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                    <h1>Internal Tools</h1>
+
                                                                <h1>1. Interlab Study</h1>
−
                        <h2>Number of mutations and mutated sequences</h2>
+
                                                                <hr>
−
                        <p>Expected number of mutations in a single sequence:
+
                                                                 
−
                            $$p_{m} = \frac{N_{mutations}}{L_{Sequence}} = N_{generations} \cdot r_{mutation} = t_{total} \cdot \Phi \cdot r_{mutation}$$
+
<p>As a first step to contribute to this year's iGEM competition we decided to participate in iGEM's fourth International Interlaboratory Study along with many other teams from around the world. This study is organized by iGEM's measurement committee in an effort to establish standardized, reliable and repeatable measurement tools for the iGEM community and the synthetic biology community as a whole. This year's iGEM InterLab study is about establishing a standardized protocol for the measurement of GFP using a plate reader. To start things off we needed a plate reader that is qualified to measure GFP fluorescence. Namely, Tecan Infinite M200 Pro plate reader. Additionally, we needed competent <i>E.coli</i> DH5&#945;. These were prepared from glycerol stocks. Together with the material iGEM had provided we were ready for work. Throughout our experiments, we tested 8 plasmids (2 controls and 6 test devices) by measuring the OD<sub>600</sub> and the fluorescence of the cells carrying the constructs.
−
                        </p>
+
The workflow can be separated into four segments. The first segment is the transformation of all plasmids into competent  DH5&#945; cells.  
−
                        <p>The expected share of sequences that shows at least one mutation in \(L_{Sequence}\) bp is the probability that \(L_{sequence}\) basepairs stay unchanged when \(\frac{N_{mutations}}{L_{Sequence}}\) mutations are expected:
+
                                                            <h2>1.1 Transformation of 8 test devices into competent DH5&#945; cells</h2>
−
                        $$p_{M} = \frac{N_{mutated}}{N_{Sequences}} = 1 - p(N_{mutations}=0) = 1 - (1-p_{m})^{L_{Sequence}} $$
+
                                                               
−
                        </p>
+
<p>The transformation was successful for all the plasmids and resulted in a sufficient amount of colonies on all plates. Therefore, two colonies from each plate were picked and inoculated in LB medium containing chloramphenicol over night for approximately 16-18 hours at 37°C and 220 rpm.</p>
−
                        <p>With this equation we can also calculate the number of sequences \(N_{Sequences}\) that have to be sequenced in order to find a mutated one with a probability of \(p(N_{mutated} > 0)\).
+
−
                        $$ N_{Sequences} = \frac{p(N_{mutated} > 0)}{p_{M}} $$
+
−
                        </p>
+
−
                        <p>The probability to find at least one mutated sequence under the given conditions is
+
−
                        $$p(N_{mutated}>0) = 1 - (1-p_{M})^{N_{sequences}}$$
+
−
                        which gives
+
−
                        $$N_{Sequences} = \frac{ln(1-p(N_{mutated}>0))}{ln(1-p_{M})}$$
+
−
                        </p>
+
−
                        <p>Set \(\Phi\) to zero to use the number of generations for the calculation. If \(\Phi\) and the number of generations are given, \(\Phi\) is used.</p>
+
−
                        <p>Consider \(L_{Sequence}\) as the number of basepairs that is expected to be mutated. If half of the sequence you are interested in, is highly conserved choose a lower \(L_{Sequence}\).</p>
+
  
 +
                                                        <h2>1.2 Measurement of LUDOX-HS40 OD600 Reference Point</h2>
 +
                                                               
 +
<p>The second segment is the measurement of the LUDOX-HS40 solution to obtain a ratiometric conversion factor which allows us to transform the absorbance data into a standard OD<sub>600</sub> measurement. This is necessary because plate readers in general do not have a 1 cm path length which means that measurements are volume dependent. By doing so, we obtained following data.</p>
  
