Team:Warwick/Parts

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             <li> <a href = "/Team:Warwick/Project"> PROJECT </a> </li>
             <li> <a href = "/Team:Warwick/Project"> PROJECT </a> </li>
             <li> <a href = "/Team:Warwick/Team"> TEAM </a> </li>
             <li> <a href = "/Team:Warwick/Team"> TEAM </a> </li>
-
             <li> <a href = "/Team:Warwick/Parts"> <span> PARTS </span> </a> </li>
+
             <li> <a href = "/Team:Warwick/Parts"> PARTS </a> </li>
             <li> <a href = "/Team:Warwick/Modelling"> MODELLING </a> </li>
             <li> <a href = "/Team:Warwick/Modelling"> MODELLING </a> </li>
             <li> <a href = "/Team:Warwick/Notebook"> NOTEBOOK </a> </li>
             <li> <a href = "/Team:Warwick/Notebook"> NOTEBOOK </a> </li>
             <li> <a href = "/Team:Warwick/Human"> POLICY & PRACTICES </a> </li>
             <li> <a href = "/Team:Warwick/Human"> POLICY & PRACTICES </a> </li>
             <!--<li> <a href = "/Team:Warwick/Measurements"> MEASUREMENTS </a> </li>-->
             <!--<li> <a href = "/Team:Warwick/Measurements"> MEASUREMENTS </a> </li>-->
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             <li> <a href = "/Team:Warwick/Interlab"> INTERLAB </a> </li>
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             <li> <a href = "/Team:Warwick/Interlab"> <span> INTERLAB </span> </a> </li>
             <li> <a href = "/Team:Warwick/Attributions"> ATTRIBUTIONS </a> </li>
             <li> <a href = "/Team:Warwick/Attributions"> ATTRIBUTIONS </a> </li>
             <li> <a href = "/Team:Warwick/Judging"> JUDGING </a> </li>
             <li> <a href = "/Team:Warwick/Judging"> JUDGING </a> </li>
         </div>
         </div>
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<div id="secondaryMenu">
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<li> <a href = "/Team:Warwick/Parts/Aptazyme"> APTAZYME </a> </li>
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<li> <a href = "/Team:Warwick/Parts/IRES"> IRES </a> </li>
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<li> <a href = "/Team:Warwick/Parts/sirna"> siRNA </a> </li>
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<li> <a href = "/Team:Warwick/Parts/Neomycin"> NEOMYCIN </a> </li>
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<li> <a href = "/Team:Warwick/Parts/T7"> T7 </a> </li>
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<li> <a href = "/Team:Warwick/Parts/RdRp"> RdRp </a> </li>
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<li> <a href = "/Team:Warwick/Parts/P2a"> P2A </a> </li>
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<li> <a href = "/Team:Warwick/Parts/MS2"> MS2 </a> </li>
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<li> <a href = "/Team:Warwick/Parts/3promoter"> PROMOTERS </a> </li>
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<li> <a href = "/Team:Warwick/Parts/Testing"> TESTING MODULES </a> </li>
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<li> <a href = "/Team:Warwick/Parts/bb"> EXISTING BIOBRICK </a> </li>
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</div>
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             <!-- THIS IS WHERE YOUR MAIN BODY GOES -->
             <!-- THIS IS WHERE YOUR MAIN BODY GOES -->
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<p> Below is an interactive schematic of our system, hover your mouse over any part to see a brief description, and click to follow through to the Registry and see a more in depth description, and information regarding sequences and results. </p>
 
-
<div id="pop1" class="popbox">
 
-
    <h2>5' Promoter</h2>
 
-
    <p>This is derived from the 5i UTR of the HCV virus strain 1b isolate Con1. The secondary structure of this sequence acts as a binding site and initiates replication of the system by RdRp. It also includes the first 16 amino acids of the first gene of HCV as this has been shown to increase the efficacy of binding of the RdRp to the 5' promoter.</p>
 
