| |
| - | <td> Dye Decolourising Peroxidase BSU38260 </td>
| |
| - | <td> <em>Bacillus subtilis</em> </td>
| |
| - | <td> Miniprep,
| |
| - | <br>51 ng/uL,
| |
| - | <br>15/07/14, TO.</td>
| |
| - | <td> 1251 bp,
| |
| - | <br><a href="">Genomic Sequence</a><strong>[Add sequence! Sent from Lisbon, see DT]</strong> </td>
| |
| - | <td> Expression vector <a href="http://www.addgene.org/browse/sequence_vdb/2549/ ">pET-21a (+) <a href=" http://biochem.web.utah.edu/hill/links/pET21.pdf">(ampR)</a>, initially cloned between <em>NdeI</em> and <em>BamHI</em>. </td>
| |
| - | <td>Plasmid provided by Lisbon; with <a href=" http://www.ncbi.nlm.nih.gov/pubmed/23820555">Paper</a>. </td>
| |
| - | </tr>
| |
| - | <tr> <!--(6) pPpDyP / BBa_K1336005-->
| |
| - | <td> 6 </td>
| |
| - | <td> pPpDyP / <a href="/Team:UCL/biobricks#BBa_K1336005">BBa_K1336005</a> </td>
| |
| - | <td> Dye Decolourising Peroxidase PP_3248 </td>
| |
| - | <td> <em>Pseudomonas putida</em> </td>
| |
| - | <td> Miniprep,
| |
| - | <br>55 ng/uL,
| |
| - | <br>15/07/14, TO. </td>
| |
| - | <td> 861 bp <strong>[Check! Not 864 bp?]</strong>,
| |
| - | <br><a href="">Genomic Sequence</a><strong>[Add sequence! Sent from Lisbon, see DT]</strong> </td>
| |
| - | <td> Expression vector <a href="http://www.addgene.org/browse/sequence_vdb/2549/ ">pET-21a (+) <a href=" http://biochem.web.utah.edu/hill/links/pET21.pdf">(ampR)</a>, initially cloned between <em>NdeI</em> and <em>BamHI</em>. </td>
| |
| - | <td>Plasmid provided by Lisbon; with <a href=" http://www.ncbi.nlm.nih.gov/pubmed/23820555">Paper</a>. </td>
| |
| - | </tr>
| |
| - |
| |
| - | <!--Distribution BioBricks; PAGES TO BE MADE!-->
| |
| - | <tr> <!--(7) BBa_J04450-->
| |
| - | <td> 7 </td>
| |
| - | <td> <a href="http://parts.igem.org/Part:BBa_J04450">BBa_J04450</a> </td>
| |
| - | <td> RFP Coding Device</td>
| |
| - | <td> Spring 2014 BioBrick Distribution.
| |
| - | <br><a href="http://parts.igem.org/partsdb/get_part.cgi?part=BBa_J04450">Plate 4, Well 4B</a>. <strong>[Check! DT?]</strong>. </td>
| |
| - | <td> (1) Miniprep,
| |
| - | <br>333 ng/uL,
| |
| - | <br>01/07/14, TO.
| |
| - | <br>
| |
| - | <br>(2) Miniprep,
| |
| - | <br>38 ng/uL (NanoDrop, dodgy!)
| |
| - | <br>01/07/14, TO. </td>
| |
| - | <td> <a href="http://parts.igem.org/cgi/sequencing/one_blast.cgi?id=21336">1069 bp</a>,
| |
| - | <br><a href="">Genomic Sequence</a><strong>[Add sequence! Made from combined BioBricks?]</strong> </td>
| |
| - | <td> Plasmid Backbone: <a href="">pSB1C3</a>, i.e. chloramphenicol resistant (camR). </td>
| |
| - | <td> LacI-, and CAP-, sensitive; can fail if system contains LacI or CAP protein!