−
              <form action="" id="form1">
+
                                                                <div class="container-fluid">
−
                <section>
+
                                                                <div class="row"  style="padding-top: 30px;padding-bottom: 30px;">
−
                <h2>Get your mutations</h2>
+
                                                                <div class="col-lg-offset-4 col-lg-4 col-md-offset-4 col-md-4 col-sm-offset-4 col-sm-4 col-xs-offset-4 col-xs-4">
−
                <ul class="input-list style-1 clearfix">
+
−
                  <li>
+
−
                    <label>
+
−
                          Mutation rate \(r_{mutation} [bp/generation]\)  
+
−
                        <input type="number" pattern="[0-9]+([,\.][0-9]+)?" id="mr" value="0" lang='en-150' step="any">
+
−
                        </label>
+
−
                  </li>
+
−
                <li>
+
−
                        <label>
+
−
                        Flow troughLagoon \(\Phi_{lagoon} [Volumes/h]\)
+
−
                        <input type="number" pattern="[0-9]+([,\.][0-9]+)?" id="phi" value="0" lang='en-150' step="any">
+
−
                        </label>
+
−
                    </li>
+
−
                <li>
+
−
                        <label>
+
−
                          Total time<br> in lagoon \(t_{total} [h]\)
+
−
                        <input type="number" pattern="[0-9]+([,\.][0-9]+)?" id="tt" value="0" lang='en-150' step="any">
+
−
                        </label>
+
−
                    </li>
+
−
                <li>
+
−
                        <label>
+
−
                          Number<br> of generations \(N_{generations}\)
+
−
                        <input type="number" pattern="[0-9]+([,\.][0-9]+)?" id="ng" value="0" lang='en-150' step="any">
+
−
                        </label>
+
−
                    </li>
+
−
                    <li>
+
−
                        <label>
+
−
                          Length of sequence that can mutate \(L_{Sequence} [bp]\)<br>
+
−
                        <input type="number" pattern="[0-9]+([,\.][0-9]+)?" id="ls" value="0" lang='en-150' step="any">
+
−
                        </label>
+
−
                    </li>
+
−
                    <li>
+
−
                        <label>
+
−
                        Number of sequences that are sequenced \(N_{Sequences}\)<br>
+
−
                        <input type="number" pattern="[0-9]+([,\.][0-9]+)?" id="ns" value="0" lang='en-150' step="any">
+
−
                        </label>
+
−
                    </li>
+
−
                    <li>
+
−
                      <label>
+
−
                        Probability to get at least one mutated result  \(p(N_{mutated}>0) \)
+
−
                        <br>
+
−
                        <input type="number" pattern="[0-9]+([,\.][0-9]+)?" id="pm" value="0.9" lang='en-150' step="any">
+
−
                        </label>
+
−
                    </li>
+
−
                    <li>
+
−
                    <div style="padding-top: 30px;">
+
−
                      <input type="button" value="Submit" id="input_form" onclick="return number_mutations();">   
+
−
                        </div>
+
−
                    </li>
+
−
                </ul>
+
−
              </section>
+
−
                </form>
+
−
              </div>
+
−
                <div class="container-fluid" style="margin-top: 10px;" id="container2">
+
−
                            <p id="warnings_mutations"></p>
+
−
                            <br>
+
−
                            <p id="number_generations"></p>
+
−
                            <p>\(p_{m} =\) <span id="amount_mutations"></span> %(bp/bp).</p>
+
−
                            <p>\(N_{mutations} =\) <span id="number_mutations"></span> bp per sequence.</p>
+
−
                            <p>The share of sequences that shows at least one mutation in \(L_{Sequence}\) bp is \(p_{M}=\) <span id="amount_mutated"></span> % of sequences</p>
+
−
                            <p id="probability_positive_sequencing"></p>
+
−
                            <p id="number_to_seq"></p>
+
−
                   
+
−
                   
+
−
                <div class="container-fluid" style="margin-top: 10px;" id="container1">
+
−
                    <h2>Diff tool</h2>
+
−
                        <p>Marks differences in two strings, ignores newlines.
+
−
                    </p>
+
  
