Difference between revisions of "Team:Vilnius-Lithuania/InterLab"

 
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<h1 class="text-wall-heading">InterLab</h1>
 
<h1 class="text-wall-heading">InterLab</h1>
 
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<div class="text-wall-area-box">
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     <h2 class="text-wall-area-box-heading">Lorem ipsum, dolor sit amet consectetur adipisicing</h2>
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     <h2 class="text-wall-area-box-heading">Studying Fluorescence</h2>
 
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     <div class="scroll-area">
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         <p class="text-content">Cell-free systems are becoming an increasingly popular in vitro tool to study biological processes as it is accompanied by less intrinsic and extrinsic noise. Relying on fundamental concepts of synthetic biology, we apply a bottom-up forward engineering approach to create a novel cell-free system for unorthodox protein-evolution. The core of this system is cell-sized liposomes that serve as excellent artificial membrane models. By encapsulating genetic material and full in vitro protein transcription and translation systems within the liposomes, we create reliable and incredibly efficient nanofactories for the production of target proteins. Even though there are many alternative proteins that can be synthesized, our main focus is directed towards membrane proteins, which occupy approximately one third of living-cells’ genomes. Considering their significance, membrane proteins are spectacularly understudied since synthesis and thus characterization of them remain prevailing obstacles to this day. We aim to utilize liposomes as nanofactories for directed evolution of membrane proteins. Furthermore, by means of directed membrane protein-evolution, a universal exposition system will be designed in order to display any protein of interest on the surface of the liposome. This way, a system is built where a phenotype of a particular protein is expressed on the outside while containing its genotype within the liposome. To prove the concept, small antibody fragments will be displayed to create a single-chain variable fragment (scFv) library for rapid screening of any designated target.</p>
+
         <p class="text-content">The goal of this year’s InterLab Study was to identify and minimize the sources of systematic variability in fluorescence measurements by normalizing to absolute cell count or colony-forming units (CFUs) instead of optical density (OD).</p>
 +
  <p class="text-content">Participating in the fifth iGEM InterLab Study was a great opportunity to start this year’s competition as well as acquire some valuable knowledge which we implemented into practice during the project.</p>
  
 
         </p>
 
         </p>
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         <h1>Description</h1>
 
