Difference between revisions of "Team:Newcastle/Notebook/LabBook"

 
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display: none
 
display: none
 
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<header class="jumbotron hero-spacer">
 
<header class="jumbotron hero-spacer">
 
<h1>Lab Book</h1>
 
<h1>Lab Book</h1>
−
<p>All experiments and lab work are fully documented here. Click on the buttons below to filter the logs.</p>
+
<p>All experiments and lab work are fully documented here. Click on the buttons below to filter the entries.</p>
 
<p>
 
<p>
 
<button class="dropbtn" onclick="hide('entry')">Hide All</button>
 
<button class="dropbtn" onclick="hide('entry')">Hide All</button>
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<p>
 
<p>
−
Month:
+
<b>Month:</b>
−
<button class="dropbtn" onclick="show('April')">April</button>
+
<button class="dropbtn filter" onclick="hide('May'); show('April'); hide('June')">April</button>
−
<button class="dropbtn" onclick="show('May')">May</button>
+
<button class="dropbtn filter" onclick="show('May'); hide('April'); hide('June')">May</button>
−
<button class="dropbtn" onclick="show('June')">June</button>
+
<button class="dropbtn filter" onclick="hide('May'); hide('April'); show('June')">June</button>
−
<button class="dropbtn" onclick="show('July')">July</button>
+
<button class="dropbtn filter" onclick="show('July')">July</button>
−
<button class="dropbtn" onclick="show('August')">August</button>
+
<button class="dropbtn filter" onclick="show('August')">August</button>
 
</p>
 
</p>
  
 
<p>
 
<p>
−
Aspect of the project:
+
<b>Aspect of the project:</b>
−
<button class="dropbtn" onclick="hide('SynPro'); hide('LHRA'); show('CFPS'); show('titles')">CFPS</button>
+
<button class="dropbtn filter" onclick="show('Gen'); hide('SynPro'); hide('LHRA'); hide('CFPS'); show('titles')">General Lab Work</button>
−
<button class="dropbtn" onclick="show('SynPro'); hide('LHRA'); hide('CFPS'); show('titles')">Synthetic Promoters</button>
+
<button class="dropbtn filter" onclick="hide('Gen'); hide('SynPro'); hide('LHRA'); show('CFPS'); show('titles')">CFPS</button>
−
<button class="dropbtn" onclick="hide('SynPro'); show('LHRA'); hide('CFPS'); show('titles')">Liquid Handling Robots Assembly Automation</button>
+
<button class="dropbtn filter" onclick="hide('Gen'); show('SynPro'); hide('LHRA'); hide('CFPS'); show('titles')">Synthetic Promoters</button>
 +
<button class="dropbtn filter" onclick="hide('Gen'); hide('SynPro'); show('LHRA'); hide('CFPS'); show('titles')">Assembly Automation</button>
 +
<button class="dropbtn filter" onclick="">Biosensor Modules</button>
 +
<button class="dropbtn filter" onclick="">Interlab Study</button>
 
 
  
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</header>
 
</header>
 +
 +
 +
  
 
<!-- ---------------------------------------------------------------------------------------------- -->
 
<!-- ---------------------------------------------------------------------------------------------- -->
 +
<!-- WC 03/04/17 -->
 +
 +
  
  
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<li>Insert work here</li>
 
<li>Insert work here</li>
 
</div>
 
</div>
−
<div class="entry GenCBCB April">
+
<div class="entry Gen April">
−
<h4>General (CBCB)</h4>
+
<h4>General</h4>
 
<li>Insert work here</li>
 
<li>Insert work here</li>
 
</div>
 
</div>
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<div class="entry CFPS April">
 
<div class="entry CFPS April">
 
<h4>CFPS:</h4>
 
<h4>CFPS:</h4>
−
<li>Transformed competent DH5α <i>E. coli</i> cells with <a href="http://parts.igem.org/Part:BBa_J04450">BBa_J04450</a> in a <a href="http://parts.igem.org/Part:pSB1AT3">pSB1AT3</a> plasmid backbone which will be used for the initial testing of <i>E coli</i> BL21-based CFPS systems <div class="img"><img src="https://static.igem.org/mediawiki/2017/5/58/T--Newcastle--BB_pSB1AT3_J04450.png"></div></li>
+
<li>Transformed competent DH5α <i>E. coli</i> cells with <a href="http://parts.igem.org/Part:BBa_J04450">BBa_J04450</a> in a <a href="http://parts.igem.org/Part:pSB1AT3">pSB1AT3</a> plasmid backbone which will be used for the initial testing of <i>E. coli</i> BL21-based CFPS systems
−
<ul><li><a href="https://static.igem.org/mediawiki/2017/1/1f/T--Newcastle--ecoli_transformation_bb.pdf" width="5px">Transformation protocol</a></li><li>Performed by Bradley Brown</li></ul>
+
<div class="img" style="height:300px"><a href="http://beta.labgeni.us/registries/parts_registry/?part=BBa_J04450"><img src="https://static.igem.org/mediawiki/2017/5/58/T--Newcastle--BB_pSB1AT3_J04450.png" height="100%"></a></div>
 +
 +
<ul>
 +
<li>Selected for plasmid by growth on ampicilin LB agar plates</li>
 +
<li><a href="https://static.igem.org/mediawiki/2017/1/1f/T--Newcastle--ecoli_transformation_bb.pdf" width="5px">Transformation protocol</a></li>
 +
</ul></li>
 