 +
                                                                  <a href="#" target="_blank" ><img src="https://static.igem.org/mediawiki/2017/4/48/T--Heidelberg--LUDOX-HS40.PNG"  alt="#"></a>
 +
                                                                    <p>Figure 1.1: Measurement of the LUDOX-HS40 and water in four replicates and calculation of the ratiometric conversion factor</p>
 +
                                                                 
  
−
              <form action="" id="form2">
+
                                                                </div>
−
                <section>
+
                                                                </div>
−
                <ul class="input-list style-1 clearfix">
+
                                                                </div>
−
                  <li>
+
                                                           
−
                    <label>
+
                                                            <p>
−
                        String 1
+
                                                                With these replicates measured, we obtained a ratiometric conversion factor of 4.30 for our specific plate reader when measured 100 &#956;L of the provided LUDOX-HS40 to water.</p>
−
                        <br>
+
                                                            <h2>1.3 Graphing a Fluorescein fluorescence standard curve</h2>     
−
                        <textarea id="str1" lang='en-150' rows=8 cols=50></textarea>
+
                                                               
−
                        </label>
+
                                                           
−
                  </li>
+
<p>In the third segment we were instructed to measure a standard curve for fluorescence of Fluorescein which can be used to correct our cell based readings to an equivalent Fluorescein concentration. Following the instructions, we could obtain following results for the serial dilution of Fluorescein in phosphate buffered saline.
−
                <li>
+
                                                            <div class="container-fluid">
−
                        <label>
+
                                                                <div class="row" style="padding-top: 30px;padding-bottom: 30px;">
−
                        String 2
+
                                                                <div class="col-lg-offset-1 col-md-offset-1 col-sm-offset-1 col-xs-offset-1 col-lg-11 col-md-11 col-sm-11 col-xs-11">
−
                        <br>
+
−
                            <textarea id="str2" lang='en-150' rows=8 cols=50></textarea>
+
−
                        </label>
+
−
                    </li>
+
−
               
+
−
                    <li>
+
−
                    <div style="padding-top: 30px;">
+
−
                      <input type="button" value="Submit" id="input_form2" onclick="return differences();">     
+
−
                        </div>
+
−
                    </li>
+
−
                </ul>
+
−
              </section>
+
−
                </form>
+
−
                    </div>
+
−
                    <div style="overflow-x:scroll !important; overflow-y:hidden !important; max-height: 100px !important;">
+
−
                        <p>Comparison:</p>
+
−
                        <p id="outstr1" style="display:inline !important; overflow: hidden !important; white-space: nowrap !important; font-family: monospace !important;"></p>
+
−
                        <br>
+
−
                        <p id="outstr2" style="display:inline !important; overflow: hidden !important; white-space: nowrap !important; font-family: monospace !important;"></p>
+
−
                    </div>
+
−
                    <p id="diffinfo"></p>
+
−
              </div>
+
−
            </div>
+
−
        </div>
+
−
    </div>
+
−
</div>
+
  
−
                                         
+
                                                                  <a href="#" target="_blank" ><img src="https://static.igem.org/mediawiki/2017/a/a0/T--Heidelberg--FluoresceinSerialDilution.PNG" alt="#"></a>
−
<script src="https://ajax.googleapis.com/ajax/libs/jquery/3.2.1/jquery.min.js"></script>
+
                                                                  <div class="caption">
−
<script src="https://cdn.plot.ly/plotly-latest.min.js"></script>
+
                                                                 
