Team:Warwick/Interlab

From 2014.igem.org

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<h1 id="Introduction"> Introduction </h1> <br>
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<h1 id="Introduction"> Introduction </h1><br><br>
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It is suggested that a team conduct the interlab study as a preamble to the main event. However, we are the first example of an iGEM team at Warwick, and coalesced rather late in the day. Hence we decided just to dedicate three members of the team to pursuing it in parallel to our other endeavours, as a side quest. It was primarily undertaken by <b> Dan Goss </b>, <b> Waqar Yousaf </b> and <b> Chelsey Tye </b>, with support from advisers <b> Will Rostain </b> and <b> Sian Davies </b>.
It is suggested that a team conduct the interlab study as a preamble to the main event. However, we are the first example of an iGEM team at Warwick, and coalesced rather late in the day. Hence we decided just to dedicate three members of the team to pursuing it in parallel to our other endeavours, as a side quest. It was primarily undertaken by <b> Dan Goss </b>, <b> Waqar Yousaf </b> and <b> Chelsey Tye </b>, with support from advisers <b> Will Rostain </b> and <b> Sian Davies </b>.
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<h1 id="Timeline"> The experimental timeline </h1><br><br>
 +
 
 +
Over a period of about 1 month, we engineered the three devices from the brief via transformation, miniprep, digestion, ligation, and all the protocols you would expect (more on that below). What follows is a timetable of our experimental work in the wet lab:
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<table style="width:100%">
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<tr>
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<th> Date </th>
 +
<th> Protocols and measurements </th>
 +
</tr>
 +
 
 +
<tr>
 +
<td> 05/08/2014 </td>
 +
<td> <b> Transformed </b> all parts from kit plates, including an <a href="http://parts.igem.org/Part:BBa_J04450"> RFP-producing control </a> </td>
 +
</tr>
 +
 
 +
<tr>
 +
<td> 06/08/2014 </td>
 +
<td> <b> Isolated/inoculated </b> one colony from each and and grew them up overnight </td>
 +
</tr>
 +
 
 +
<tr>
 +
<td> 07/08/2014 </td>
 +
<td> <b> Miniprepped </b> the overnights to secure sufficient plasmid DNA for digest </td>
 +
</tr>
 +
 
 +
<tr>
 +
<td> 08/08/2014 </td>
 +
<td> <b> Restriction digested </b> parts and linearised plasmid backbones for assembly </td>
 +
</tr>
 +
 
 +
<tr>
 +
<td> 08/08/2014 </td>
 +
<td> <b> Ligated </b> digested parts to produce devices 2 and 3 </td>
 +
</tr>
 +
 
 +
<tr>
 +
<td> 08/08/2014 </td>
 +
<td> <b> Transformed </b> ligation products over weekend </td>
 +
</tr>
 +
 
 +
<tr>
 +
<td> 11/08/2014 </td>
 +
<td> <b> Inoculated </b> colonies of ligation products </td>
 +
</tr>
 +
 
 +
<tr>
 +
<td> 12/08/2014 </td>
 +
<td> <b> Miniprepped </b> ligation products to ascertain plasmid DNA of devices 2/3 </td>
 +
</tr>
 +
 
 +
<tr>
 +
<td> 12/08/2014 </td>
 +
<td> <b> Gel electrophoresis </b> assay of these devices, with positive results </td>
 +
</tr>
 +
 
 +
<tr>
 +
<td> ... </td>
 +
<td> Hiatus period (focusing on other work!) </td>
 +
</tr>
 +
 
 +
<tr>
 +
<td> 20/08/2014 </td>
 +
<td> <b> Transformation </b> of all devices from plasmid DNA for measurement </td>
 +
</tr>
 +
 
 +
<tr>
 +
<td> 21/08/2014 </td>
 +
<td> <b> Inoculated </b> colonies of each device (three biological replicates for each) </td>
 +
</tr>
 +
 
 +
<tr>
 +
<td> 22/08/2014 </td>
 +
<td> <b> Refreshed </b> cultures in M9 minimal media in the morning </td>
 +
</tr>
 +
 
 +
<tr>
 +
<td> 22/08/2014 </td>
 +
<td> <b> Measured optical density and fluorescence </b> with plate reader overnight </td>
 +
</tr>
 +
 
 +
<tr>
 +
<td> 25/08/2014 </td>
 +
<td> <b> Collected </b> data from plate reader, but gain was set to 100 so had to repeat </td>
 +
</tr>
 +
 
 +
<tr>
 +
<td> 26/08/2014 </td>
 +
<td> <b> Inoculated </b> colonies of each device (three biological replicates for each) </td>
 +
</tr>
 +
 
 +
<tr>
 +
<td> 27/08/2014 </td>
 +
<td> <b> Refreshed </b> cultures in M9 minimal media in the morning </td>
 +
</tr>
 +
 