| |
| - | <br>RFP Coding Device contains: LacI (<a href="http://parts.igem.org/wiki/index.php/Part:BBa_R0010">R0010</a>), strong RBS (<a href="http://parts.igem.org/wiki/index.php/Part:BBa_B0034">B0034</a>), mRFP1 (<a href="http://parts.igem.org/wiki/index.php/Part:BBa_E1010">E1010</a>), and double terminator (<a href="http://parts.igem.org/wiki/index.php/Part:BBa_B0015">B0015</a> = <a href="http://parts.igem.org/wiki/index.php/Part:BBa_B0010">B0010</a>+<a href="http://parts.igem.org/wiki/index.php/Part:BBa_B0012">B0012</a>).</td>
| |
| - | </tr>
| |
| - | <tr> <!--(8) BBa_R0010-->
| |
| - | <td> 8 </td>
| |
| - | <td> <a href="http://parts.igem.org/Part:BBa_R0010">BBa_R0010</a> </td>
| |
| - | <td> Promoter (LacI regulated)</td>
| |
| - | <td> Spring 2014 BioBrick Distribution.
| |
| - | <br><a href="http://parts.igem.org/partsdb/get_part.cgi?part=BBa_R0010">Plate 3, Well 4G</a>. <strong>[Check! DT?]</strong>. </td>
| |
| - | <td> Miniprep,
| |
| - | <br>329.1 ng/uL,
| |
| - | <br>01/07/14, TO. </td>
| |
| - | <td> 200 bp,
| |
| - | <br><a href="">Genomic Sequence</a><strong>[Add sequence!]</strong> </td>
| |
| - | <td> Plasmid Backbone: <a href="">pSB1C3</a>, i.e. chloramphenicol resistant (camR).
| |
| - | <br><a href="http://beta.labgeni.us/registries/parts_registry/?part=BBa_R0010">Plasmid / Vector Map</a>. </td>
| |
| - | <td> This part is an inverting regulator sensitive to LacI and CAP. In the absence of LacI and CAP proteins, this part promotes transcription; in their presence, the part inhibits transcription. LacI can be inhibited by <a href="openwetware.org/wiki/IPTG">IPTG</a>.</td>
| |
| - | </tr>
| |
| - | <tr> <!--(9) BBa_R0011-->
| |
| - | <td> 9 </td>
| |
| - | <td> <a href="http://parts.igem.org/Part:BBa_R0011">BBa_R0011</a> </td>
| |
| - | <td> Promoter (LacI regulated, lambda pL hybrid)</td>
| |
| - | <td> Spring 2014 BioBrick Distribution.
| |
| - | <br><a href="http://parts.igem.org/partsdb/get_part.cgi?part=BBa_R0011">Plate 2, Well 6D</a> (Inconsistent sequencing!). <strong>[Check! DT? / Maybe use Spring 2013 Distribution, Plate 5, Well 6G.]</strong>. </td>
| |
| - | <td> Miniprep,
| |
| - | <br>38 ng/uL (NanoDrop, dodgy!),
| |
| - | <br>01/07/14, TO. </td>
| |
| - | <td> 55 bp,
| |
| - | <br><a href="">Genomic Sequence</a><strong>[Add sequence!]</strong> </td>
| |
| - | <td> Plasmid Backbone: <a href="">pSB1C3</a>, i.e. chloramphenicol resistant (camR).
| |
| - | <br><a href="http://beta.labgeni.us/registries/parts_registry/?part=BBa_R0011">Plasmid / Vector Map</a>. </td>
| |
| - | <td> Inverting regulatory region controlled by LacI (BBa_C0010, BBa_C0012, etc.) The PLlac 0-1 promoter is a hybrid regulatory region consisting of the promoter P(L) of phage lambda with the cI binding sites replaced with lacO1.</td>
| |
| - | </tr>
| |
| - | <tr> <!--(10) BBa_K314103-->
| |
| - | <td> 10 </td>
| |
| - | <td> <a href="http://parts.igem.org/Part:BBa_K314103">BBa_K314103</a> </td>
| |
| - | <td> Lac induced expression cassette </td>
| |
| - | <td> Spring 2014 BioBrick Distribution.