−
<script src="https://cdnjs.cloudflare.com/ajax/libs/numjs/0.13.1/numjs.js"></script>
+
                                                                    <p>Figure 1.2: Measurement of a standard curve with four replicates by serial dilution of Fluorescein in phosphate buffered saline</p>
−
<script src="http://ajax.googleapis.com/ajax/libs/jquery/1.8.2/jquery.min.js"></script>
+
                                                                  </div>
−
<script src="http://evanplaice.github.io/jquery-csv/src/jquery.csv.min.js"></script>
+
−
<script src="https://2017.igem.org/common/MathJax-2.5-latest/MathJax.js?config=TeX-AMS-MML_HTMLorMML"></script>
+
−
<script>
+
−
//example that contains everything needed
+
−
function dummy(){
+
−
    //get the values
+
−
    var mr = Number(document.getElementById('mr').value);
+
−
   
+
−
    //initialise variables
+
−
    var a_mutations = 0;
+
−
   
+
−
    //calculate
+
−
    if(phi==0){
+
−
        a_mutations = ng * mr;
+
−
    }
+
−
   
+
−
    //return the results
+
−
    $("#amount_mutated").html((Math.round(100000 * a_mutated))/1000);
+
−
    $("#number_generations").html(ng_out);
+
  
−
    //end the function
+
                                                                </div>
−
    return false;
+
                                                                </div>
−
} 
+
                                                                <div class="row" style="padding-top: 30px;padding-bottom: 30px;">
−
   
+
                                                                <div class="col-lg-offset-1 col-md-offset-1 col-sm-offset-1 col-xs-offset-1 col-lg-11 col-md-11 col-sm-11 col-xs-11">
−
function number_mutations(){
+
−
   
+
−
    //get the values
+
−
    var mr = Number(document.getElementById('mr').value);
+
−
    var phi = Number(document.getElementById('phi').value);
+
−
    var ng = Number(document.getElementById('ng').value);
+
−
    var tt = Number(document.getElementById('tt').value);
+
−
    var ls = Number(document.getElementById('ls').value);
+
−
    var ns = Number(document.getElementById('ns').value);
+
−
    var pm = Number(document.getElementById('pm').value);
+
−
 
+
−
    //initialise variables
+
−
    var a_mutations = 0;
+
−
    var n_mutations = '?';
+
−
    var a_mutated = '?';
+
−
    var ng_out = "";
+
−
    var number_to_seq_out = "";
+
−
    var number_to_seq = 0;
+
−
    var probability_positive_sequencing = ''
+
−
    var warnings = ""
+
−
   
+
−
    //Check, if given values make sense:
+
−
   
+
−
    if(mr>1){
+
−
        warnings += "A mutation rate that is above one mutation per basepair, per generation does not make sense. 100 % should be enough, right? ";
+
−
        mr = 1;
+
−
    }
+
−
   
+
−
    if(pm>1){
+
−
        warnings += "A probability of 100 % should be enough, right?. ";
+
−
        pm = 1;
+
−
    }
+
−
   
+
−
    //calculate
+
−
    if(phi*tt==0){
+
−
        a_mutations = 1 - Math.pow((1 - mr), ng);
+
−
        ng_out = "";
+
−
    }
+
−
    else{
+
−
        a_mutations = 1 - Math.pow((1 - mr), tt*phi);
+
−
        ng = tt*phi;
+
−
        ng_out = "The conditions result in " + ng + " generations.";
+
−
    }
+
−
   
+
−
    n_mutations = ls * a_mutations;
+
−
    a_mutated = 1 - Math.pow((1-a_mutations), ls);
+
−
   
+
−
    if(ns!=0){
+
−
        probability_positive_sequencing = ns*a_mutated;
+
−
        $("#probability_positive_sequencing").html("Sequencing " + ns + " sequences results in a " + ((Math.round(probability_positive_sequencing*100))/100) + "% probabiltiy of finding at least one mutated sequence.");
+
−
    }
+
−
    else{
+
−
        $("#probability_positive_sequencing").html("");
+
−
    }
+
−
   