-
</div>
 
-
<div id="pop2" class="popbox">
 
-
    <h2>IRES</h2>
 
-
    <p>This
 
-
acts as an initiation for eukaryotic
+
<div id="toc-wrapper" style='width:16%; float:left; position:fixed; text-indent:-21px;'><table id="toc" class="toc" style='width:100%; font-size:12px; color:green;'><tr><td>
-
ribosomes and begins translation of the
+
<ul>
 +
<li style="list-style-type: none;"><a href="#Introduction"><span class="tocnumber"> 1. </span><span class="toctext"> Introduction </span></a></li>
 +
<li style="list-style-type: none;"><a href="#Timeline"><span class="tocnumber"> 2. </span><span class="toctext"> The experimental timeline </span></a></li>
 +
<li style="list-style-type: none;"><a href="#Protocols"><span class="tocnumber"> 3. </span><span class="toctext"> Protocols and methodology </span></a></li>
 +
<li style="list-style-type: none;"><a href="#Measurements"><span class="tocnumber"> 4. </span><span class="toctext"> Measurements and results </span></a></li>
 +
</ul>
-
following protein sequence. We
+
</td></tr></table><script>if (window.showTocToggle) { var tocShowText = "show"; var tocHideText = "hide"; showTocToggle(); } </script></div>
-
compared two different IRESs: the
+
<div id="main_content" style='min-height:100%; width: 80%; float: right; border-left:2px black solid; padding-left:10px'>
-
classical EMCV IRES used in many papers,which has been shown to be compatible
 