| |
| - | <br><a href="http://parts.igem.org/partsdb/get_part.cgi?part=BBa_K314103">Plate 1, Well 4D</a>. <strong>[Check! DT?]</strong>. </td>
| |
| - | <td> Miniprep,
| |
| - | <br>334 ng/uL,
| |
| - | <br>01/07/14, TO. </td>
| |
| - | <td> 1638 bp,
| |
| - | <br><a href="">Genomic Sequence</a><strong>[Add sequence!]</strong> </td>
| |
| - | <td> Plasmid Backbone: <a href="">pSB1C3</a>, i.e. chloramphenicol resistant (camR).
| |
| - | <br><a href="http://beta.labgeni.us/registries/parts_registry/?part=BBa_K314103">Plasmid / Vector Map</a>. </td>
| |
| - | <td> Lactose (<a href="openwetware.org/wiki/IPTG">IPTG</a>) inducible protein expression insert includes f1 origin (<a href="http://parts.igem.org/Part:BBa_K314110">K314110</a>), a Lac I generator (<a href="http://parts.igem.org/Part:BBa_K314111">K314111</a>), a lactose inducible promoter (<a href="http://parts.igem.org/Part:BBa_R0011">R0011</a>), and the Elowitz standard RBS (<a href="http://parts.igem.org/Part:BBa_B0034">B0034</a>).</td>
| |
| - | </tr>
| |
| - | <tr> <!--(11) BBa_K206000-->
| |
| - | <td> 11 </td>
| |
| - | <td> <a href="http://parts.igem.org/Part:BBa_K206000">BBa_K206000</a> </td>
| |
| - | <td> pBAD Strong Promoter </td>
| |
| - | <td> Spring 2014 BioBrick Distribution.
| |
| - | <br><a href="http://parts.igem.org/partsdb/get_part.cgi?part=BBa_K206000">Plate 3, Well 14A</a>. <strong>[Check! DT?]</strong>. </td>
| |
| - | <td> Miniprep,
| |
| - | <br>144 ng/uL,
| |
| - | <br>01/07/14, TO. </td>
| |
| - | <td> 130 bp,
| |
| - | <br><a href="">Genomic Sequence</a><strong>[Add sequence!]</strong> </td>
| |
| - | <td> Plasmid Backbone: <a href="">pSB1C3</a>, i.e. chloramphenicol resistant (camR).
| |
| - | <br><a href="http://beta.labgeni.us/registries/parts_registry/?part=BBa_K206000">Plasmid / Vector Map</a> </td>
| |
| - | <td> pBAD is an <em>E. coli</em> promoter that is induced by L-arabinose. In the absence of arabinose, the repressor protein AraC (<a href="http://parts.igem.org/Part:I13458">BBa_I13458</a>) binds to the AraI1 operator site of pBAD and the upstream operator site AraO2, blocking transcription; in its presence, transcription is permitted.</td>
| |
| - | </tr>
| |
| - | <tr> <!--(12) BBa_B0034-->
| |
| - | <td> 12 </td>
| |
| - | <td> <a href="http://parts.igem.org/Part:BBa_B0034">BBa_B0034</a> </td>
| |
| - | <td> RBS </td>
| |
| - | <td> Spring 2014 BioBrick Distribution.
| |
| - | <br><a href="http://parts.igem.org/partsdb/get_part.cgi?part=BBa_B0034">Plate 4, Well 1N</a>. <strong>[Check! DT?]</strong>. </td>
| |
| - | <td> Miniprep,
| |
| - | <br>156.5 ng/uL,
| |
| - | <br>01/07/14, TO. </td>
| |
| - | <td> 12 bp,
| |
| - | <br><a href="">Genomic Sequence</a><strong>[Add sequence!]</strong> </td>
| |
| - | <td> Plasmid Backbone: <strong><a href="">pSB1A2</a></strong>, i.e. ampicillin resistant (ampR).