+
−
    if(pm!=0){
+
−
        number_to_seq = Math.max(1, Math.round((Math.log(1-pm))/(Math.log(1-a_mutated))));
+
−
        if(isNaN(number_to_seq)){
+
−
            if(pm==1){
+
−
                number_to_seq_out = "To be 100 % sure, sequence infinitely many sequences.";
+
−
            }
+
−
            else{
+
−
                number_to_seq_out = "Unluckily we were unable to calculate the number of sequences to sequence to have a " + (pm*100) + "% probability for a positive result.";
+
−
            }
+
−
        }
+
−
        else{
+
−
            number_to_seq_out = "Sequence " + number_to_seq + " clones to have a " + (pm*100) + " % probability to have at least one mutated under the results.";
+
−
        }
+
−
    }
+
−
   
+
−
    //return the results
+
−
    $("#amount_mutations").html((Math.round(1000000 * a_mutations))/10000);
+
−
    $("#number_mutations").html((Math.round(n_mutations*100))/100);
+
−
    $("#amount_mutated").html((Math.round(100000 * a_mutated))/1000);
+
−
    $("#number_generations").html(ng_out);
+
−
    $("#number_to_seq").html(number_to_seq_out);
+
−
    $("#warnings_mutations").html(warnings);
+
  
 +
                                                                  <a href="#" target="_blank" ><img src="https://static.igem.org/mediawiki/2017/5/57/T--Heidelberg--FluoresceinStandardCurve.png"  alt="#"></a>
 +
                                                                  <div class="caption">
 +
                                                                   
 +
                                                                    <p>Figure 1.3: Fluorescence is plotted against the Fluorescein concentration</p>
 +
                                                                  </div>
  
−
    //end the function
+
                                                                </div>
−
    return false;
+
                                                                </div>
−
}
+
                                                                <div class="row" style="padding-top: 30px;padding-bottom: 30px;"> 
 +
                                                                <div class="col-lg-offset-1 col-md-offset-1 col-sm-offset-1 col-xs-offset-1 col-lg-11 col-md-11 col-sm-11 col-xs-11">
  
−
function glucose(){
+
                                                                  <a href="#" target="_blank" ><img src="https://static.igem.org/mediawiki/2017/f/fa/T--Heidelberg--FluoresceinStandardCurveLogScale.png"  alt="#"></a>
−
    //get the values
+
                                                                  <div class="caption">
−
    var initial_concentration = Number(document.getElementById('initial_concentration').value);
+
                                                                   
−
    var minimal_concentration = Number(document.getElementById('minimal_concentration').value);
+
                                                                    <p>Figure 1.4: Fluorescence is plotted against the Fluorescein concentration on a logarithmic scale</p>
−
    var final_ecoli_concentration = Number(document.getElementById('final_ecoli_concentration').value);
+
                                                                  </div>
−
    var beginning_ecoli_concentration = Number(document.getElementById('beginning_ecoli_concentration').value);
+
−
    var pace = Boolean(document.getElementById('pace').value);
+
−
    var doubling_time = Number(document.getElementById('doubling_time').value);
+
−
    var time_induction = Number(document.getElementById('time_induction').value);
+
−
    var flow_lagoon = Number(document.getElementById('flow_lagoon').value);
+
−
   
+
−
    //initialise variables
+
−
    var time_turbidostat = 0;
+
−
    var time_lagoon = 0;
+
−
   
+
−
    //calculate
+
−
    if(pace){
+
−
        phi = Math.LN2/doubling_time; //vol/h, not L/h!
+
−
        time_turbidostat = 1/phi; // as in the esvelt supplementaries?
+
−
        time_lagoon = 1/flow_lagoon;
+
−
    }
+
−
   
+
−
    //return the results
+
−
    $("#amount_mutated").html((Math.round(100000 * a_mutated))/1000);
+
−
    $("#number_generations").html(ng_out);
+
  
−
    //end the function
+
                                                                </div>
−
    return false;
+
                                                                </div>
−
} 
+
                                                                </div>
−
   
+
−
function differences(){
+
−
    //get the values
+
−
    var str1 = document.getElementById('str1').value;
+
−
    var str2 = document.getElementById('str2').value;
+
−
str1 = str1.replace(/(\r\n|\n|\r)/gm,"");
+
−
str2 = str2.replace(/(\r\n|\n|\r)/gm,"");
+
−
   