 +
<tr>
 +
<td> 27/08/2014 </td>
 +
<td> <b> Measured optical density and fluorescence </b> with plate reader overnight </td>
 +
</tr>
-
<h1 id="Timeline"> The experimental timeline </h1>  
+
<tr>
 +
<td> 28/08/2014 </td>
 +
<td> <b> Collected </b> data from plate reader and imported into Excel for analysis </td>
 +
</tr>
-
Timeline of when protocols were run and measurements were taken is listed below
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<tr>
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<td> 29/08/2014 </td>
 +
<td> The end! </td>
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</tr>
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</table>
  <li>05/08/2014 Transformed all parts from kit plates, including an RFP-producing control</li>
  <li>05/08/2014 Transformed all parts from kit plates, including an RFP-producing control</li>

Revision as of 10:38, 16 October 2014

Introduction



The interlab study is an attempt by iGEM HQ to conduct a comparative analysis of the different methods employed by the varied and diverse teams internationally to arrive at useful data. A key problem in science is ascertaining absolute measurements; there is no point in one measuring the fluorescence of some given part, only to arrive at arbitrary units whose meaning to other scientists is near zero. Standards must be set, in order to embue our results with any useful meaning. The interlab study is a step towards that ultimate goal of blanket standardisation in the synthetic biological context.

It is suggested that a team conduct the interlab study as a preamble to the main event. However, we are the first example of an iGEM team at Warwick, and coalesced rather late in the day. Hence we decided just to dedicate three members of the team to pursuing it in parallel to our other endeavours, as a side quest. It was primarily undertaken by Dan Goss , Waqar Yousaf and Chelsey Tye , with support from advisers Will Rostain and Sian Davies .



The experimental timeline



Over a period of about 1 month, we engineered the three devices from the brief via transformation, miniprep, digestion, ligation, and all the protocols you would expect (more on that below). What follows is a timetable of our experimental work in the wet lab:
Date Protocols and measurements
05/08/2014 Transformed all parts from kit plates, including an RFP-producing control
06/08/2014 Isolated/inoculated one colony from each and and grew them up overnight
07/08/2014 Miniprepped the overnights to secure sufficient plasmid DNA for digest
08/08/2014 Restriction digested parts and linearised plasmid backbones for assembly
08/08/2014 Ligated digested parts to produce devices 2 and 3
08/08/2014 Transformed ligation products over weekend
11/08/2014 Inoculated colonies of ligation products
12/08/2014 Miniprepped ligation products to ascertain plasmid DNA of devices 2/3
12/08/2014 Gel electrophoresis assay of these devices, with positive results
... Hiatus period (focusing on other work!)
20/08/2014 Transformation of all devices from plasmid DNA for measurement
21/08/2014 Inoculated colonies of each device (three biological replicates for each)
22/08/2014 Refreshed cultures in M9 minimal media in the morning
22/08/2014 Measured optical density and fluorescence with plate reader overnight
25/08/2014 Collected data from plate reader, but gain was set to 100 so had to repeat
26/08/2014 Inoculated colonies of each device (three biological replicates for each)
27/08/2014 Refreshed cultures in M9 minimal media in the morning
27/08/2014 Measured optical density and fluorescence with plate reader overnight
28/08/2014 Collected data from plate reader and imported into Excel for analysis
29/08/2014 The end!
  • 05/08/2014 Transformed all parts from kit plates, including an RFP-producing control
  • 06/08/2014 Isolated/inoculated one colony from each and and grew them up overnight
  • 07/08/2014 Miniprepped the overnights to secure sufficient plasmid DNA for digest
  • 08/08/2014 Restriction digested parts and linearised plasmid backbones for assembly
  • 08/08/2014 Ligated digested parts to produce devices 2 and 3
  • 08/08/2014 Transformed ligation products over weekend
  • 11/08/2014 Inoculated colonies of ligation products
  • 12/08/2014 Miniprepped ligation products to ascertain plasmid DNA of devices 2/3
  • 12/08/2014 Gel electrophoresis assay of these devices, with positive results
  • Interlab hiatus period
  • 20/08/2014 Transformation of all devices from plasmid DNA for measurement
  • 21/08/2014 Inoculated colonies of each device (three biological replicates for each)
  • 22/08/2014 Refreshed cultures in M9 minimal media in the morning
  • 22/08/2014 Measured optical density and fluorescence with plate reader overnight
  • 25/08/2014 Collected data from plate reader, but gain was set to 100 so had to repeat
  • 26/08/2014 Inoculated colonies of each device (three biological replicates for each)
  • 27/08/2014 Refreshed cultures in M9 minimal media in the morning
  • 27/09/2014 Measured optical density and fluorescence with plate reader overnight
  • 28/08/2014 Collected data from plate reader and imported into Excel for analysis

    • The remit of the interlab study boils down to constructing and characterising, albeit minimally, three devices. They share a lot of similarities, and the objective is obviously not to create some wacky new form of life, but to measure well characterised and well understood parts in order to measure the measuring equipment, as it were. I will quickly describe the nature of these devices.

    Protocols and methodology

    Many of the protocols mentioned above which we used were harvested straight from the iGEM website, but we also used content from previous iGEM teams and instructions packaged with kits. The specific materials and procedures can be accessed through clicking the relevant hyperlink

  • Transformation
  • Growing (which I refer to mostly as inoculation)
  • Miniprep
  • Restriction digest
  • Ligation
  • Gel electrophoresis
  • 3A Assembly basically comprises digestion and ligation

    • Measurements and results

    Retrieved from "http://2014.igem.org/Team:Warwick/Interlab"