| |
| - | <br><a href="http://beta.labgeni.us/registries/parts_registry/?part=BBa_B0034">Plasmid / Vector Map</a> </td>
| |
| - | <td>RBS based on Elowitz (1999) repressilator.</td>
| |
| - | </tr>
| |
| - | <tr> <!--(13) BBa_K518012-->
| |
| - | <td> 13 </td>
| |
| - | <td> <a href="http://parts.igem.org/Part:BBa_K518012">BBa_K518012</a> </td>
| |
| - | <td> RBS + RFP + double Terminator </td>
| |
| - | <td> Spring 2014 BioBrick Distribution.
| |
| - | <br><a href="http://parts.igem.org/partsdb/get_part.cgi?part=BBa_K518012">Plate 1, Well 18C</a>. <strong>[Check! DT?]</strong>. </td>
| |
| - | <td> (1) Miniprep,
| |
| - | <br>49 ng/uL,
| |
| - | <br>01/07/14, TO.
| |
| - | <br>
| |
| - | <br>(2) Miniprep,
| |
| - | <br> 219.2 ng/uL,
| |
| - | <br> 08/08/14, YKH. </td>
| |
| - | <td> 828 bp,
| |
| - | <br><a href="">Genomic Sequence</a><strong>[Add sequence!]</strong> </td>
| |
| - | <td> Plasmid Backbone: <a href="">pSB1C3</a>, i.e. chloramphenicol resistant (camR).
| |
| - | <br><a href="http://beta.labgeni.us/registries/parts_registry/?part=BBa_K518012">Plasmid / Vector Map</a> </td>
| |
| - | <td> This Coding Device contains: RBS.3 (medium) (<a href="http://parts.igem.org/wiki/index.php/Part:BBa_B0032">B0032</a>), mRFP1 (<a href="http://parts.igem.org/wiki/index.php/Part:BBa_E1010">E1010</a>), and double terminator (<a href="http://parts.igem.org/wiki/index.php/Part:BBa_B0014">B0014</a> = <a href="http://parts.igem.org/wiki/index.php/Part:BBa_B0012">B0012</a>+<a href="http://parts.igem.org/wiki/index.php/Part:BBa_B0011">B0011</a>).</td>
| |
| - | </tr>
| |
| - | <tr> <!--(14) BBa_B0012-->
| |
| - | <td> 14 </td>
| |
| - | <td> <strong>[CHECK: BAD PART !]</strong>
| |
| - | <br><a href="http://parts.igem.org/Part:BBa_B0012">BBa_B0012 (2)</a> </td>
| |
| - | <td> Transcription Terminator for <em>E. coli</em> RNA polymerase </td>
| |
| - | <td> Spring 2014 BioBrick Distribution.
| |
| - | <br><a href="http://parts.igem.org/partsdb/get_part.cgi?part=BBa_K518012">Plate 2, Well 2B</a>. <strong>[Check! DT?]</strong>. </td>
| |
| - | <td> Miniprep,
| |
| - | <br>128 ng/uL,
| |
| - | <br>01/07/14, TO. </td>
| |
| - | <td> 41 bp,
| |
| - | <br><a href="">Genomic Sequence</a><strong>[Add sequence!]</strong> </td>
| |
| - | <td> Plasmid Backbone: <a href="">pSB1C3</a>, i.e. chloramphenicol resistant (camR).
| |
| - | <br><a href="http://beta.labgeni.us/registries/parts_registry/?part=BBa_B0012">Plasmid / Vector Map</a> </td>
| |
| - | <td>TE from coliphage T7. <strong>This is a bad terminator (Experience: Fails)</strong>. It is a promoter in the reverse direction.</td>
| |
| - | </tr>
| |
| - | <tr> <!--(#) p-->
| |
| - | <td> # </td>
| |
| - | <td> ID </td>
| |
| - | <td> Name / Function </td>
| |
| - | <td> Source </td>
| |
| - | <td> State / Concentration / Date Made </td>
| |
| - | <td> Gene Size / Sequence </td>
| |
| - | <td> Initial Plasmid / Vector </td>
| |
| - | <td> Comments </td>
| |
| - | </tr>
| |
| - | </tbody>
| |
| - | </table>
| |
| | | | |
| | <p><h2>EXPERIMENTS. INSERT HERE.</h2> | | <p><h2>EXPERIMENTS. INSERT HERE.</h2> |
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