+
−
    //initialise variables
+
−
    var diff = [];
+
−
    var outstr1 = "";
+
−
    var outstr2 = "";
+
−
    var diffs = 0;
+
−
    var lendiffs = 0;
+
−
    var sharedlen = 0;
+
−
    var diffinfo = "";
+
−
    //calculate
+
−
    var i = 0;
+
−
    while(i < str1.length){
+
−
      if(i==str2.length){
+
−
          break;
+
−
      }
+
−
        if(str1[i]==str2[i]){
+
−
            outstr1 += str1[i];
+
−
            outstr2 += str2[i];
+
−
        }
+
−
        else{
+
−
            diffs += 1;
+
−
            outstr1 += '<span style="color: red">' + str1[i] + '</span>';
+
−
            outstr2 += '<span style="color: red">' + str2[i] + '</span>';
+
−
        }
+
−
        i++; 
+
−
    }
+
−
    sharedlen = i;
+
−
    while(i < str1.length){
+
−
        outstr1 = outstr1 + '<span style="color: blue">' + str1[i] + '</span>';
+
−
        i++;
+
−
        lendiffs++;
+
−
    }
+
−
    while(i < str2.length){
+
−
        outstr2 = outstr2 + '<span style="color: blue">' + str2[i] + '</span>';
+
−
        i++;
+
−
        lendiffs++;
+
−
    }
+
−
    diffinfo = "The strings are different for " + diffs + "  from " + sharedlen + " positions (" + (0.01*Math.round(10000*(diffs/sharedlen))) + "%)and their length differs by " + lendiffs + " positions.";
+
−
   
+
−
    //return the results
+
−
    $("#outstr1").html(outstr1);
+
−
    $("#outstr2").html(outstr2);
+
−
    $("#diffinfo").html(diffinfo);
+
  
−
    //end the function
+
                                                           
−
    return false;
+
<p>These data give us an approximate linear regression line when plotted in a logarithmic scale and show an expected exponential course when plotted in a linear form.</p>
−
}
+
                                                            <h2>1.4 Measurement of OD<sub>600</sub> and Fluorescence of Transformed Cells</h2>
 +
                                                         
 +
<p>In the last of the four segments the OD<sub>600</sub> and the fluorescence of the transformed cells were measured according to the protocol after 0, 2, 4 and 6 hours. Measurements of all these time points gave us the following data and calculations which were conducted with the values we obtained from the standard curves and the reference point. </p>
 +
                                                           
 +
<div class="container-fluid">
 +
                                                                <div class="row" style="padding-top: 30px;padding-bottom: 30px;"> 
 +
                                                                <div class="col-lg-offset-1 col-md-offset-1 col-sm-offset-1 col-xs-offset-1 col-lg-11 col-md-11 col-sm-11 col-xs-11">
  
 +
                                                                  <a href="#" target="_blank" ><img src="https://static.igem.org/mediawiki/2017/2/2e/T--Heidelberg--RawPlateReadings.PNG"  alt="#"></a>
 +
                                                                  <div class="caption">
 +
                                                                   
 +
                                                                   
 +
<p>Figure 1.5: Raw data obtained from measurements</p>
 +
                                                                  </div>
 +
 +
                                                                </div>
 +
                                                                </div>
 +
                                                                <div class="row" style="padding-top: 30px;padding-bottom: 30px;"> 
 +
                                                                <div class="col-lg-offset-1 col-md-offset-1 col-sm-offset-1 col-xs-offset-1 col-lg-11 col-md-11 col-sm-11 col-xs-11">
 +
 +
                                                                  <a href="#" target="_blank" ><img src="https://static.igem.org/mediawiki/2017/8/8e/T--Heidelberg--ExperimentalValues0-2.PNG"  alt="#"></a>
 +
                                                                  <div class="caption">
 +
                                                                   