-
with replicons and the NKRF IRES derived from the 3’UTR of
+
<h1 id="Introduction"> Introduction </h1><br><br>
-
the mammalian NF-kappaB repressing
+
<p>
 +
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.
 +
</p><br><br>
-
factor, which we also chose to test because during
+
<p>
 +
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>. All consent is given in accordance with the <a href="http://www.wtfpl.net/"> WTFPL </a> license!
 +
</p><br><br>
-
investigation regarding the efficacy and
+
<h1 id="Timeline"> The experimental timeline </h1><br><br>
-
strength of the EMCV IRES, the NKRF derived
+
<p> 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. </p>
-
IRES was shown to be 30-fold more  
+
<p> Therefore, 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: </p>
-
efficient however had never been previously used in Huh7.</p>
+
<table style="width:100%">
-
</div>
+
-
<div id="pop3" class="popbox">
+
-
    <h2>MS2 Box</h2>
+
-
    <p>The MS2 box acts as a binding
+
-
site for the MS2 coat protein, formed
+
<tr>
 +
<th> Date </th>
 +
<th> Protocols and measurements </th>
 +
</tr>
-
downstream in the replicon, which
+
<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>
-
represses translation hence preventing
+
<tr>
 +
<td> 06/08/2014 </td>
 +
<td> <b> Isolated/inoculated </b> one colony from each and and grew them up overnight </td>
 +
</tr>
-
exponential growth of the replicon.</p>
+
<tr>
-
</div>
+
<td> 07/08/2014 </td>
-
<div id="pop4" class="popbox">
+
<td> <b> Miniprepped </b> the overnights to secure sufficient plasmid DNA for digest </td>
-
    <h2>Neomycin Resistance</h2>
+
</tr>
-
    <p>This was included in order to select
+
-
for the cells which had been
+
<tr>
 +
<td> 08/08/2014 </td>
 +
<td> <b> Restriction digested </b> parts and linearised plasmid backbones for assembly </td>
 +
</tr>
-
successfully transfected with the
+
<tr>
 +
<td> 08/08/2014 </td>
 +
<td> <b> Ligated </b> digested parts to produce devices 2 and 3 </td>
 +
</tr>
-
replicon. This is commonly used in
+
<tr>
 +
<td> 08/08/2014 </td>
 +
<td> <b> Transformed </b> ligation products over weekend </td>
 +
</tr>
-
human cell studies and allows survival
+
<tr>
 +
<td> 11/08/2014 </td>
 +
<td> <b> Inoculated </b> colonies of ligation products </td>
 +
</tr>
-
of eukaryotic cells in the prescence of
+
<tr>
 +
<td> 12/08/2014 </td>
 +
<td> <b> Miniprepped </b> ligation products to ascertain plasmid DNA of devices 2/3 </td>
 +
</tr>
-
geneticin.</p>
+
<tr>
-
</div>
+
<td> 12/08/2014 </td>
-
<div id="pop5" class="popbox">
+
<td> <b> Gel electrophoresis </b> assay of these devices, with positive results </td>
-
    <h2>Aptazyme</h2>
+
</tr>
-
    <p>This is an RNA enzyme which self-
+
-
cleaves in the presence of
+
<tr>
 +
<td> ... </td>
 +
<td> Hiatus period (focusing on other work!) </td>
 +
</tr>
-
theophylline. Theophylline is not
+
<tr>
 +
<td> 20/08/2014 </td>
 +
<td> <b> Transformation </b> of all devices from plasmid DNA for measurement </td>
 +
</tr>
-
endogenous to mammalian cells
+
<tr>
 +
<td> 21/08/2014 </td>
 +
<td> <b> Inoculated </b> colonies of each device (three biological replicates for each) </td>
 +
</tr>
-
hence acts as a selective “off-switch”
+
<tr>
 +
<td> 22/08/2014 </td>
 +
<td> <b> Refreshed </b> cultures in M9 minimal media in the morning </td>
 +
</tr>
-
in an instance such as, Hepatitis C
+
<tr>
 +
<td> 22/08/2014 </td>
 +
<td> <b> Measured optical density and fluorescence </b> with plate reader overnight </td>
 +
</tr>
-
infection or tumour formation which
+
<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>
-
is exacerbated by lack of DPP-IV.</p>
+
<tr>
-
</div>
+
<td> 26/08/2014 </td>
-
<div id="pop6" class="popbox">
+
<td> <b> Inoculated </b> colonies of each device (three biological replicates for each) </td>
-
    <h2>siRNA</h2>
+
</tr>
-
    <p>This is the functional element of our replicon and shows a
+
-
potential area for RNA experimentation. This sequence was
+
<tr>
 +
<td> 27/08/2014 </td>
 +
<td> <b> Refreshed </b> cultures in M9 minimal media in the morning </td>
 +
</tr>
-
specifically designed using RNA fold and investigating the minimum
+
<tr>
 +
<td> 27/08/2014 </td>
 +
<td> <b> Measured optical density and fluorescence </b> with plate reader overnight </td>
 +
</tr>
-
free energy bond formation, in order to form a secondary structure
+
<tr>
 +
<td> 28/08/2014 </td>
 +
<td> <b> Collected </b> data from plate reader and imported into Excel for analysis </td>
 +
</tr>
-
in the positive sense that would not include a length of double
+
<tr>
 +
<td> 29/08/2014 </td>
 +
<td> The end! </td>
 +
</tr>
-
stranded RNA longer than 16 bases and hence would not be
+
</table>
-
recognised by Dicer and our replicon would remain intact for further
+
<h1 id="Protocols"> Protocols and methodology </h1>
-
replication. However, in the negative sense the secondary structure
+
<p> 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: </p> <br>
-
will form a hairpin recognised by Dicer which will result in cleavage
+
<p>
 +
<ul>
 +
<li> <a href="http://parts.igem.org/Help:Protocols/Transformation">Transformation </a> </li>
 +
<li> <a href="http://parts.igem.org/Help:3A_Assembly_Kit/Growing">Growing (which I refer to mostly as inoculation)</a> </li>
 +
<li> <a href="http://www.qiagen.com/gb/resources/resourcedetail?id=331740ca-077f-4ddd-9e5a-2083f98eebd5&lang=en">Miniprep </a></li>
 +
<li> <a href="http://parts.igem.org/Help:Protocols/Restriction_Digest">Restriction digest </a></li>
 +
<li> <a href="http://parts.igem.org/Help:Protocols/Ligation">Ligation </a></li>
 +
<li> <a href="https://2010.igem.org/Team:Newcastle/Gel_electrophoresis">Gel electrophoresis </a></li>
 +
<li> 3A Assembly basically comprises digestion and ligation </li>
 +
</ul>
 +
</p>
-
and release of the siRNA from the RNA strand allowing it to bind in a  
+
<p> To acquire the measurements necessary, we used a microplate reader, rather than a flow cytometer or something similar. To do this, we used the <a href="http://www.tecan.com/platform/apps/product/index.asp?MenuID=2575&ID=4890&Menu=1&Item=21.2.10.2"> Tecan Infinite F500 </a>, seen below. </p> <br>
-
complimentary fashion to the 3’UTR of DPP-IV mRNA and cause RNA
+
<img style = "width:70%;" align="middle" src="https://static.igem.org/mediawiki/2014/2/2f/Warwick_Interlab_Tecan.jpg"> <br>
-
silencing hence reducing the amount of DPP-IV protein present in the  
+
<p> This machine is very flexible, supporting various different plate sizes (from 6- to 1536-well plates!) and various modes of measurement (supporting absorbance wavelength from 230-1000nm and excitation wavelengths from 230-900nm). It is configured in to take the readings we were after using the following setup: </p> <br>