</div>
 
</div>
 
</ul>
 
</ul>
Line 92: Line 137:
 
<div class="entry CFPS April">
 
<div class="entry CFPS April">
 
<h4>CFPS:</h4>
 
<h4>CFPS:</h4>
−
<li>Insert work here</li>
+
<li>Confirmed successful transformation of plasmid into cells by visualisation of red colonies on the LB agar AMP plate:
 +
<div class="img" style="height:300px"><img src="https://static.igem.org/mediawiki/2017/9/92/T--Newcastle--BB_pSB1AT3_J04450_plate.png" height="100%"></div>
 
</div>
 
</div>
 
</ul>
 
</ul>
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</ul>
 
</ul>
 +
 +
 +
 +
 +
 +
 +
 +
<!-- ---------------------------------------------------------------------------------------------- -->
 +
<!-- WC 10/04/17 -->
 +
 +
 +
 +
 +
  
 
<div class="entry April titles" id="April_2">
 
<div class="entry April titles" id="April_2">
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<div class="entry CFPS April">
 
<div class="entry CFPS April">
 
<h4>CFPS:</h4>
 
<h4>CFPS:</h4>
−
<li>Insert work here
+
<li>Four Overnight cultures of J04450 in pSB1AT3 from transformation plates from 05/06/17
−
<div style="background-color:red; width:200px; height:150px">Image</div></li>
+
<ul>
 +
<li>5 mL LB broth with 100 μg/mL ampicilin, incubated overnight at 37<sup>o</sup>C and 250 RPM</li>
 +
</ul>
 +
</li>
 
</div>
 
</div>
 
</ul>
 
</ul>
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<h4>11/04/17:</h4></div>
 
<h4>11/04/17:</h4></div>
 
<ul>
 
<ul>
−
 
+
<div class="entry CFPS April">
 +
<h4>CFPS:</h4>
 +
<li>Used 1 mL of overnight culture from 10/04/17 to prepare glycerol stock of DH5α with pSB1AT3-J04450 plasmid
 +
<ul>
 +
<li><a href="">Glycerol stocks protocol</a></li>
 +
</ul>
 +
</li>
 +
<li>Used remaining overnight cultures for miniprep to extract pSB1AT3-J04450 plasmid DNA
 +
<ul>
 +
<li><a href="">Qiagen MiniPrep Kit Protocol</a></li>
 +
</ul>
 +
</li>
 +
</div>
 
</ul>
 
</ul>
  
Line 123: Line 198:
 
<h4>12/04/17:</h4></div>
 
<h4>12/04/17:</h4></div>
 
<ul>
 
<ul>
−
 
+
<div class="entry CFPS April">
 +
<h4>CFPS:</h4>
 +
<li>Measured concentration of minipreped pSB1AT3-J04450 plasmid DNA using a nanodrop
 +
<ul>
 +
<li><b>BB_MP1:</b> 153.2 μg/mL</li>
 +
<li><b>BB_MP2:</b> 172.1 μg/mL</li>
 +
<li><b>BB_MP3:</b> 165.9 μg/mL</li>
 +
<li><b>BB_MP4:</b> 172.1 μg/mL</li>
 +
</ul>
 +
</li>
 +
</div>
 