         <h1>Description</h1>
 
         <p></p>
 
         <p></p>
 +
        At the beginning of the InterLab study we completed three distinct calibration protocols. At first, we performed the <strong>LUDOX Protocol</strong> in order to obtain a conversion factor to transform absorbance (Abs<sub>600</sub>) from the plate reader into a comparable OD<sub>600</sub> measurement as would be obtained with a spectrophotometer. Next, we completed the <strong>Microsphere Protocol</strong> as it allows a standard curve of particle concentration which is used to convert Abs<sub>600</sub> measurements to an estimated number of cells. Finally, by completing the <strong>Fluorescein Protocol</strong> we generated a standard fluorescence curve which is used to compare fluorescence output of different test devices. Completion of the calibrations ensured that we take cell measurements under the same conditions. It is worth mentioning that prior calibration, we prepared competent E. coli DH5-alpha cells and transformed them according to the standard transformation protocol. During all of the experiments we tested 8 plasmids: 2 controls and 6 test devices (Tab 1).
 +
 +
<div class="image-container">
 +
        <p><strong>Tab. 1</strong> Parts received and tested during iGEM’s fifth InterLab Study</p>
 +
          <table>
 +
        <thead>
 +
          <tr>
 +
          <th><strong>Device</strong></th>
 +
          <th><Strong>Part Number</Strong></th>
 +
          <th><strong>Features</strong></th>
 +
          </tr>
 +
        </thead>
 +
        <tbody>
 +
          <tr>
 +
          <td>Negative control</td>
 +
          <td><a href="http://parts.igem.org/Part:BBa_R0040">  BBa_R0040  </td>
 +
            <td>Medium strength promoter, promoter is constitutive and repressed by TetR
 +
                    </td>
 +
          </tr>
 +
          <tr>
 +
                <td>Positive Control</td>
 +
                <td><a href="http://parts.igem.org/Part:BBa_I20270">  BBa_I20270  </td>
 +
                <td>J23151 inserted in the Promoter MeasKit</td>
 +
              </tr>
 +
              <tr>
 +
                    <td>Test Device 1</td>
 +
                    <td><a href="http://parts.igem.org/Part:BBa_J364000">  BBa_J364000  </td>
 +
                    <td>GFP expressing constitutive device</td>
 +
                  </tr>
 +
                  <tr>
 +
                        <td>Test Device 2</td>
 +
                        <td><a href="http://parts.igem.org/Part:BBa_J364001">  BBa_J364001  </td>
 +
                        <td>GFP expressing constitutive device</td>
 +
                      </tr>
 +
                      <tr>
 +
                            <td>Test Device 3</td>
 +
                            <td><a href="http://parts.igem.org/Part:BBa_J364002">  BBa_J364002  </td>
 +
                            <td>GFP expressing constitutive device</td>
 +
                          </tr>
 +
                          <tr>
 +
                                <td>Test Device 4</td>
 +
                                <td><a href="http://parts.igem.org/Part:BBa_J364007">  BBa_J364007  </td>
 +
                                <td>Expresses GFP under the control of a constitutive promoter from the Anderson collection</td>
 +
                              </tr>
 +
                              <tr>
 +
                                    <td>Test Device 5</td>
 +
                                    <td><a href="http://parts.igem.org/Part:BBa_J364008">  BBa_J364008  </td>
 +
                                    <td>Expresses GFP under the control of a constitutive promoter from the Anderson collection</td>
 +
                                    </tr>
 +
                                    <tr>
 +
                                          <td>Test Device 6</td>
 +
                                          <td><a href="http://parts.igem.org/Part:BBa_J364009">  BBa_J364009  </td>
 +
                                          <td>Expresses GFP under the control of a constitutive promoter from the Anderson collection</td>
 +
                                        </tr>
 +
        </tbody>
 +
        </table>
 +
</div>
 +
 +
<p></p>
 
         <p></p>
 
         <p></p>
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         <h2>What is SynORI?</h2>
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         <h1>Results and Discussion</h1>
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         <p>SynORI stands for synthetic origin of replication. It is a framework designed to make working with single
+
         <p></p>
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            and multi-plasmid systems precise, easy and on top of that - more functional.</p>
+
<h3>1. MEASUREMENT OF LUDOX CL-X OD<sub>600</sub> REFERENCE POINT</h3>
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        <p>The SynORI framework enables scientists to build a multi-plasmid system in a standardized manner by:</p>
+
              
−
        <ol>
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−
            <li>Selecting the number of plasmid groups</li>
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−
            <li>Choosing the copy number of each group</li>
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             <li>Picking the type of copy number control (specific to one group or regulating all of them at once).</li>
+
  
−
         </ol>
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         <p></p>
−
        </p>
+
<p>Using LUDOX CL-X as a single point reference allowed us to obtain a ratiometric conversion factor to transform absorbance data into a standard OD<sub>600</sub> measurement. This is crucial to ensure that plate reader measurements are not volume dependent. After this calibration part we obtained a radiometric conversion factor (Tab. 2) which will be used in further Interlab study measurements.</p>
  