 +
                                                                   
 +
<p>Figure 1.6: Raw data and measurements for the 0 and 2 hour time points</p>
 +
                                                                  </div>
 +
 +
                                                                </div>
 +
                                                                </div>
 +
                                                                <div class="row" style="padding-top: 30px;padding-bottom: 30px;"> 
 +
                                                                <div class="col-lg-offset-1 col-md-offset-1 col-sm-offset-1 col-xs-offset-1 col-lg-11 col-md-11 col-sm-11 col-xs-11">
 +
 +
                                                                  <a href="#" target="_blank" ><img src="https://static.igem.org/mediawiki/2017/4/4d/T--Heidelberg--ExperimentalValues4-6.PNG"  alt="#"></a>
 +
                                                                  <div class="caption">
 +
                                                                   
 +
                                                                   
 +
<p>Figure 1.7: Raw data and measurements for the 4 and 6 hour time points</p>
 +
                                                                  </div>
 +
 +
                                                                </div>
 +
                                                                </div>
 +
                                                                </div>
 +
<h2>2. Discussion</h2>
 +
<p>The protocol was easy to follow and the constructs were nicely expressed which allowed reliable and comparable measurements. In the process of obtaining transformed DH5&#945; cells we did not encounter any problems with transformation as bacteria showed exceptional growth. The specific ratiometric conversion factor of our plate reader could be determined without further adjustments using the LUDOX-HS40 solution. When measuring the Fluorescein standard curve on a logarithmic scale the data points showed a linear distribution. The final step, the measurement of the fluorescence emission and optical density of the cells, resulted in the raw data shown in figure 1.5. To make this data visually accessible the mean values for OD and fluorescence were calculated and plotted against their respective time points which is seen in the following graphs.
 +
 +
 +
                                                         
 +
                                                                <div class="row" style="padding-top: 30px;padding-bottom: 30px;"> 
 +
                                                                <div class="col-lg-offset-1 col-md-offset-1 col-sm-offset-1 col-xs-offset-1 col-lg-11 col-md-11 col-sm-11 col-xs-11">
 +
 +
                                                                  <a href="#" target="_blank" ><img src="https://static.igem.org/mediawiki/2017/e/eb/T--Heidelberg--ODGraph.png"  alt="#"></a>
 +
                                                                  <div class="caption">
 +
                                                                   
 +
                                                                   
 +
<p>Figure 1.8: Calculated means from both replicates for the OD<sub>600</sub> after each time point</p>
 +
                                                                  </div>
 +
                                                                    </div>
 +
                                                                </div>
 +
                                                                <div class="row" style="padding-top: 30px;padding-bottom: 30px;"> 
 +
                                                                <div class="col-lg-offset-1 col-md-offset-1 col-sm-offset-1 col-xs-offset-1 col-lg-11 col-md-11 col-sm-11 col-xs-11">
 +
 +
                                                                  <a href="#" target="_blank" ><img src="https://static.igem.org/mediawiki/2017/0/07/T--Heidelberg--FluorescenceGraph.png"  alt="#"></a>
 +
                                                                  <div class="caption">
 +
                                                                   
 +
                                                                   
 +
<p>Figure 1.9: Calculated means from both replicates for the fluorescence after each time point</p>
 +
                                                                  </div>
 +
                                                                    </div>
 +
                                                                    </div>
 +
                                                                 
 +
<p>Conclusively, these data suggest that the Interlab study was successful and that the provided protocol can be shared in the community for standardized fluorescence measurement with a plate reader.</p>
 +
</div>
 +
</div>
 +
        </div>                                       
  
−
</script>
 
  
  
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Revision as of 07:42, 26 September 2017


Team:Heidelberg/InterLab - 2017.igem.org

Team:Heidelberg/InterLab

WikitemplateA home - 2014.igem.org

 