-
cell.  We used siRNA sequences from various papers to design this
+
<p>
 +
<ul>
 +
<li> 96 well plate (see diagram below) </li>
 +
<li> Temperature: 37 degrees Celsius </li>
 +
<li> Absorbance measurement wavelength: 600nm (OD) </li>
 +
<li> Excitation wavelength: 465nm (GFP) </li>
 +
<li> Emission wavelength: 530nm (GFP) </li>
 +
<li> GFP gain: 35 </li>
 +
</ul>
 +
</p> <br>
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hairpin.</p>
+
<p> We then ran the programme ‘GFPandOD_overnight’, for which one cycle runs like so: </p><br><br>
-
</div>
+
-
<div id="pop7" class="popbox">
+
-
    <h2>MS2 Coat Protein</h2>
+
-
    <p>This coding sequence forms
+
-
a protein which binds to the MS2 box, hairpin
+
<p>
 +
<ol>
 +
<li> Orbital shaking (amplitude 2.5mm, frequency 246.7rpm) for 200s </li>
 +
<li> Wait for 20s </li>
 +
<li> Repeat shaking for 200s </li>
 +
<li> Wait for 2s </li>
 +
<li> Repeat shaking for 200s </li>
 +
<li> Take measurements (takes about four minutes)  </li>
 +
</ol>
 +
</p> <br>
-
type structure, and represses translation of the  
+
<p> This whole process takes 15 minutes per cycle, and would run 80 cycles, coming to 20 hours, unless interrupted. Generally the optical density (that is, the growth of cells) has maxed out earlier than that. I stopped my measurements after 14 hours because growth had reached a plateau. </p><br>
-
replicon.</p>
+
<p> In terms of the protocol for preparing the samples and the plate for the reader, the first step was to concoct some M9 minimal media in which to refresh inoculated overnights before putting them into the plate reader. For 200ml of M9, combine the ingredients given, paying attention to amounts, concentrations and preparation advice given, and make up with H2O: </p><br>
-
</div>
+
-
<div id="pop8" class="popbox">
+
-
    <h2>P2A</h2>
+
-
    <p>This is derived from the Porcine
+
-
Teschovirus-1 genome and cleaves with high
+
<p>
 +
<ul>
 +
<li> 20μl, 1M CaCl2 (A) </li>
 +
<li> 400μl, 1M MgSO4 (A) </li>
 +
<li> 200μl, 10mM FeSO4¬ (F) </li>
 +
<li> 40ml, 5x M9 salts (A) </li>
 +
<li> 3.2ml, 50% glycerol  (A) </li>
 +
<li> 8ml, casamino acids 5% (A) </li>
 +
<li> 20μl, 10mg/ml thiamine (F) </li>
 +
<li> 2ml, 2mg/ml uracil (F) </li>
 +
<li> 2ml 3mg/ml leucine (F) </li>
 +
<li> 50μl, 8-10M pH 7.4 NaOH </li>
 +
</ul>
 +
</p> <br>
-
efficiency and leaving no scar following the
+
<p> <b> NB. </b> Here <b> A </b> = autoclaved and <b> F </b> = filter sterilised. </p><br>
-
formation of a polyprotein MS2 coat protein,  
+
<p> The final solution should be 7.4pH, so usually best to make up with water, and then add NaOH carefully.
 +
Now that you have M9, the protocol is quite simple: </p><br>
-
P2A and RdRp. This is required as the HCV
+
<p>
 +
<ol>
 +
<li> Inoculate three colonies (biological replicates) from each device overnight in 5ml LB broth, as given in the above ‘growing’ protocol </li>
 +
<li> We need to refresh the samples in M9. First transfer broth to falcon tubes and spin down </li>
 +
<li> Pipette away or decant the supernatant and resuspend the pellet in M9 </li>
 +
<li> Prepare 1:50 concentration of sample in 1ml of M9 </li>
 +
<li> Put refreshed samples in a shaking incubator at 37 degrees Celsius for six hours </li>
 +
<li> Retrieve samples, get a 96 well plate. Any given well should contain 10μl of sample solution and be made up to 200μl with M9. Prepare three wells per sample (technical replicates) </li>
 +
<li> In the top row, put 200μl of M9 in every well; these act as the blanks </li>
 +
<li> Where possible, avoid putting samples in wells along the boundary of the plate </li>
 +
<li> Into all unused wells, pipette 200μl of distilled water </li>
 +
<li> Put the plate into the machine and run the programme defined above </li>
 +
</ol>
 +
</p> <br>
-
genome which has one open reading frame
+
<p> Hence if my devices are D1, D2 and D3, biological replicates denoted by A, B and C, and technical replicates denoted by #1, #2 and #3, then the table below describes exactly my 96 well plate: </p><br>
-
from which one long polyprotein is produced.  
+
<img style = "width:60%;" src="https://static.igem.org/mediawiki/2014/4/4a/Warwick_Interlab_Plate.jpg"><br>
-
RdRp is the final protein in this polygenic
+
<p> The point of three technical replicates is that it is easy to spot anomalies – they are the odd ones out. With regards to optical density, most samples were ending up at around 1, so to eliminate any anomalous data I used a difference threshold of 20% of this. That is, if any member of a triplet of technical replicates was more than 0.2 away from both other values at end of play, that datum would be excluded. This test led to no exclusions. </p>
-
mRNA and hence does not have a start codon,  
+
<p> Then I considered instead the GFP measurements, and applied a similar protocol, using a 20% difference threshold specific to the datum being considered. If any overflowing was registered, as was the case for many incarnations of device 1, I considered the last full row of data, before any overflowing took place. In this fashion, I excluded from my data set D3 A #3, D3 B #1 and D3 C #1. Excluding these values prior to taking averages allowed me to then consider, by comparison of the final averages for each set of three technical replicates (unless one had been excluded), whether the inclusion of any biological replicates should be disputed. This analysis led me to discard all data related to device 3 (D3) since none of its three final averages were compatible with each other by my 20% test. I therefore held off calculating the standard deviation of these data. </p><br>
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adding a start codon could be disastrous for
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<p> Any excluded data has a red background on the <b> Excel databook </b>, which can be found <a href"https://static.igem.org/mediawiki/2014/e/ef/Warwick_Interlab_Measurements.xls"> here </a>. </p><br>
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the function of RdRp.</p>
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<h1 id="Measurements"> Measurements and results </h1>  
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    <h2>RdRp</h2>
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    <p>This is a polymerase derived from Hepatitis C Virus Strain
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1b isolate Con1 which catalyses the replication of RNA from an RNA template, reffered to as NS5B in
 
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the contact of the HCV genome. Heterelogous expression of NS5B has been achieved in insect and
 
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bacterial hosts, with RNA-dependent RNA synthesis initiated de novo (Behrens et al., 1996; Lohmann
 
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et al., 1997). Structural studies indicate the hydrophobic C-terminal 21 amino acid residues cause
 
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(Moradpour et al., 2004). In prokaryotic cells the final 21 amino acids are expendable. RdRp initiates
 
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viral RNA synthesis with nucleotidyl transfer activity found within palm motifs A and C, with several
 
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activity has been demonstrated ''in vitro'', with synthesis of full length HCV RNA (Lohmann et al.,
 
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1997; Ferrari et al., 1999). 5’ and 3’ untranslated regions (UTRs) of the HCV genome contains
 
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    <p>Each unique RNA dependent RNA polymerase (RdRP) initiates de novo replication of a RNA strand by interacting with RdRP-specific RNA sequences, henceforth called RdRP/RNA promoters. The RdRP chosen for our project is taken from the Hepatitis C virus (HCV) and it recognizes a limited set of such sequences. All of them possess a few common characteristics: an initiation cytidylate at the 3’ end, where the replication starts; and a stable secondary structure – single stranded tail and a stem of various length. For our project in addition to the indigenous to HCV RdRP promoters, we designed alternative promoter sequences previously identified by Heinz et. Al. as templates for replication by the HCV RdRP.</p>
 
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Revision as of 21:50, 17 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 . All consent is given in accordance with the WTFPL license!



The experimental timeline



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.

Therefore, 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!

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:


To acquire the measurements necessary, we used a microplate reader, rather than a flow cytometer or something similar. To do this, we used the Tecan Infinite F500 , seen below.



This machine is very flexible, supporting various different plate sizes (from 6- to 1536-well plates!) and various modes of measurement (supporting absorbance wavelength from 230-1000nm and excitation wavelengths from 230-900nm). It is configured in to take the readings we were after using the following setup:


  • 96 well plate (see diagram below)
  • Temperature: 37 degrees Celsius
  • Absorbance measurement wavelength: 600nm (OD)
  • Excitation wavelength: 465nm (GFP)
  • Emission wavelength: 530nm (GFP)
  • GFP gain: 35


We then ran the programme ‘GFPandOD_overnight’, for which one cycle runs like so:



  1. Orbital shaking (amplitude 2.5mm, frequency 246.7rpm) for 200s
  2. Wait for 20s
  3. Repeat shaking for 200s
  4. Wait for 2s
  5. Repeat shaking for 200s
  6. Take measurements (takes about four minutes)


This whole process takes 15 minutes per cycle, and would run 80 cycles, coming to 20 hours, unless interrupted. Generally the optical density (that is, the growth of cells) has maxed out earlier than that. I stopped my measurements after 14 hours because growth had reached a plateau.


In terms of the protocol for preparing the samples and the plate for the reader, the first step was to concoct some M9 minimal media in which to refresh inoculated overnights before putting them into the plate reader. For 200ml of M9, combine the ingredients given, paying attention to amounts, concentrations and preparation advice given, and make up with H2O:


  • 20μl, 1M CaCl2 (A)
  • 400μl, 1M MgSO4 (A)
  • 200μl, 10mM FeSO4¬ (F)
  • 40ml, 5x M9 salts (A)
  • 3.2ml, 50% glycerol (A)
  • 8ml, casamino acids 5% (A)
  • 20μl, 10mg/ml thiamine (F)
  • 2ml, 2mg/ml uracil (F)
  • 2ml 3mg/ml leucine (F)
  • 50μl, 8-10M pH 7.4 NaOH


NB. Here A = autoclaved and F = filter sterilised.


The final solution should be 7.4pH, so usually best to make up with water, and then add NaOH carefully. Now that you have M9, the protocol is quite simple:


  1. Inoculate three colonies (biological replicates) from each device overnight in 5ml LB broth, as given in the above ‘growing’ protocol
  2. We need to refresh the samples in M9. First transfer broth to falcon tubes and spin down
  3. Pipette away or decant the supernatant and resuspend the pellet in M9
  4. Prepare 1:50 concentration of sample in 1ml of M9
  5. Put refreshed samples in a shaking incubator at 37 degrees Celsius for six hours
  6. Retrieve samples, get a 96 well plate. Any given well should contain 10μl of sample solution and be made up to 200μl with M9. Prepare three wells per sample (technical replicates)
  7. In the top row, put 200μl of M9 in every well; these act as the blanks
  8. Where possible, avoid putting samples in wells along the boundary of the plate
  9. Into all unused wells, pipette 200μl of distilled water
  10. Put the plate into the machine and run the programme defined above


Hence if my devices are D1, D2 and D3, biological replicates denoted by A, B and C, and technical replicates denoted by #1, #2 and #3, then the table below describes exactly my 96 well plate:



The point of three technical replicates is that it is easy to spot anomalies – they are the odd ones out. With regards to optical density, most samples were ending up at around 1, so to eliminate any anomalous data I used a difference threshold of 20% of this. That is, if any member of a triplet of technical replicates was more than 0.2 away from both other values at end of play, that datum would be excluded. This test led to no exclusions.

Then I considered instead the GFP measurements, and applied a similar protocol, using a 20% difference threshold specific to the datum being considered. If any overflowing was registered, as was the case for many incarnations of device 1, I considered the last full row of data, before any overflowing took place. In this fashion, I excluded from my data set D3 A #3, D3 B #1 and D3 C #1. Excluding these values prior to taking averages allowed me to then consider, by comparison of the final averages for each set of three technical replicates (unless one had been excluded), whether the inclusion of any biological replicates should be disputed. This analysis led me to discard all data related to device 3 (D3) since none of its three final averages were compatible with each other by my 20% test. I therefore held off calculating the standard deviation of these data.


Any excluded data has a red background on the Excel databook , which can be found here .


Measurements and results