 +
<div class="image-container">
 +
<p><strong>Tab. 2</strong> LUDOX CL-X measurement. Obtained ratiometric conversion factor is 3,419</p>
 +
<table>
 +
    <thead>
 +
    <tr>
 +
      <th><strong></strong></th>
 +
      <th><Strong>LUDOX CL-X</Strong></th>
 +
      <th><strong>H<small>2</small>O</strong></th>
 +
    </tr>
 +
    </thead>
 +
    <tbody>
 +
    <tr>
 +
      <td>Negative control</td>
 +
      <td><a href="http://parts.igem.org/Part:BBa_R0040">  BBa_R0040  </td>
 +
      <td>Medium strength promoter, promoter is constitutive and repressed by TetR
 +
              </td>
 +
    </tr>
 +
    <tr>
 +
          <td>Positive Control</td>
 +
          <td><a href="http://parts.igem.org/Part:BBa_I20270">  BBa_I20270  </td>
 +
          <td>J23151 inserted in the Promoter MeasKit</td>
 +
        </tr>
 +
        <tr>
 +
              <td>Test Device 1</td>
 +
              <td><a href="http://parts.igem.org/Part:BBa_J364000">  BBa_J364000  </td>
 +
              <td>GFP expressing constitutive device</td>
 +
              </tr>
 +
              <tr>
 +
                    <td>Test Device 2</td>
 +
                    <td><a href="http://parts.igem.org/Part:BBa_J364001">  BBa_J364001  </td>
 +
                    <td>GFP expressing constitutive device</td>
 +
                  </tr>
 +
                  <tr>
 +
                      <td>Test Device 3</td>
 +
                      <td><a href="http://parts.igem.org/Part:BBa_J364002">  BBa_J364002  </td>
 +
                      <td>GFP expressing constitutive device</td>
 +
                      </tr>
 +
                      <tr>
 +
                            <td>Test Device 4</td>
 +
                            <td><a href="http://parts.igem.org/Part:BBa_J364007">  BBa_J364007  </td>
 +
                            <td>Expresses GFP under the control of a constitutive promoter from the Anderson collection</td>
 +
                          </tr>
 +
                          <tr>
 +
                                <td>Test Device 5</td>
 +
                                <td><a href="http://parts.igem.org/Part:BBa_J364008">  BBa_J364008  </td>
 +
                                <td>Expresses GFP under the control of a constitutive promoter from the Anderson collection</td>
 +
                              </tr>
 +
                              <tr>
 +
                                    <td>Test Device 6</td>
 +
                                    <td><a href="http://parts.igem.org/Part:BBa_J364009">  BBa_J364009  </td>
 +
                                    <td>Expresses GFP under the control of a constitutive promoter from the Anderson collection</td>
 +
                                  </tr>
 +
    </tbody>
 +
  </table>
 +
</div>
 
         <p></p>
 
         <p></p>
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        <p>The framework also includes a possibility of adding a selection system that reduces the usage of antibiotics
 
−
            (only 1 antibiotic for up to 5 different plasmids!) and an active partitioning system to make sure that low
 
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            copy number plasmid groups are not lost during the division.
 
−
        </p>
 
 
         <p></p>
 
         <p></p>
−
        <div class="img-cont">
+
<h3>2. GRAPHING A SILICA MICROSPHERE ABSORBANCE (Abs<sub>600</sub>) STANDARD CURVE</h3>
−
            <img src="https://static.igem.org/mediawiki/parts/8/84/Collect.png" alt="img">
+
−
            <div class="img-label">
+
−
            </div>
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−
        </div>
+
−
        <h2>Applications</h2>
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−
        <p>
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            <h5>Everyday lab work</h5>
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            <p>
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−
                A multi-plasmid system that is easy to assemble and control. With our framework the need to limit your
+
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                research to a particular plasmid copy number just because there are not enough right replicons to
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                choose from, is eliminated. With SynORI you can easily create a vector with a desired copy number that
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−
                suits your needs.</li>
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−
            </p>
+
−
            <h5>Biological computing</h5>
+
−
            <p>
+
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                The ability to choose a wide range of copy number options and their control types will make the
+
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                synthetic biology engineering much more flexible and predictable. Introduction of plasmid copy number
+
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                regulation is equivalent to adding a global parameter to a computer system. It enables the coordination
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                of multiple gene group expression.
+
−
            </p>
+
−
            <h5>Smart assembly of large protein complexes</h5>
+
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            <p>
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                The co-expression of multi-subunit complexes using different replicons brings incoherency to an already
+
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                chaotic cell system. This can be avoided by using SynORI, as in this framework every plasmid group uses
+
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                the same type of control, and in addition can act in a group-specific manner.</p>
+
  