</ul>
 
</ul>
  
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</ul>
 
</ul>
 +
 +
 +
<!-- ---------------------------------------------------------------------------------------------- -->
 +
<!-- WC 17/04/17 -->
 +
 +
 +
<div class="entry April titles" id="April_3">
 +
<hr><h4>Week Commencing: 17/04/17</h4></div>
 +
<div class="entry April titles" id="170417">
 +
<h4>17/04/17:</h4></div>
 +
<ul>
 +
 +
</ul>
 +
 +
<div class="entry April titles" id="180417">
 +
<h4>18/04/17:</h4></div>
 +
<ul>
 +
 +
</ul>
 +
 +
<div class="entry April titles" id="190417">
 +
<h4>19/04/17:</h4></div>
 +
<ul>
 +
 +
</ul>
 +
 +
<div class="entry April titles" id="200417">
 +
<h4>20/04/17:</h4></div>
 +
<ul>
 +
 +
</ul>
 +
 +
<div class="entry April titles" id="210417">
 +
<h4>21/04/17:</h4></div>
 +
<ul>
 +
<div class="entry CFPS April">
 +
<h4>CFPS:</h4>
 +
<li>Tested S30 cell-free circular <i>E. coli</i> commercial kit from promega
 +
<ul>
 +
<table border="2" style="font-size:16px">
 +
<th>Reaction ID</th><th>Plasmid DNA used</th><th>Stock DNA concentration <br />(ng/μL)</th><th>DNA amount in reaction (μg)</th><th>DNA volume in reaction (μL)</th>
 +
<tr>
 +
<td>Neg1</td><td>N/a</td><td>0</td><td>0</td><td>0</td>
 +
</tr>
 +
<tr>
 +
<td>Neg2</td><td>N/a</td><td>0</td><td>0</td><td>0</td>
 +
</tr>
 +
<tr>
 +
<td>Neg3</td><td>N/a</td><td>0</td><td>0</td><td>0</td>
 +
</tr>
 +
<tr>
 +
<td>Luc1</td><td>pBEST-Luc (Promega)</td><td>1000</td><td>2</td><td>2</td>
 +
</tr>
 +
<tr>
 +
<td>Luc2</td><td>pBEST-Luc (Promega)</td><td>1000</td><td>2</td><td>2</td>
 +
</tr>
 +
<tr>
 +
<td>RFPa1</td><td>pSB1AT3-mRFP1 (BB_MP2)</td><td>172.1</td><td>1</td><td>5.81</td>
 +
</tr>
 +
<tr>
 +
<td>RFPa2</td><td>pSB1AT3-mRFP1 (BB_MP2)</td><td>172.1</td><td>1</td><td>5.81</td>
 +
</tr>
 +
<tr>
 +
<td>RFPa3</td><td>pSB1AT3-mRFP1 (BB_MP2)</td><td>172.1</td><td>1</td><td>5.81</td>
 +
</tr>
 +
<tr>
 +
<td>RFPb1</td><td>pSB1AT3-mRFP1 (BB_MP4)</td><td>172.1</td><td>1.7</td><td>9.88</td>
 +
</tr>
 +
<tr>
 +
<td>RFPb2</td><td>pSB1AT3-mRFP1 (BB_MP4)</td><td>172.1</td><td>1.7</td><td>9.88</td>
 +
</tr>
 +
<tr>
 +
<td>RFPb3</td><td>pSB1AT3-mRFP1 (BB_MP4)</td><td>172.1</td><td>1.7</td><td>9.88</td>
 +
</tr>
 +
</table>
 +
<li>Reactions were prepared according to the <a href="https://www.promega.co.uk/-/media/files/resources/protocols/technical-bulletins/0/e-coli-s30-extract-system-for-circular-dna-protocol.pdf?la=en">manufactuer's protocol</a></li>
 +
<li>Reactions were made up to 50 μL and pipetted into wells of a flat-bottom black 96 well plate</li>
 +
<li>Reactions were incubated in a plate reader (BMG Fluostar Optima) for ~4 hours with fluorescence readings every 15 mins - gain was set at 900</li>
 +
<li>For the Luc1 & 2 and Neg1 & 2 reactions, after four hours the plate was removed and 50 μL Luciferase assay reagent was added to the reactions before luminescence was measured by the plate reader
 +
<ul>
 +
<li>The gain was calculated as 40% of Luc1</li>
 +
</ul>
 +
</li>
 +
<li>Raw results can be accessed <a href="#">here</a></li>
 +
<table>
 +
<tr>
 +
<td><div class="img" style="height:600px; border-style:solid"><img src="https://static.igem.org/mediawiki/2017/b/b9/T--Newcastle--BB_S30_initial_test_210417.png" height="70%">
 +
<p><font class="image_title">Time course of commercial S30 circular CFPS systems expressing pSB1AT3-mRFP1:</font> <font class="image_caption">Data points are averages of triplicate repeats and error bars show plus and minus standard error. Red circles are reactions with no plasmid DNA, black squares are reactions with 1 μg plasmid DNA, and blue triangles are reactions with 1.7 μg plasmid DNA.</font>
 +
</p>
 +
</div>
 +
</td>
 +
<td>
 +