WikitemplateA home

From 2014.igem.org

1. Interlab Study

As a first step to contribute to this year's iGEM competition we decided to participate in iGEM's fourth International Interlaboratory Study along with many other teams from around the world. This study is organized by iGEM's measurement committee in an effort to establish standardized, reliable and repeatable measurement tools for the iGEM community and the synthetic biology community as a whole. This year's iGEM InterLab study is about establishing a standardized protocol for the measurement of GFP using a plate reader. To start things off we needed a plate reader that is qualified to measure GFP fluorescence. Namely, Tecan Infinite M200 Pro plate reader. Additionally, we needed competent E.coli DH5α. These were prepared from glycerol stocks. Together with the material iGEM had provided we were ready for work. Throughout our experiments, we tested 8 plasmids (2 controls and 6 test devices) by measuring the OD600 and the fluorescence of the cells carrying the constructs. The workflow can be separated into four segments. The first segment is the transformation of all plasmids into competent DH5α cells.

1.1 Transformation of 8 test devices into competent DH5α cells

The transformation was successful for all the plasmids and resulted in a sufficient amount of colonies on all plates. Therefore, two colonies from each plate were picked and inoculated in LB medium containing chloramphenicol over night for approximately 16-18 hours at 37°C and 220 rpm.

1.2 Measurement of LUDOX-HS40 OD600 Reference Point

The second segment is the measurement of the LUDOX-HS40 solution to obtain a ratiometric conversion factor which allows us to transform the absorbance data into a standard OD600 measurement. This is necessary because plate readers in general do not have a 1 cm path length which means that measurements are volume dependent. By doing so, we obtained following data.

#

Figure 1.1: Measurement of the LUDOX-HS40 and water in four replicates and calculation of the ratiometric conversion factor

With these replicates measured, we obtained a ratiometric conversion factor of 4.30 for our specific plate reader when measured 100 μL of the provided LUDOX-HS40 to water.

1.3 Graphing a Fluorescein fluorescence standard curve

In the third segment we were instructed to measure a standard curve for fluorescence of Fluorescein which can be used to correct our cell based readings to an equivalent Fluorescein concentration. Following the instructions, we could obtain following results for the serial dilution of Fluorescein in phosphate buffered saline.

#

Figure 1.2: Measurement of a standard curve with four replicates by serial dilution of Fluorescein in phosphate buffered saline

#

Figure 1.3: Fluorescence is plotted against the Fluorescein concentration

#

Figure 1.4: Fluorescence is plotted against the Fluorescein concentration on a logarithmic scale

These data give us an approximate linear regression line when plotted in a logarithmic scale and show an expected exponential course when plotted in a linear form.

1.4 Measurement of OD600 and Fluorescence of Transformed Cells

In the last of the four segments the OD600 and the fluorescence of the transformed cells were measured according to the protocol after 0, 2, 4 and 6 hours. Measurements of all these time points gave us the following data and calculations which were conducted with the values we obtained from the standard curves and the reference point.

#

Figure 1.5: Raw data obtained from measurements

#

Figure 1.6: Raw data and measurements for the 0 and 2 hour time points

#

Figure 1.7: Raw data and measurements for the 4 and 6 hour time points

2. Discussion

The protocol was easy to follow and the constructs were nicely expressed which allowed reliable and comparable measurements. In the process of obtaining transformed DH5α cells we did not encounter any problems with transformation as bacteria showed exceptional growth. The specific ratiometric conversion factor of our plate reader could be determined without further adjustments using the LUDOX-HS40 solution. When measuring the Fluorescein standard curve on a logarithmic scale the data points showed a linear distribution. The final step, the measurement of the fluorescence emission and optical density of the cells, resulted in the raw data shown in figure 1.5. To make this data visually accessible the mean values for OD and fluorescence were calculated and plotted against their respective time points which is seen in the following graphs.

#

Figure 1.8: Calculated means from both replicates for the OD600 after each time point

#

Figure 1.9: Calculated means from both replicates for the fluorescence after each time point

Conclusively, these data suggest that the Interlab study was successful and that the provided protocol can be shared in the community for standardized fluorescence measurement with a plate reader.

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