−
            <h5>Metabolic engineering</h5>
+
<p></p>
−
            <p>
+
<p>Monodisperse silica microspheres exhibit size and optical characteristics similar to cells, with the additional benefit that the number of particles in a solution is known. Therefore, this measurement allowed us to construct a standard curve which can be used to convert Abs<sub>600</sub> measurements to an estimated number of cells.
−
                A big challenge for heterologous expression of multiple gene pathways is to accurately adjust the
+
</p>
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                levels of each enzyme to achieve optimal production efficiency. Precise promoter tuning in
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<div class="image-container">
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                transcriptional control and synthetic ribosome binding sites in translational control are already
+
<img src="https://static.igem.org/mediawiki/2018/3/31/T--Vilnius-Lithuania--1_InterLab.png"/>
−
                widely used to maintain expression levels. In addition to current approaches, our framework allows a
+
      <p><strong>Fig. 1</strong> LUDOX CL-X measurement. Obtained ratiometric conversion factor is 3,419.</p>
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                simultaneous multiple gene control. Furthermore, an inducible regulation that we offer, can make the
+
</div>
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                search for perfect conditions a lot easier.
+
  
 +
<div class="image-container">
 +
<img src="https://static.igem.org/mediawiki/2018/b/bc/T--Vilnius-Lithuania--2_InterLab.png"/>
 +
      <p><strong>Fig. 2</strong> Particle standard curve generated by measuring the absorbance of serial dilutions of silica microspheres (known amount of particles per volume) displayed in a log scale to demonstrate a linear relationship between particle count per volume and absorbance.</p>
 +
<div class="image-container">
 +
       
 +
<p>During this calibration part we obtained two particle standard curves which are important for proper cell measurement. However, we can observe a curve in the log scale graph (Fig.  1), although it should have a 1:1 slope. We assume that this inconsistency could have been due to pipetting errors or an oversaturated detector.
 +
</p>
 +
        <p></p>
  
 +
<h3>3. GRAPHING A FLUORESCEIN FLUORESCENCE STANDARD CURVE</h3>
  
−
            </p>
+
<p>In the last part of the calibration we prepared a dilution series of fluorescein in four replicates and measured the fluorescence. During this calibration part we generated a standard curve of fluorescence for fluorescein concentration.</p>
 +
          <p></p>
 +
<div class="image-container">
 +
<img src="https://static.igem.org/mediawiki/2018/b/b0/T--Vilnius-Lithuania--3_InterLab.png">
 +
      <p><strong>Fig. 3</strong> Standard curve of fluorescein generated by measuring the fluorescence of serial dilution stock (µM). Fluorescence is plotted against the fluorescein concentration.</p>
 +
</div>
  