<div class="img" style="height:600px; border-style:solid"><img src="https://static.igem.org/mediawiki/2017/b/b6/T--Newcastle--BB_Initial_com_testing_luc.png" height="70%">
 +
<p><font class="image_title">End point luminescence readings for commercial S30 cicular CFPS systems expressing pBEST-Luc:</font> <font class="image_caption">Data points are averages of duplicate reactions and error bars show plus and minus standard error. 'Neg' reactions contained no plasmid DNA. 'Luc' reactions contained 2 μg pBEST-Luc. Readings were taken after 3.75 hours incubation at 37<sup>o</sup>C. The Y axis is in log<sub>10</sub>.</font>
 +
</p>
 +
</div>
 +
</td>
 +
</tr>
 +
</table>
 +
</ul>
 +
</li>
 +
</div>
 +
</ul>
 +
 +
 +
 +
 +
 +
 +
 +
 +
<!-- ---------------------------------------------------------------------------------------------- -->
 +
<!-- WC 24/04/17 -->
 +
 +
 +
 +
 +
 +
 +
 +
<div class="entry April titles" id="April_4">
 +
<hr><h4>Week Commencing: 17/04/17</h4></div>
 +
<div class="entry April titles" id="240417">
 +
<h4>24/04/17:</h4></div>
 +
<ul>
 +
<div class="entry CFPS April">
 +
<h4>CFPS:</h4>
 +
<li>Streak plate of <i>E. coli</i> BL21 cells from glycerol stock onto plain LB agar plate</li>
 +
<li>Obtainined a streak plate of <i>B. subtilis</i> 168 from a lab member (Dr. Wendy Smith)</li>
 +
<li>Cells will be used to prepare CFPS cell extract</li>
 +
</div>
 +
</ul>
 +
 +
<div class="entry April titles" id="250417">
 +
<h4>25/04/17:</h4></div>
 +
<ul>
 +
<div class="entry CFPS April">
 +
<h4>CFPS:</h4>
 +
<li>Overnight cultures of <i>E. coli</i> BL21 and <i>B.subtilis</i> 168 from streak plates (24/04/17) were prepared
 +
<ul>
 +
<li>5 mL LB agar broth, incubated overnight at 37<sup>o</sup>C and 250 RPM</li>
 +
</ul>
 +
</li>
 +
<li>Prepared amino acid stock solutions
 +
<ul>
 +
<table>
 +
<tr>
 +
<td>
 +
<li>The following amino acids were dissolved in 250 μL KOH (5 M):
 +
<ul>
 +
<li>Aspartic Acid: 125 mg</li>
 +
<li>Cysteine: 74.5 mg</li>
 +
<li>Glutamic acid: 134.5 mg</li>
 +
<li>Glutamine: 91.5 mg</li>
 +
<li>Glycine: 79 mg</li>
 +
<li>Histidine: 127.5 mg</li>
 +
<li>Isoleucine: 123.5 mg</li>
 +
<li>Leucine: 83.5 mg</li>
 +
<li>Lysine: 87.5 mg</li>
 +
<li>Methionine: 93 mg</li>
 +
<li>Phenylalanine: 71 mg</li>
 +
<li>Proline: 112 mg</li>
 +
<li>Serine: 104.5 mg</li>
 +
<li>Threonine: 114.5 mg</li>
 +
<li>Tryptophan: 85 mg</li>
 +
<li>Tyrosine: 108.5 mg</li>
 +
<li>Valine: 76 mg</li>
 +
</ul>
 +
</li>
 +
<li>The following amino acids were dissolved in 500 μL KOH (5 M):
 +
<ul>
 +
<li>Alanine: 182 mg</li>
 +
<li>Arginine: 202 mg</li>
 +
<li>Asparagine-monohydrate: 282 mg</li>
 +
</ul>
 +
</li>
 +
<li>Note that some amino acids may not fully dissolve and stay in solution</li>
 +
<li>Stock solutions were stored at -20<sup>o</sup>C</li>
 +
</td>
 +
<td>
 +
<div class="img" style="width:300px; border-style:solid; padding: 0px 0px 0px 0px;"><img style="vertical-align:center" src="https://static.igem.org/mediawiki/2017/c/c8/T--Newcastle--BB_stock_amino_acids.png" width="100%">
 +
</div>
 +
</td>
 +
</tr>
 +
</table>
 +
</li>
 +
</div>
 +
</ul>
 +
 +
<div class="entry April titles" id="26417">
 +
<h4>26/04/17:</h4></div>
 +
<ul>
 +
<div class="entry CFPS April">
 +
<h4>CFPS:</h4>
 +
<li>Used 1 mL of overnight culture from 25/04/17 to prepare glycerol stocks of <i>E. coli</i> BL21 and <i>B. subtilis</i> 168
 +
<ul>
 +
<li><a href="">Glycerol stocks protocol</a></li>
 +
</ul>
 +
</li>
 +
<li>Growth curves for <i>E. coli</i> BL21 and <i>B. subtilis</i> 168
 +
<ul>
 +
<li>Inoculated 2 * 200 mL LB broth in a sterile 2 L conical flask with 2 mL of <i>E. coli</i> BL21 overnight culture in one flask, and <i>B. subtilis</i> 168 in the other</i>
 +
<li>Incubated at 37<sup>o</sup>C and 250 RPM</li>
 +
<li>OD<sub>600</sub> readings with a Fenway spectrophotometer - plain LB broth used as a blank
 +
<ul>
 +
<li>Readings at 0 mins, 60 mins, then every 30 mins</li>
 +
</ul>
 +
</li>
 +
<table>
 +
<tr>
 +
<td>
 +
<table border="2" style="font-size:16px">
 +
<th rowspan=2>Time (mins)</th><th colspan=2 style="text-align:center">Strain</th>
 +
<tr>
 +
<td><i>E. coli</i> BL21</td><td><i>B. subtilis</i> 168</td>
 +
</tr>
 +
<tr>
 +
<td>0</td><td>0.043</td><td>0.034</td>
 +
</tr>
 +
<tr>
 +
<td>60</td><td>0.109</td><td>0.045</td>
 +
</tr>
 +
<tr>
 +
<td>90</td><td>0.305</td><td>0.068</td>
 +
</tr>
 +
<tr>
 +
<td>120</td><td>0.636</td><td>0.136</td>
 +
</tr>
 +
<tr>
 +
<td>150</td><td>1.090</td><td>0.282</td>
 +
</tr>
 +
<tr>
 +
<td>180</td><td>1.575</td><td>0.677</td>
 +
</tr>
 +
<tr>
 +
<td>210</td><td>1.887</td><td>1.198</td>
 +
</tr>
 +
<tr>
 +
<td>245</td><td>2.228</td><td>1.601</td>
 +
</tr>
 +
<tr>
 +
<td>270</td><td>2.372</td><td>1.843</td>
 +
</tr>
 +
<tr>
 +
<td>300</td><td>2.592</td><td>2.099</td>
 +
</tr>
 +
<tr>
 +
<td>330</td><td>2.687</td><td>2.114</td>
 +
</tr>
 +
</table>
 +
</td>
 +
<td>
 +
<div class="img" style="width:600px; border-style:solid"><img src="https://static.igem.org/mediawiki/2017/0/0e/T--Newcastle--BB_BL21_168_growth_curves_260417.png" width="100%">
 +
<p><font class="image_title">Growth curves for <i>E. coli</i> BL21 and <i>B. subtilis</i> 168</font> <font class="image_caption"></font>
 +
</p>
 +
</div>
 +
</td>
 +
</tr>
 +
<tr>
 +
<td colspan=2><center>
 +
<div class="img" style="width:500px; border-style:solid; padding: 0px 0px 0px 0px;"><img src="https://static.igem.org/mediawiki/2017/0/03/T--Newcastle--BB_cuvettes_growth_curve.jpg" width="100%">
 +
</div></center>
 +
</td>
 +
</tr>
 +
</table>
 +
 +
 +
</div>
 +
</ul>
 +
 +
<div class="entry April titles" id="270417">
 +
<h4>27/04/17:</h4></div>
 +
<ul>
 +
 +
</ul>
 +
 +
<div class="entry April titles" id="280417">
 +
<h4>28/04/17:</h4></div>
 +
<ul>
 +
 +
</ul>
 +
 +
 +
 +
<!-- ---------------------------------------------------------------------------------------------- -->
 +
<!-- WC 01/05/17 -->
 +
 +
 +
 +
 +
 +
 +
 +
<div class="entry May titles">
 +
<hr><h3>May:</h3></div>
 +
 +
 +
<div class="entry May titles" id="May_1">
 +
<hr><h4>Week Commencing: 01/05/17</h4></div>
 +
<div class="entry May titles" id="010517">
 +
<h4>01/05/17:</h4></div>
 +
<ul>
 +
 +
</ul>
 +
 +
<div class="entry May titles" id="020517">
 +
<h4>02/05/17:</h4></div>
 +
<ul>
 +
 +
</ul>
 +
 +
<div class="entry May titles" id="030517">
 +
<h4>03/05/17:</h4></div>
 +
<ul>
 +
<div class="entry CFPS May">
 +
<h4>CFPS:</h4>
 +
<li>prepared 400 mL of CFPS wash buffer (14 mM magnesium glutamate, 60 mM potassium glutamate, 10 mM TRIS-acetate)
 +
<ul>
 +
<li><a href="#">CFPS wash buffer preparation Protocol</a></li>
 +
</ul>
 +
</li>
 +
</div>
 +
</ul>
 +
 +
<div class="entry May titles" id="040517">
 +
<h4>04/05/17:</h4></div>
 +
<ul>
 +
 +
</ul>
 +
 +
<div class="entry May titles" id="050517">
 +
<h4>05/05/17:</h4></div>
 +
<ul>
 +
 +
</ul>
 +
 +
 +
 +
 +
<!-- ---------------------------------------------------------------------------------------------- -->
 +
<!-- WC 08/05/17 -->
 +
 +
 +
 +
 +
 +
 +
<div class="entry May titles" id="May_2">
 +
<hr><h4>Week Commencing: 08/05/17</h4></div>
 +
<div class="entry May titles" id="080517">
 +
<h4>08/05/17:</h4></div>
 +
<ul>
 +
<div class="entry CFPS May">
 +
<h4>CFPS:</h4>
 +
<li>prepared overnight cultures (5 mL LB broth) of <i>E. coli</i> BL21 and <i>B. subtilis</i> 168</li>
 +
</div>
 +
</ul>
 +
 +
<div class="entry May titles" id="090517">
 +
<h4>09/05/17:</h4></div>
 +
<ul>
 +
<div class="entry CFPS May">
 +
<table>
 +
<tr>
 +
<td>