 +
<div class="image-container">
 +
<img src="https://static.igem.org/mediawiki/2018/d/d8/T--Vilnius-Lithuania--4_InterLab.png">
 +
      <p><strong>Fig. 4</strong> A standard curve of fluorescein generated by measuring the fluorescence of serial dilution stock (µM). Fluorescence is plotted against the fluorescein concentration on a logarithmic scale.
 +
</p>
 +
</div>
 +
      <p>During this calibration part we generated a standard curve of fluorescein. Standard curves (linear and on a logarithmic scale) have a 1:1 slope which ensures us that there were no significant mistakes during this calibration part and the data can be used for cell measurement. This allows us to successfully convert cell based readings to an equivalent fluorescein concentration.</p>
 +
 +
<h1>Cell Measurements</h1>
 +
<p></p>
 +
<p>For cell measurements we used the same settings that we used in our calibration measurements. At first, according to the standard protocol we transformed cells with 8 different plasmids (Tab. 1). We picked 2 colonies from each transformation plates and inoculated in 5-10 mL LB medium + Chloramphenicol. We grew the cells overnight (16-18 hours) at 37 °C and 220 rpm. After that we diluted the cultures to a target Abs<sub>600</sub> of 0.02. We took samples from these diluted cultures prior to incubation and after 6 hours of incubation measured Abs600 (Fig.  5) and fluorescence (Fig.  6). </p>
 +
 +
<p></p>
 +
<div class="image-container">
 +
<img src="https://static.igem.org/mediawiki/2018/1/1d/T--Vilnius-Lithuania--5_InterLab.png">
 +
      <p><strong>Fig. 5</strong> Graph comparing the raw Abs<sub>600</sub> prior incubation and at hour 6 for each colony using each control/device</p>
 +
</div>
 +
<p></p>
 +
 +
<div class="image-container">
 +
<img src="https://static.igem.org/mediawiki/2018/b/bb/T--Vilnius-Lithuania--6_InterLab.png">
 +
      <p><strong>Fig. 6 </strong>Graph comparing the raw fluorescence prior to incubation and at hour 6 for each colony using each control/device</p>
 +
</div>
 +
 +
<p>Comparing absorbance and fluorescence of cells prior to incubation and after 6 hours we can observe that absorbance as well as fluorescence were more intense after 6 h of incubation as it was expected.
 +
Based on the assumption that one bacterial cell gives rise to one colony, colony forming units per 1 mL of an OD<sub>600</sub> = 0.1 culture was calculated by counting the colonies on each plate with fewer than 300 colonies and multiplying the colony count by the Final Dilution Factor on each plate The results are shown in Tab. 3.</p>
 +
 +
 +
<div class="image-container">
 +
<p> <strong>Tab. 3</strong> Colony forming units (CFU) per 1 mL of an OD<sub>600</sub> = 0.1culture</p>
 +
 +
<p><table>
 +
    <thead>
 +
    <tr>
 +
      <th><strong>Samples</strong></th>
 +
      <th><Strong>CFU/ml in Starting Sample</Strong></th>
 +
    </tr>
 +
    </thead>
 +
    <tbody>
 +
        </tr>
 +
    <tr>
 +
        <td>1.1 Positive Control</td>
 +
        <td>0.132667 * 10^8</td>
 +
    </tr> <tr>
 +
            <td>1.2 Positive Control</td>
 +
        <td>0.086667 * 10^8</td>
 +
  </tr> <tr>
 +
            <td>1.3 Positive Control</td>
 +
        <td>0.271333 * 10^8</td>
 +
    </tr> <tr>
 +
            <td>2.1 Positive Control</td>
 +
        <td>0.448667 * 10^8</td>
 +
    </tr>  <tr>
 +
            <td>2.2 Positive Control</td>
 +
        <td>0.394667 * 10^8</td>
 +
    </tr>  <tr>
 +
            <td>2.3 Positive Control</td>
 +
        <td>0.659667 * 10^8</td>
 +
    </tr>  <tr>
 +
            <td>3.1 Negative Control</td>
 +
        <td>0.236 * 10^8</td>
 +
    </tr>  <tr>
 +
            <td>3.2 Negative Control</td>
 +
        <td>0.722 * 10^8</td>
 +
    </tr>  <tr>
 +
            <td>3.3 Negative Contro</td>
 +
        <td>0.346667 * 10^8</td>
 +
    </tr>  <tr>
 +
            <td>4.1 Negative control</td>
 +
        <td>0.494 * 10^8</td>
 +
    </tr>  <tr>
 +
            <td>4.2 Negative control</td>
 +
        <td>0.279 * 10^8</td>
 +
    </tr>  <tr>
 +
            <td>4.3 Negative control</td>
 +
        <td>0.395 * 10^8</td>
 +
    </tr>                                                                                                       
 +
    </tbody>
 +
  </table></p>
 +
 +
</div>
  
−
        </p>
 
−
        <p>
 
−
        </p>
 
−
        <table style="width:100%">
 
−
<thead>
 
−
<td align='center'>Species sign in ODE system</td>
 
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<td align='center'>Species</td>
 
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<td align='center'>Initial concentration (M)</td>
 
−
</thead>
 
−
<tbody>
 
−
<tr>
 
−
<td align='center'>A</td>
 
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<td align='center'>pDNA+RNA I+RNAII early</td>
 
−
<td align='center'>0</td>
 
−
</tr>
 
−
<tr>
 
−
<td align='center'>B</td>
 
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<td align='center'>pDNA+RNA II short</td>
 
−
<td align='center'>0</td>
 
−
</tr>
 
−
<tr>
 
−
<td align='center'>RNAI</td>
 
−
<td align='center'>RNA I</td>
 
−
<td align='center'>1E-6</td>
 
−
</tr>
 
−
<tr>
 
−
<td align='center'>D</td>
 
−
<td align='center'>pDNA+RNA II long</td>
 
−
<td align='center'>0</td>
 
−
</tr>
 
−
<tr>
 
−
<td align='center'>E</td>
 
−
<td align='center'>pDNA+RNAII primer</td>
 
−
<td align='center'>0</td>
 
−
</tr>
 
−
<tr>
 
−
<td align='center'>F</td>
 
−
<td align='center'>RNA II long</td>
 
−
<td align='center'>0</td>
 
−
</tr>
 
−
<tr>
 
−
<td align='center'>G</td>
 
−
<td align='center'>pDNA</td>
 
−
<td align='center'>4E-8*</td>
 
−
</tr>
 
−
<tr>
 
−
<td align='center'>H</td>
 
−
<td align='center'>pDNA+RNA II+RNA I late</td>
 
−
<td align='center'>0</td>
 
−
</tr>
 
−
<tr>
 
−
<td align='center'>RNA II</td>
 
−
<td align='center'>RNA II</td>
 
−
<td align='center'>0</td>
 
−
</tr>
 
−
<tr>
 
−
<td align='center'>J</td>
 
−
<td align='center'>RNAI+RNAII</td>
 
−
<td align='center'>0</td>
 
−
</tr>
 
−
</tbody>
 
−
</table>
 
 
     </div>
 
     </div>
 
</div>
 
</div>
 
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Latest revision as of 21:42, 4 November 2018

InterLab

Studying Fluorescence

The goal of this year’s InterLab Study was to identify and minimize the sources of systematic variability in fluorescence measurements by normalizing to absolute cell count or colony-forming units (CFUs) instead of optical density (OD).

Participating in the fifth iGEM InterLab Study was a great opportunity to start this year’s competition as well as acquire some valuable knowledge which we implemented into practice during the project.

Description

At the beginning of the InterLab study we completed three distinct calibration protocols. At first, we performed the LUDOX Protocol in order to obtain a conversion factor to transform absorbance (Abs600) from the plate reader into a comparable OD600 measurement as would be obtained with a spectrophotometer. Next, we completed the Microsphere Protocol as it allows a standard curve of particle concentration which is used to convert Abs600 measurements to an estimated number of cells. Finally, by completing the Fluorescein Protocol we generated a standard fluorescence curve which is used to compare fluorescence output of different test devices. Completion of the calibrations ensured that we take cell measurements under the same conditions. It is worth mentioning that prior calibration, we prepared competent E. coli DH5-alpha cells and transformed them according to the standard transformation protocol. During all of the experiments we tested 8 plasmids: 2 controls and 6 test devices (Tab 1).

Tab. 1 Parts received and tested during iGEM’s fifth InterLab Study

Device Part Number Features
Negative control BBa_R0040 Medium strength promoter, promoter is constitutive and repressed by TetR
Positive Control BBa_I20270 J23151 inserted in the Promoter MeasKit
Test Device 1 BBa_J364000 GFP expressing constitutive device
Test Device 2 BBa_J364001 GFP expressing constitutive device
Test Device 3 BBa_J364002 GFP expressing constitutive device
Test Device 4 BBa_J364007 Expresses GFP under the control of a constitutive promoter from the Anderson collection
Test Device 5 BBa_J364008 Expresses GFP under the control of a constitutive promoter from the Anderson collection
Test Device 6 BBa_J364009 Expresses GFP under the control of a constitutive promoter from the Anderson collection

Results and Discussion

1. MEASUREMENT OF LUDOX CL-X OD600 REFERENCE POINT

Using LUDOX CL-X as a single point reference allowed us to obtain a ratiometric conversion factor to transform absorbance data into a standard OD600 measurement. This is crucial to ensure that plate reader measurements are not volume dependent. After this calibration part we obtained a radiometric conversion factor (Tab. 2) which will be used in further Interlab study measurements.

Tab. 2 LUDOX CL-X measurement. Obtained ratiometric conversion factor is 3,419

LUDOX CL-X H2O
Negative control BBa_R0040 Medium strength promoter, promoter is constitutive and repressed by TetR
Positive Control BBa_I20270 J23151 inserted in the Promoter MeasKit
Test Device 1 BBa_J364000 GFP expressing constitutive device
Test Device 2 BBa_J364001 GFP expressing constitutive device
Test Device 3 BBa_J364002 GFP expressing constitutive device
Test Device 4 BBa_J364007 Expresses GFP under the control of a constitutive promoter from the Anderson collection
Test Device 5 BBa_J364008 Expresses GFP under the control of a constitutive promoter from the Anderson collection
Test Device 6 BBa_J364009 Expresses GFP under the control of a constitutive promoter from the Anderson collection

2. GRAPHING A SILICA MICROSPHERE ABSORBANCE (Abs600) STANDARD CURVE

Monodisperse silica microspheres exhibit size and optical characteristics similar to cells, with the additional benefit that the number of particles in a solution is known. Therefore, this measurement allowed us to construct a standard curve which can be used to convert Abs600 measurements to an estimated number of cells.

Fig. 1 LUDOX CL-X measurement. Obtained ratiometric conversion factor is 3,419.

Fig. 2 Particle standard curve generated by measuring the absorbance of serial dilutions of silica microspheres (known amount of particles per volume) displayed in a log scale to demonstrate a linear relationship between particle count per volume and absorbance.

During this calibration part we obtained two particle standard curves which are important for proper cell measurement. However, we can observe a curve in the log scale graph (Fig. 1), although it should have a 1:1 slope. We assume that this inconsistency could have been due to pipetting errors or an oversaturated detector.

3. GRAPHING A FLUORESCEIN FLUORESCENCE STANDARD CURVE

In the last part of the calibration we prepared a dilution series of fluorescein in four replicates and measured the fluorescence. During this calibration part we generated a standard curve of fluorescence for fluorescein concentration.

Fig. 3 Standard curve of fluorescein generated by measuring the fluorescence of serial dilution stock (µM). Fluorescence is plotted against the fluorescein concentration.

Fig. 4 A standard curve of fluorescein generated by measuring the fluorescence of serial dilution stock (µM). Fluorescence is plotted against the fluorescein concentration on a logarithmic scale.

During this calibration part we generated a standard curve of fluorescein. Standard curves (linear and on a logarithmic scale) have a 1:1 slope which ensures us that there were no significant mistakes during this calibration part and the data can be used for cell measurement. This allows us to successfully convert cell based readings to an equivalent fluorescein concentration.

Cell Measurements

For cell measurements we used the same settings that we used in our calibration measurements. At first, according to the standard protocol we transformed cells with 8 different plasmids (Tab. 1). We picked 2 colonies from each transformation plates and inoculated in 5-10 mL LB medium + Chloramphenicol. We grew the cells overnight (16-18 hours) at 37 °C and 220 rpm. After that we diluted the cultures to a target Abs600 of 0.02. We took samples from these diluted cultures prior to incubation and after 6 hours of incubation measured Abs600 (Fig. 5) and fluorescence (Fig. 6).

Fig. 5 Graph comparing the raw Abs600 prior incubation and at hour 6 for each colony using each control/device

Fig. 6 Graph comparing the raw fluorescence prior to incubation and at hour 6 for each colony using each control/device

Comparing absorbance and fluorescence of cells prior to incubation and after 6 hours we can observe that absorbance as well as fluorescence were more intense after 6 h of incubation as it was expected. Based on the assumption that one bacterial cell gives rise to one colony, colony forming units per 1 mL of an OD600 = 0.1 culture was calculated by counting the colonies on each plate with fewer than 300 colonies and multiplying the colony count by the Final Dilution Factor on each plate The results are shown in Tab. 3.

Tab. 3 Colony forming units (CFU) per 1 mL of an OD600 = 0.1culture

Samples CFU/ml in Starting Sample
1.1 Positive Control 0.132667 * 10^8
1.2 Positive Control 0.086667 * 10^8
1.3 Positive Control 0.271333 * 10^8
2.1 Positive Control 0.448667 * 10^8
2.2 Positive Control 0.394667 * 10^8
2.3 Positive Control 0.659667 * 10^8
3.1 Negative Control 0.236 * 10^8
3.2 Negative Control 0.722 * 10^8
3.3 Negative Contro 0.346667 * 10^8
4.1 Negative control 0.494 * 10^8
4.2 Negative control 0.279 * 10^8
4.3 Negative control 0.395 * 10^8

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