 +
<h4>CFPS:</h4>
 +
<li><b>CFPS cell extract preparation</b>
 +
<ul>
 +
 +
 +
<li>Inoculated 200 mL LB broth in a 2 L conical flask with 2 mL of <i>E. coli</i> culture (from 09/09/17)</li>
 +
<li>Inoculated another 200 mL LB broth in a 2 L conical flask with 2 mL of <i>B. subtilis</i> culture</li>
 +
<li>Shake incubated the flasks at 37<sup>o</sup>C until late exponential phase was reached (OD<sub>600</sub> ~ 2.5 for <i>E. coli</i> and ~2.0 for </i>B. subtilis</i></li>
 +
<li>Cells were harvested by centrifugation at 4,500 RPM and 4<sup>o</sup>C for 20 mins</li>
 +
</td>
 +
<td>
 +
<div class="img" style="width:250px; border-style:solid; padding: 0px 0px 0px 0px"><img src="https://static.igem.org/mediawiki/2017/d/dd/T--Newcastle--BB_cell_extract_harvest_1.jpg" width="100%">
 +
</div>
 +
</td>
 +
</tr>
 +
 +
 +
</ul>
 +
</li>
 +
</table>
 +
</div>
 +
 +
 +
</ul>
 +
 +
<div class="entry May titles" id="100517">
 +
<h4>10/05/17:</h4></div>
 +
<ul>
 +
 +
</ul>
 +
 +
<div class="entry May titles" id="110517">
 +
<h4>11/05/17:</h4></div>
 +
<ul>
 +
 +
</ul>
 +
 +
<div class="entry May titles" id="120517">
 +
<h4>12/05/17:</h4></div>
 +
<ul>
 +
 +
</ul>
 +
 +
 +
 +
 +
<!-- ---------------------------------------------------------------------------------------------- -->
 +
<!-- WC 15/05/17 -->
 +
 +
 +
 +
 +
<div class="entry May titles" id="May_3">
 +
<hr><h4>Week Commencing: 15/05/17</h4></div>
 +
<div class="entry May titles" id="150517">
 +
<h4>15/05/17:</h4></div>
 +
<ul>
 +
 +
</ul>
 +
 +
<div class="entry May titles" id="160517">
 +
<h4>16/05/17:</h4></div>
 +
<ul>
 +
 +
</ul>
 +
 +
<div class="entry May titles" id="170517">
 +
<h4>17/05/17:</h4></div>
 +
<ul>
 +
 +
</ul>
 +
 +
<div class="entry May titles" id="180517">
 +
<h4>18/05/17:</h4></div>
 +
<ul>
 +
 +
</ul>
 +
 +
<div class="entry May titles" id="190517">
 +
<h4>19/05/17:</h4></div>
 +
<ul>
 +
 +
</ul>
 +
 +
 +
 +
 +
 +
<!-- ---------------------------------------------------------------------------------------------- -->
 +
<!-- WC 22/05/17 -->
 +
 +
 +
 +
 +
<div class="entry May titles" id="May_4">
 +
<hr><h4>Week Commencing: 22/05/17</h4></div>
 +
<div class="entry May titles" id="220517">
 +
<h4>22/05/17:</h4></div>
 +
<ul>
 +
 +
</ul>
 +
 +
<div class="entry May titles" id="230517">
 +
<h4>23/05/17:</h4></div>
 +
<ul>
 +
 +
</ul>
 +
 +
<div class="entry May titles" id="240517">
 +
<h4>24/05/17:</h4></div>
 +
<ul>
 +
 +
</ul>
 +
 +
<div class="entry May titles" id="250517">
 +
<h4>25/05/17:</h4></div>
 +
<ul>
 +
 +
</ul>
 +
 +
<div class="entry May titles" id="260517">
 +
<h4>26/05/17:</h4></div>
 +
<ul>
 +
 +
</ul>
 +
 +
 +
 +
 +
 +
 +
 +
<!-- ---------------------------------------------------------------------------------------------- -->
 +
<!-- WC 29/05/17 -->
 +
 +
 +
 +
 +
<div class="entry May titles" id="May_5">
 +
<hr><h4>Week Commencing: 29/05/17</h4></div>
 +
<div class="entry May titles" id="290517">
 +
<h4>29/05/17:</h4></div>
 +
<ul>
 +
 +
</ul>
 +
 +
<div class="entry May titles" id="300517">
 +
<h4>30/05/17:</h4></div>
 +
<ul>
 +
 +
</ul>
 +
 +
<div class="entry May titles" id="310517">
 +
<h4>31/05/17:</h4></div>
 +
<ul>
 +
 +
</ul>
 +
 +
 +
 +
 +
 +
<!-- ---------------------------------------------------------------------------------------------- -->
 +
<!-- WC 29/05/17 (June)-->
 +
 +
 +
 +
 +
 +
 +
 +
<div class="entry June titles">
 +
<hr><h3>June:</h3></div>
 +
 +
 +
<div class="entry June titles" id="June_1">
 +
<hr><h4>Week Commencing: 29/05/17</h4></div>
 +
<div class="entry June titles" id="010617">
 +
<h4>01/06/17:</h4></div>
 +
<ul>
 +
 +
</ul>
 +
 +
<div class="entry June titles" id="020617">
 +
<h4>02/06/17:</h4></div>
 +
<ul>
 +
 +
</ul>
 +
 +
 +
 +
 
 
  
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</div>
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                    <a href="https://2017.igem.org/Team:Newcastle/Notebook" class="btn btn-primary" style="color: white">Back to Notebooks</a>
 +
                </div>
 
</body>
 
</body>
  

Latest revision as of 10:41, 14 June 2017

Our wiki is currently under construction, please bear with us while we make it functional

Lab Book

All experiments and lab work are fully documented here. Click on the buttons below to filter the entries.

Month:

Aspect of the project:

April:

Week Commencing: 03/04/17

03/04/17:

    Synthetic Promoters:

  • Insert work here

    • Liquid Handling Robots Assembly Automation

  • Insert work here

    • General

  • Insert work here

04/04/17:

05/04/17:

    CFPS:

  • Transformed competent DH5α E. coli cells with BBa_J04450 in a pSB1AT3 plasmid backbone which will be used for the initial testing of E. coli BL21-based CFPS systems

06/04/17:

    CFPS:

  • Confirmed successful transformation of plasmid into cells by visualisation of red colonies on the LB agar AMP plate:

07/04/17:

Week Commencing: 10/04/17

10/04/17:

    CFPS:

  • Four Overnight cultures of J04450 in pSB1AT3 from transformation plates from 05/06/17
    • 5 mL LB broth with 100 μg/mL ampicilin, incubated overnight at 37oC and 250 RPM

11/04/17:

12/04/17:

    CFPS:

  • Measured concentration of minipreped pSB1AT3-J04450 plasmid DNA using a nanodrop
    • BB_MP1: 153.2 μg/mL
    • BB_MP2: 172.1 μg/mL
    • BB_MP3: 165.9 μg/mL
    • BB_MP4: 172.1 μg/mL

13/04/17:

14/04/17:

Week Commencing: 17/04/17

17/04/17:

18/04/17:

19/04/17:

20/04/17:

21/04/17:

    CFPS:

  • Tested S30 cell-free circular E. coli commercial kit from promega
      Reaction IDPlasmid DNA usedStock DNA concentration
      (ng/μL)      		DNA amount in reaction (μg)DNA volume in reaction (μL)
      Neg1N/a000
      Neg2N/a000
      Neg3N/a000
      Luc1pBEST-Luc (Promega)100022
      Luc2pBEST-Luc (Promega)100022
      RFPa1pSB1AT3-mRFP1 (BB_MP2)172.115.81
      RFPa2pSB1AT3-mRFP1 (BB_MP2)172.115.81
      RFPa3pSB1AT3-mRFP1 (BB_MP2)172.115.81
      RFPb1pSB1AT3-mRFP1 (BB_MP4)172.11.79.88
      RFPb2pSB1AT3-mRFP1 (BB_MP4)172.11.79.88
      RFPb3pSB1AT3-mRFP1 (BB_MP4)172.11.79.88
    • Reactions were prepared according to the manufactuer's protocol
    • Reactions were made up to 50 μL and pipetted into wells of a flat-bottom black 96 well plate
    • Reactions were incubated in a plate reader (BMG Fluostar Optima) for ~4 hours with fluorescence readings every 15 mins - gain was set at 900
    • For the Luc1 & 2 and Neg1 & 2 reactions, after four hours the plate was removed and 50 μL Luciferase assay reagent was added to the reactions before luminescence was measured by the plate reader
      • The gain was calculated as 40% of Luc1
    • Raw results can be accessed here

      • Time course of commercial S30 circular CFPS systems expressing pSB1AT3-mRFP1: Data points are averages of triplicate repeats and error bars show plus and minus standard error. Red circles are reactions with no plasmid DNA, black squares are reactions with 1 μg plasmid DNA, and blue triangles are reactions with 1.7 μg plasmid DNA.

      End point luminescence readings for commercial S30 cicular CFPS systems expressing pBEST-Luc: Data points are averages of duplicate reactions and error bars show plus and minus standard error. 'Neg' reactions contained no plasmid DNA. 'Luc' reactions contained 2 μg pBEST-Luc. Readings were taken after 3.75 hours incubation at 37oC. The Y axis is in log10.

Week Commencing: 17/04/17

24/04/17:

    CFPS:

  • Streak plate of E. coli BL21 cells from glycerol stock onto plain LB agar plate
  • Obtainined a streak plate of B. subtilis 168 from a lab member (Dr. Wendy Smith)
  • Cells will be used to prepare CFPS cell extract

25/04/17:

    CFPS:

  • Overnight cultures of E. coli BL21 and B.subtilis 168 from streak plates (24/04/17) were prepared
    • 5 mL LB agar broth, incubated overnight at 37oC and 250 RPM
  • Prepared amino acid stock solutions
    • The following amino acids were dissolved in 250 μL KOH (5 M):
      • Aspartic Acid: 125 mg
      • Cysteine: 74.5 mg
      • Glutamic acid: 134.5 mg
      • Glutamine: 91.5 mg
      • Glycine: 79 mg
      • Histidine: 127.5 mg
      • Isoleucine: 123.5 mg
      • Leucine: 83.5 mg
      • Lysine: 87.5 mg
      • Methionine: 93 mg
      • Phenylalanine: 71 mg
      • Proline: 112 mg
      • Serine: 104.5 mg
      • Threonine: 114.5 mg
      • Tryptophan: 85 mg
      • Tyrosine: 108.5 mg
      • Valine: 76 mg
    • The following amino acids were dissolved in 500 μL KOH (5 M):
      • Alanine: 182 mg
      • Arginine: 202 mg
      • Asparagine-monohydrate: 282 mg
    • Note that some amino acids may not fully dissolve and stay in solution
    • Stock solutions were stored at -20oC
      		•

26/04/17:

    CFPS:

  • Used 1 mL of overnight culture from 25/04/17 to prepare glycerol stocks of E. coli BL21 and B. subtilis 168
  • Growth curves for E. coli BL21 and B. subtilis 168
    • Inoculated 2 * 200 mL LB broth in a sterile 2 L conical flask with 2 mL of E. coli BL21 overnight culture in one flask, and B. subtilis 168 in the other
    • Incubated at 37oC and 250 RPM
    • OD600 readings with a Fenway spectrophotometer - plain LB broth used as a blank
      • Readings at 0 mins, 60 mins, then every 30 mins
      • Time (mins)Strain
      E. coli BL21B. subtilis 168
      00.0430.034
      600.1090.045
      900.3050.068
      1200.6360.136
      1501.0900.282
      1801.5750.677
      2101.8871.198
      2452.2281.601
      2702.3721.843
      3002.5922.099
      3302.6872.114

      Growth curves for E. coli BL21 and B. subtilis 168

27/04/17:

28/04/17:

May:

Week Commencing: 01/05/17

01/05/17:

02/05/17:

03/05/17:

04/05/17:

05/05/17:

Week Commencing: 08/05/17

08/05/17:

    CFPS:

  • prepared overnight cultures (5 mL LB broth) of E. coli BL21 and B. subtilis 168

09/05/17:

    CFPS:

  • CFPS cell extract preparation
    • Inoculated 200 mL LB broth in a 2 L conical flask with 2 mL of E. coli culture (from 09/09/17)
    • Inoculated another 200 mL LB broth in a 2 L conical flask with 2 mL of B. subtilis culture
    • Shake incubated the flasks at 37oC until late exponential phase was reached (OD600 ~ 2.5 for E. coli and ~2.0 for B. subtilis
    • Cells were harvested by centrifugation at 4,500 RPM and 4oC for 20 mins
    		•

10/05/17:

11/05/17:

12/05/17:

Week Commencing: 15/05/17

15/05/17:

16/05/17:

17/05/17:

18/05/17:

19/05/17:

Week Commencing: 22/05/17

22/05/17:

23/05/17:

24/05/17:

25/05/17:

26/05/17:

Week Commencing: 29/05/17

29/05/17:

30/05/17:

31/05/17:

June:

Week Commencing: 29/05/17

01/06/17:

02/06/17: