Difference between revisions of "Team:Edinburgh UG/Measurement"

 
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             <h2 class="header-subsection"> Protocol </h2>
 
             <h2 class="header-subsection"> Protocol </h2>
 
             <p> <strong> Material needed </strong></p>
 
             <p> <strong> Material needed </strong></p>
−
             <ol type="1" style="list-style-type: none; list-style-image: none; font-size: 17px;">
+
             <ol type="1" style="list-style-image: none; font-size: 17px;">
 
                 <li>Escherichia coli strain for cloning purpose (e.g. TOP10, DH5α)</li>
 
                 <li>Escherichia coli strain for cloning purpose (e.g. TOP10, DH5α)</li>
 
                 <li>Any T7 polymerase expressing E. coli strain (e.g. BL21 (DE3))</li>
 
                 <li>Any T7 polymerase expressing E. coli strain (e.g. BL21 (DE3))</li>
 
                 <li>Biobricks: pSB4K5 plasmid backbone</li>
 
                 <li>Biobricks: pSB4K5 plasmid backbone</li>
−
                 <li>Biobricks: T7-LacO-regulated Cre/Dre/VCre/SCre/Vika recombinase generators (BBa_K2406080, BBa_K2406081, BBa_K2406082, BBa_K2406083, BBa_K2406084) </li>
+
                 <li>Biobricks: T7-LacO-regulated Cre/Dre/VCre/SCre/Vika recombinase generators (BBa_K2406080, BBa_K2406081, BBa_K2406082,
−
                 <li>Biobricks: Measurement device (BBa_K2406051, BBa_K2406053, BBa_K2406055, BBa_K2406057, BBa_K2406059, BBa_K2406061, BBa_K2406063, BBa_K2406065, BBa_K2406067, BBa_K2406069, BBa_K2406071, BBa_K2406073, BBa_K2406075, BBa_K2406077, BBa_K2406079)</li>
+
                    BBa_K2406083, BBa_K2406084) </li>
 +
                 <li>Biobricks: Measurement device (BBa_K2406051, BBa_K2406053, BBa_K2406055, BBa_K2406057, BBa_K2406059, BBa_K2406061,
 +
                    BBa_K2406063, BBa_K2406065, BBa_K2406067, BBa_K2406069, BBa_K2406071, BBa_K2406073, BBa_K2406075, BBa_K2406077,
 +
                    BBa_K2406079)
 +
                </li>
 
                 <li>Chloramphenical (stock of 25 mg/mL in EtOH</li>
 
                 <li>Chloramphenical (stock of 25 mg/mL in EtOH</li>
 
                 <li>Kanamycin (stock of 50 mg/mL in dH2O)</li>
 
                 <li>Kanamycin (stock of 50 mg/mL in dH2O)</li>
Line 63: Line 67:
 
                 <li>Microcentrifuge (for eppendorf tubes)</li>
 
                 <li>Microcentrifuge (for eppendorf tubes)</li>
 
                 <li>Centrifuge (for falcon tubes)</li>
 
                 <li>Centrifuge (for falcon tubes)</li>
−
                 <li>Incubator at 37ºC </li>
+
                 <li>Incubator at 37ºC </li>
 
             </ol><br><br>
 
             </ol><br><br>
  
 
             <p> <strong> Method </strong></p>
 
             <p> <strong> Method </strong></p>
 
             <p> Day 1: </p>
 
             <p> Day 1: </p>
−
              
+
             <ol type="1" style="list-style-image: none; font-size: 17px;">
 +
                <li>Transform E. coli DH5α with the following inducible recombinase generators: <br>
 +
                    <ol type="a" style="list-style-image: none; font-size: 17px;">
 +
                        <li>T7-LacO-Cre (BBa_K2406080) </li>
 +
                        <li>T7-LacO-Dre (BBa_K2406081) </li>
 +
                        <li>T7-LacO-Vika (BBa_K2406082) </li>
 +
                        <li>T7-LacO-VCre (BBa_K2406083) </li>
 +
                        <li>T7-LacO-SCre (BBa_K2406084) </li>
 +
                    </ol><br>
 +
                    <strong> Plate on LB agar with chloramphenicol </strong>
 +
                </li>
 +
                <li>Transform E. coli DH5α with the following measurement constructs: <br>
 +
                    <ol type="a" style="list-style-image: none; font-size: 17px;">
 +
                        <li>Rox-term-Rox (BBa_K2406051) </li>
 +
                        <li>Vox-term-Vox (BBa_K2406053)</li>
 +
                        <li>Lox-term-Lox (BBa_K2406055</li>
 +
                        <li>Vlox-term-Lox (BBa_K2406057) </li>
 +
                        <li>Vlox-term-Vlox (BBa_K2406059) </li>
 +
                        <li>Slox-term-Lox (BBa_K2406061) </li>
 +
                        <li>Slox-term-Vlox (BBa_K2406063) </li>
 +
                        <li>Slox-term-Slox (BBa_K2406065) </li>
 +
                        <li>Vox-term-Vlox (BBa_K2406067) </li>
 +
                        <li>Vox-term-Slox (BBa_K2406069) </li>
 +
                        <li>Rox-term-Slox (BBa_K2406071) </li>
 +
                        <li>Rox-term-Vox (BBa_K2406073) </li>
 +
                        <li>Lox-term-Vox (BBa_K2406075) </li>
 +
                        <li>Vlox-term-Rox (BBa_K2406077) </li>
 +
                        <li>Lox-term-Rox (BBa_K2406079) </li>
 +
                    </ol><br>
 +
                    <strong> Plate on LB agar with chloramphenicol </strong>
 +
                </li>
 +
                <li>
 +
                    If the users do not have miniprepped DNA of pSB4K5 backbone, transform E. coli DH5α with it as well, and plate on LB agar
 +
                    with kanamycin.
 +
                </li>
 +
                <li>
 +
                    Incubate all the LB agar plates with transformants overnight at 37ºC.
 +
                </li>
 +
            </ol><br><br>
  
 +
            <p> Day 2: </p>
 +
            <ol start="5" type="1" style="list-style-image: none; font-size: 17px;">
 +
                <li>Inoculate the twenty transformed parts (pSB4K5 too if done) with 5 mL LB broth with chloramphenicol. Shake
 +
                    at 220 rpm, 37ºC overnight (16 – 18 hours)
 +
                </li>
 +
            </ol><br><br>
  
 +
            <p> Day 3: </p>
 +
            <ol start="6" type="1" style="list-style-image: none; font-size: 17px;">
 +
                <li>Miniprep the twenty parts (pSB4K5 too if done) using the miniprep kit. </li>
 +
                <li>Reserve the fifteen measurement devices for later use. For the inducible recombinase generator, mix its miniprep
 +
                    with the miniprep of pSB4K5, and perform restriction digest and heat inactivation with EcoRI and SpeI
 +
                    according to the recommendation by supplier. </li>
 +
                <li>Add T4 DNA ligase and its buffer to the heat-inactivated reaction mix (make sure that the T4 DNA ligase is
 +
                    not incompatible with the previous buffer). Incubate according to the supplier’s recommendation. </li>
 +
                <li>Transform the ligated products (a total of five, one for each recombinases) into E. coli BL21 (DE3). Plate
 +
                    on LB agar with kanamycin. </li>
 +
            </ol><br><br>
  
 +
            <p> Day 4: </p>
 +
            <ol start="10" type="1" style="list-style-image: none; font-size: 17px;">
 +
                <li>Pick out a white colony from each of the five transformations done yesterday. Inoculate the white colony
 +
                    in 10 mL of LB broth + kanamycin overnight (220 rpm, 37ºC).
 +
                </li>
  
−
             <p> We also discovered interdisciplinarity in SMORE. Our cell sorter is a fusion of biology, engineering and design,
+
             </ol><br><br>
−
                made by our biologist and product designer. This made us think: how important is interdiciplinarity in synthetic
+
−
                biology? Can we make synthetic biology more interdisciplinary, to facilitate creativity and innovation? </p>
+
−
            <p> Our human practices start by exploring interdisciplinarity in synthetic biology. We conducted a skill exchange
+
−
                survey to find out the challenges in interdisciplinary collaboration. To study the interdisciplinary status
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−
                of synthetic biology, we asked if more diverse teams perform better in iGEM? The results of both were thought-provoking,
+
−
                leading to our work in accessibility. </p>
+
  
−
             <p> References </h2>
+
             <p> Day 5: </p>
−
                 <p> 1. Klein, J.T. 1990. Interdisciplinarity: History, Theory and Practice. Detriot: Wayne State University Press.
+
            <ol start="11" type="1" style="list-style-image: none; font-size: 17px;">
−
                 </p>
+
                 <li>Inoculate 100 mL of LB broth + kanamycin with 1 mL of overnight culture. Shake at 220 rpm, 37ºC for approximately
−
                 <p> 2. Nissani, M. 1997. Ten Cheers for Interdisciplinarity: The Case for Interdisciplinary Knowledge and Research.
+
                    2 hours, or until the OD600 reaches 0.3 – 0.6 (absorbance at 600 nm).
−
                    The Social Science Journal 34(2):201–216. </p>
+
                </li>
−
                 <p> 3. Drews, J. 2000. Drug Discovery: A Historical Perspective. Science 287(5460):1960–1964. </p>
+
                <li>Transfer the culture to two 50mL falcon tubes and leave on ice for 30 minutes. </li>
 +
                <li>Transfer at least 150 mL of 0.1M MgCl2 and 150 mL of 0.1M CaCl2 onto ice. </li>
 +
                 <li>Prepare 20 mL of CaCl2/Glycerol solution by mixing 17mL of 0.1M CaCl2 with 3mL of 100% glycerol.</li>
 +
                 <li>Centrifuge at 4000x g for 5 minutes at 4ºC. </li>
 +
                <li>Pour out the supernatant and resuspend the pellet gently with 25 mL ice cold 0.1M MgCl2. </li>
 +
                <li>Incubate on ice for 30 minutes.</li>
 +
                <li>Centrifuge at 4000x g for 5 minutes at 4ºC.</li>
 +
                <li>Pour out the supernatant and resuspend the pellet gently with 25 mL ice cold 0.1M CaCl2. </li>
 +
                <li>Incubate on ice for 30 minutes. </li>
 +
                 <li>Centrifuge at 4000x g for 5 minutes at 4ºC</li>
 +
                <li>Resuspend the pellet gently with 1.25 mL of CaCl2/Glycerol solution prepared earlier.</li>
 +
                <li>Aliquot 100 μL of cell resuspension into eppendorf tubes.</li>
 +
                <li>Flash freeze the aliquots on dry ice or liquid nitrogen.</li>
 +
                <li>Store the recombinase-expressing competent cells in -80ºC freezer.</li>
 +
            </ol><br><br>
  
−
                <h2 class="header-subsection"> Skill Exchange Survey </h2>
+
            <p> Day 6: </p>
−
                 <p> We began our investigation into interdisciplinarity with a simple question: how beneficial and difficult
+
            <ol start="26" type="1" style="list-style-image: none; font-size: 17px;">
−
                     is it to work with someone from another background? In a skill exchange survey administered to our team
+
                <li>Thaw the recombinase-expressing competent cells on ice. You will need five tubes for each recombinase.</li>
−
                    and our iGEM collaborators, we developed a series of questions targeted to members involved in a “skill
+
                 <li>Transform the measurement devices into the competent cells according to this table:<br>
−
                    exchange”. We defined “skill exchange” as an event where one member taught another in an area previously
+
                     <table>
−
                    untrained for the latter. For example, one of our biologist brought our product designer into the lab
+
                        <tr>
−
                    for training in basic protocols, including miniprep and agarose gel electrophoresis. </p>
+
                            <th>Competent cells</th>
−
                <p> Overall, trainees were surprisingly positive about the learning experience. While they would often appear
+
                            <th>Measurement devices to transform</th>
−
                    uneasy when approaching with unfamiliar work, they often reported the communication with the trainer
+
                        </tr>
−
                    was straightforward, and the trainer’s expectations were reasonable. </p>
+
                        <tr>
−
                <p> Particularly, a well-defined protocol has been identified to greatly helped the trainee. The protocol provides
+
                            <td> Cre </td>
−
                    clear and consistent terminology for both parties, and it gave a reference that the trainee can depend
+
                            <td>
−
                    on, especially when they forget a task or technical terms. The trainers also reported the protocol aided
+
                                <ol start="a" type="a" style="list-style-image: none; font-size: 17px;">
−
                    them to perform demonstrations, which allowed the trainee to follow in a logical and precise manner.
+
                                    <li>Lox-term-Lox (BBa_K2406055) </li>
−
                    </p>
+
                                    <li>Vlox-term-Lox (BBa_K2406057) </li>
−
                <p> Another aspect that the participants reported as beneficial was previous cooperation experience. While it
+
                                    <li>Slox-term-Lox (BBa_K2406061) </li>
−
                    may seem obvious, the results clearly pointed out when both parties were already friends or past collaborators,
+
                                    <li>Lox-term-Vox (BBa_K2406075) </li>
−
                    they find communication easier. In other words, practice makes perfect. Even if communication is initially
+
                                    <li>Lox-term-Rox (BBa_K2406079) </li>
−
                    difficult, it is worthwhile to continuously ironing out the kinks. </p>
+
                                </ol>
−
                <p> Nonetheless, as the survey identified, difficulties in collaboration remain. A common theme was that communication
+
                            </td>
−
                    became difficult when too many technical terms were used by the trainer. Naturally, every discipline
+
                        </tr>
−
                    develops jargons. And despite the reported attempts of trainer to minimise their use of technical language,
+
                        <tr>
−
                    trainees still frequently complained that it hindered their understanding. Therefore, it is likely interdisciplinary
+
                            <td> Dre </td>
−
                    collaborators were already aware of the drawback of technical language, but it should still be consciously
+
                            <td>
−
                    and deliberately controlled. </p>
+
                                <ol start="f" type="a" style="list-style-image: none; font-size: 17px;">
−
                <p> Interestingly, there was a disconnect between trainer and trainee not before, but after the skill exchange.
+
                                    <li>Rox-term-Rox (BBa_K2406051) </li>
−
                    The last question of our survey asked both parties if the trainee can repeat the work independently.
+
                                    <li>Rox-term-Slox (BBa_K2406071) </li>
−
                    In every questionnaire, the answer from the trainer, with varying amounts of added qualifications, was
+
                                    <li>Rox-term-Vox (BBa_K2406073) </li>
−
                    positive. However, every trainee reported they would feel uncomfortable without guidance. Therefore,
+
                                    <li>Vlox-term-Rox (BBa_K2406077) </li>
−
                    our questionnaire highlighted a disconnect in confidence. To illustrate, an experienced biologist deemed
+
                                    <li>Lox-term-Rox (BBa_K2406079) </li>
−
                    miniprep trivial, but every non-biologist that was taught in miniprep reported they liked further assistance
+
                                </ol>
−
                    to master the process. </p>
+
                            </td>
−
                <p> Therefore, it is crucial to be reminded communication and collaboration are not one-time events. Instead,
+
                        </tr>
−
                    experts should consider them a long-term endeavour that requires multiple sessions before learners can
+
                        <tr>
−
                    fully impart the skill and knowledge. </p>
+
                            <td> Vika </td>
−
                <br>
+
                            <td>
−
                <p> Links: </p>
+
                                <ol start="k" type="a" style="list-style-image: none; font-size: 17px;">
−
                <a href="https://static.igem.org/mediawiki/2017/2/2b/T--Edinburgh_UG--interdisciplinarity_survey_bulgaria.pdf" style="font-size: 20px"> [Skill exchange surveys from Bulgaria] </a><br>
+
                                    <li>Vox-term-Vox (BBa_K2406053) </li>
−
                <a href="https://static.igem.org/mediawiki/2017/a/ab/T--Edinburgh_UG--interdisciplinarity_survey_edinburgh.pdf" style="font-size: 20px"> [Link for Skill exchange surveys from Edinburgh] </a><br>
+
                                    <li>Vox-term-Vlox (BBa_K2406067) </li>
−
                <a href="https://static.igem.org/mediawiki/2017/d/d2/T--Edinburgh_UG--interdisciplinarity_survey_israel.pdf" style="font-size: 20px"> [Link for Skill exchange surveys from Israel] </a>
+
                                    <li>Vox-term-Slox (BBa_K2406069) </li>
 +
                                    <li>Rox-term-Slox (BBa_K2406071) </li>
 +
                                    <li>Lox-term-Vox (BBa_K2406075) </li>
 +
                                </ol>
 +
                            </td>
 +
                        </tr>
 +
                        <tr>
 +
                            <td> VCre </td>
 +
                            <td>
 +
                                <ol start="p" type="a" style="list-style-image: none; font-size: 17px;">
 +
                                    <li>Vlox-term-Lox (BBa_K2406057) </li>
 +
                                    <li>Slox-term-Vlox (BBa_K2406063) </li>
 +
                                    <li>Vox-term-Vlox (BBa_K2406067) </li>
 +
                                    <li>Vlox-term-Rox (BBa_K2406077) </li>
 +
                                    <li>Vlox-term-Vlox (BBa_K2406059) </li>
 +
                                </ol>
 +
                            </td>
 +
                        </tr>
 +
                        <tr>
 +
                            <td> SCre </td>
 +
                            <td>
 +
                                <ol start="u" type="a" style="list-style-image: none; font-size: 17px;">
 +
                                    <li>Slox-term-Slox (BBa_K2406065) </li>
 +
                                    <li>Slox-term-Lox (BBa_K2406061) </li>
 +
                                    <li>Slox-term-Vlox (BBa_K2406063) </li>
 +
                                    <li>Vox-term-Slox (BBa_K2406069) </li>
 +
                                    <li>Rox-term-Slox (BBa_K2406071) </li>
 +
                                </ol>
 +
                            </td>
 +
                        </tr>
 +
                    </table>
 +
                </li>
 +
            </ol><br><br>
  
 +
            <p> Day 7: </p>
 +
            <ol start="28" type="1" style="list-style-image: none; font-size: 17px;">
 +
                <li>Inoculate a colony from each of the transformation into 5 mL of LB broth + chloramphenicol and kanamycin
 +
                    in the morning. <br> *With only 96 wells in each plate, it is recommended to split the 25 transformations
 +
                    into several experiments, inoculate only a portion of the transformants each time.
 +
                </li>
 +
                <li>When the OD600 reaches 0.3 – 0.6, dilute the cultures with LB broth + chloramphenicol and kanamycin until
 +
                    all the cultures have similar OD600, between 0.1 – 0.2. </li>
 +
                <li>Aliquot 100 μL of the diluted cultures into the 96-well plates. Reserve a column for loading the blank (LB
 +
                    Broth with chloramphenicol and kanamycin, without any E. coli). To half of the cultures, add 0.5 μL of
 +
                    0.1M IPTG to reach a final concentration of about 500 μM.</li>
 +
                <li>Seal the 96-well plate and put into the plate reader. Measure the RFP fluorescence intensity every 30 minutes
 +
                    over 48 hours with the following conditions:
 +
                    <ul style="list-style-image: none; list-style-type: disc;">
 +
                        <li>Excitation: 584 nm </li>
 +
                        <li>Emission: 610 nm </li>
 +
                        <li>Shaking frequency: 300 rpm </li>
 +
                        <li>Shaking mode: double orbital </li>
 +
                        <li>Incubation temperature: 37ºC</li>
 +
                    </ul>
  
−
                 <h2 class="header-subsection"> Skill Exchange Survey </h2>
+
                 </li>
−
                <p> Given interdisciplinarity is thought to benefit academic research, we like to know the status of interdiciplinarity
+
            </ol><br><br>
−
                    in synthetic biology, more specifically, in iGEM. iGEM is opened to students from all disciplines and
+
−
                    is interdisciplinary by nature. For example, human practices and modelling represent social sciences
+
−
                    and mathematical sciences, respectively. Moreover, iGEM indirectly qualifies projects in terms of medals
+
−
                    and prizes, providing a model system to measure the benefit of interdiciplinarity. </p>
+
  
−
                <p> To measure the interdisciplinarity of past iGEM teams, we recorded the discipline of each iGEM participant
 
−
                    from 2013 to 2015. It is noteworthy that a diversity in disciplines does not always imply interdisciplinarity.
 
−
                    Even in a diverse team, members may not exchange in skills or closely cooperate. But, diversity is a
 
−
                    close and practical measure. The measurement of interdiciplinarity is correlated to their tracks, and
 
−
                    medals to observe the relationship between interdisciplinarity and performance in iGEM. </p>
 
  
−
                <h2 style="font-style: italic; font-size: 20px;"> Measurement of Interdisciplinarity </h2> <br>
+
            <h2 class="header-subsection"> Results </h2>
 +
            <p> Results are summarized below. We have obtained quantitative results regarding the cross-reactivity of our four
 +
                recombinases: Dre, Vika, VCre, and SCre. Our devices detected some unexpected cross-reactivity. For example,
 +
                VCre and SCre were shown to recognize one another’s target site. Below is a “heat map” of every recombinases
 +
                activity with respect to every measurement device tested. Note grey corresponds to constructs that were not tested due to time constraints. Also included is a graph demonstrating observed
 +
                fluorescence output over time for every single measurement taken by our team. </p><br><br>
  
−
                <p> Interestingly, the measure of interdiciplinarity is a product of interdisciplinarity itself. Diversity of
+
            <div style="text-align: center;"></div><br><br>
−
                    disciplines is analogous to biodiversity, which ecologists have been studying. Here, we use an ecology-inspired
+
−
                    interdisciplinarity index: Rao-Stirling index, to quantify the following aspects of interdiciplinarity
+
−
                    [1]: </p>
+
  
−
                <ul style="list-style-type: none; list-style-image: none; font-size: 17px;">
+
<figure style="text-align: center">
−
                    <li> <strong> Variety: </strong> how many different disciplines are within each iGEM team?</li>
+
                <a target="_blank" href="https://static.igem.org/mediawiki/2017/e/e3/T--Edinburgh_UG--measurement_heatmap.png">
−
                    <li> <strong> Balance: </strong> are participants of each discipline of similar proportions? </li>
+
                <img class="same-width" src="https://static.igem.org/mediawiki/2017/e/e3/T--Edinburgh_UG--measurement_heatmap.png">
−
                    <li> <strong> Disparity: </strong> how different are the disciplines in the iGEM team? E.g. the difference
+
                <br></a>
−
                        between linguistics and synthetic biology is far greater than that between cell biology and synthetic
+
                <figcaption style="margin: 0 auto; max-width: 600px; padding-top: 20px; font-size: 16px; text-align: justify;"> Heat map showing describing the orthogonality of different recombinase systems in E. coli. Percentage of orthogonality is normalised with Rox-Rox in Dre being 100%.</figcaption>
−
                        biology.
+
            </figure> <br> <br>
−
                    </li>
+
−
                </ul>
+
−
                <br>
+
  
−
                <p>We chose the Rao-Stirling index due to its incorporation of all three aspects and its well-known uses in
+
            <div style="text-align: center;"><img class="same-width" src="https://static.igem.org/mediawiki/2017/c/ce/T--Edinburgh_UG--17_Oct.png"></div><br><br>
−
                    social sciences [2-3]. Moreover, Rao-Stirling index, similar to its ecological counterpart Rao’s quadratic
+
<div style="text-align: center;"><img class="same-width" src="https://static.igem.org/mediawiki/2017/2/26/T--Edinburgh_UG--20_Oct.png"></div><br><br>
−
                    entropy, behave mathematically in a fashion closely resembling the human intuition of diversity [4].
+
−
                    For example, the index is very sensitive to the addition of a highly different discipline and an increase
+
−
                    in overall range of values covered.</p>
+
  
 +
            <h2 class="header-subsection"> Discussion </h2>
 +
            <p> The results show that are measurement constructs function and are able to detect cross-reactivity between two
 +
                recombinases. Indeed, observed sensitivity of our devices was so high that we demonstrated significant cross-reactivity
 +
                between SCre and VCre, two recombinases originally reported as orthogonal [1]. Furthermore, we demonstrate
 +
                that there is minimal cross-reactivity between Dre [2], Vika [3], and Cre, indicating that these recombinases
 +
                can be used to catalyse distinct recombination events within one cell. </p>
 +
            <p>Our measurement protocol is also very repeatable. Any team wishing to carry out their own measurement using our
 +
                parts could simply order the relevant parts from the registry and follow our above protocol. Also, the principle
 +
                behind these measurement devices is useful for other projects. For example, if another team wanted to utilize
 +
                Gin and Tn3 invertases [4][5] in one cell, they would be wise to first test if the two systems were orthogonal.
 +
                They could achieve this by following our design plan and our wet-lab protocol. Our project would simplify
 +
                the process of designing constructs through our web-based support tool: our oligo designer. Our recombinase
 +
                target sites could be tricky to design because they contain repetitive sequences that cannot be chemically
 +
                synthesized. </p>
 +
            <p>In conclusion, we have developed a robust tool for assessing recombinase activity. Our measurement devices provided
 +
                invaluable insight into our own project by distinguishing which of our recombinases were orthogonal and which
 +
                would cross-react. The devices we have produced are valuable to the iGEM community as a whole due to our
 +
                detailed protocol, submission of standardized measurement devices, generalizable platform, and web-based
 +
                support for designing elements of the devices.</p>
  
−
                <p style="text-align: center;">Rao-Stirling Index: $$D = \sum _{ij,i \neq j} d_{ij}p_{i}p_{j}$$</p>
 
−
                <p>where D is the interdisciplinarity measure. pi and pj are proportions of discipline i and discipline j in
 
−
                    the team, respectively. dij is the difference between discipline i and discipline j, measured by Euclidean
 
−
                    distance. (Note: some versions of Rao-Stirling Index include parameters alpha and beta [3]. But, they
 
−
                    are inapplicable to this study and are not discussed here.)</p>
 
−
                <p>The measurement of disparity (dij) is less straightforward than variety (i, j) and balance (pi, pj). We devised
 
−
                    a simple framework based on the Biglan model, a well-established categorisation of disciplines, to semi-quantify
 
−
                    each discipline in the following three qualities [5]:</p>
 
  
−
                <ul style="list-style-type: none; list-style-image: none; font-size: 17px;">
+
            <h2 class="header-subsection"> References </h2>
−
                    <li> <strong> Foundational – Applicational: </strong> is the knowledge gained of pure academic interest (e.g.
+
            <p> [1] Suzuki E. and Nakayama, M. 2011. “VCre/VloxP and SCre/SloxP: new site-specific recombination systems for genome engineering.” Nucleic Acids Research 39(8):e49.</p>
−
                        philosophy), or of applicational value outside academia (e.g. software engineering)?</li>
+
            <p> [2] Anastassiadis, K., Fu, J., Patsch, C., Hu, S., Weidlich, S., Duerschke, K., Buchholz, F., Edenhofer, F., and Stewart A.F. 2009. “Dre recombinase, like Cre, is a highly efficient site-specific recombinase in E. coli, mammalian cells and mice.” Disease Models and Mechanisms: Sep-Oct; 2(9-10):508-515</p>
−
                    <li> <strong> Hard - soft: </strong> if a paradigm exists to be followed (e.g. engineering) or if qualitative
+
            <p> [3] Karimova, M., Abi-Ghanem, J., Berger, N., Surendranath, V., Pisabarro, M.T., Buchholz, F. 2013 “Vika/vox, a novel efficient and specific Cre/loxP-like site-specific recombination system”. Nucleic Acids Research 41(2):e37.</p>
−
                        discussion is the only requirement (e.g. philosophy)? </li>
+
            <p>[4] Maeser, S. and Kahmann, R. 1991 “The Gin recombinase of phage Mu can catalyse site-specific recombination in plant protoplasts.” Molecular Genetics and Genomics 230(1-2):170-176</p>
−
                    <li> <strong> Life - non-life:</strong> is the discipline concerned with living organisms (e.g. biology) or
+
            <p>[5] Olorunniji, F.J. and Stark, W.M. 2010 “Catalysis of site-specific recombination by Tn3 resolvase”. Biochemical Society Transactions 38(2):417-421</p>
−
                        is the discipline unconnected and unapplied directly to any living organisms (e.g. software engineering)?
+
−
                    </li>
+
−
                </ul>
+
−
                <br>
+
  
−
                <p>We score each discipline from 1 to 7 for each of the three qualities. Euclidean distance (dij) is calculated
+
           
−
                    by using the scores as three-dimensional coordinates. For example, a subject with scores 3, 6, 7 would
+
−
                    have a coordinate (3, 6, 7) in a (x, y, z) format. We then used the data to calculate a diversity value
+
−
                    for each team.
+
−
                </p>
+
−
 
+
−
                <h2 style="font-style: italic; font-size: 20px;"> Data Collection and Analysis </h2><br>
+
−
 
+
−
 
+
−
                <p>It should be noted that we have collected and analysed the data in the following way: </p>
+
−
                <ul style="list-style-type: disc; color: black; font-size: 17px;">
+
−
                    <li>iGEM teams with an ambiguous or incomplete record on disciplines of team members were excluded from the
+
−
                        analysis</li>
+
−
                    <li>iGEM teams that received a blocked medal were excluded from the analysis, as a blocked medal does not
+
−
                        correlate with the quality of the project</li>
+
−
                    <li>Datasets from different years were analysed separately, as the award criteria were not consistent throughout
+
−
                        2013-2015
+
−
                    </li>
+
−
                </ul>
+
−
 
+
−
                <p>We respect the achievements of every iGEM team and we promise that the sole purpose of the collection of
+
−
                    data was to understand interdisciplinarity in synthetic biology. Data was collected from publicly available
+
−
                    iGEM team websites. </p>
+
−
 
+
−
                <h2 style="font-style: italic; font-size: 20px;"> Results </h2><br>
+
−
 
+
−
                <p>Distribution of discipline diversity is grouped by medals (Gold, Silver, Bronze and None; Figure 1). Disappointingly,
+
−
                    no significant difference in interdisciplinarity was detected between medal winners in any year (p >
+
−
                    0.1 for all combinations; ANOVA).</p>
+
−
 
+
−
                <figure style="max-width: 900px; margin: 0 auto;">
+
−
                    <img src="https://static.igem.org/mediawiki/2017/5/5e/T--Edinburgh_UG--interdisciplinarity1.jpg">
+
−
                    <figcaption style="text-align: center;"> <strong>Figure 1:</strong> distribution of interdisciplinarity measure grouped by iGEM medals. Whiskers
+
−
                        represent the range of values. Box indicates median and interquartile range (25th and 75th percentile).</figcaption>
+
−
 
+
−
                </figure>
+
−
                <br><br>
+
−
                <p>This was surprising as the interdisciplinary that benefitted on our team was not reflected in the data. Why
+
−
                    is that? Here are some possibilities:</p>
+
−
 
+
−
                <ul style="list-style-image: none; list-style-type: disc; color: black; font-size: 17px;">
+
−
                    <li>While interdisciplinary may theoretically facilitate innovation and skill exchange, practical difficulties
+
−
                        in communication and cooperation remain a great obstacle, as reflected from the skill exchange survey.
+
−
                        The benefits were offset by poor integration of a diverse skillset.</li>
+
−
                    <li>A diversity in discipline fails to capture the exchange of skill and knowledge within the cooperation
+
−
                        – the essence of interdisciplinarity. </li>
+
−
                    <li>iGEM is an interdisciplinary competition with a core of synthetic biology. Thus, a focus in biologists
+
−
                        and engineers in team composition is often required for most tracks in iGEM. The optimal interdisciplinarity
+
−
                        was a balance, rather than a complete diversity.
+
−
                    </li>
+
−
                </ul>
+
−
                <p>These results were thought-provoking, as we expected the potential of interdisciplinarity can be fully realised
+
−
                    in iGEM. This expresses a dire need to improve the integration of interdisciplinarity in iGEM.</p>
+
−
                <p>How can we better promote diversity and innovation in iGEM? We went back to our skill exchange survey to
+
−
                    address the problems of interdisciplinary collaboration. We start by making SMORE a tool accessible to
+
−
                    researchers from all backgrounds and disciplines, encouraging an influx of perspectives and ideas. </p>
+
−
                <br>
+
−
 
+
−
                <a href="https://2017.igem.org/Team:Edinburgh_UG/HP/Accessibility" style="font-size: 20px;"> Take a look at how we do this on the Accessibility page. </a><br><br>
+
−
 
+
−
                <a href="https://static.igem.org/mediawiki/2017/c/c2/T--Edinburgh_UG--interdisciplinarity_python.ipynb.txt" style="font-size: 17px;"> [iPython file for calculation for Rao-Stirling Index and data visualisation] </a><br>
+
−
                <a href="https://static.igem.org/mediawiki/2017/f/fd/T--Edinburgh_UG--interdisciplinarity2.xlsx" style="font-size: 17px;"> [Diversity data] </a>
+
−
 
+
−
                <h2 class="header-subsection"> References </h2>
+
−
                <p> 1. Stirling, A. 2007. A general framework for analysing diversity in science, technology and society. Journal
+
−
                    of the Royal Society Interface 4:707–719.</p>
+
−
                <p> 2. Porter, A.L. and Rafols, I. 2009. Is science becoming more interdisciplinary? Measuring and mapping six
+
−
                    research fields over time. Scienometrics 81(3):719–745.</p>
+
−
                <p> 3. Rafols, I. and Meyer, M. 2010. Diversity and network coherence as indicators of interdisciplinarity: case
+
−
                    studies in bionanoscience. Scientometrics 82(2):263–287.</p>
+
−
                <p> 4. Schleuter, A.D., Daufresne, M., Massol, F., Argillier, C., Schleuter, D., Daufresne, M., Massol, F. and
+
−
                    Argillier, C. 2010. A user’s guide to functional diversity indices. Ecological Monographs 80(3):469–484.</p>
+
−
                <p> 5. Biglan, A. 1973. The characteristics of subject matter in different academic areas. Journal of Applied
+
−
                    Psychology 57(3):195–203.</p>
+
  
  

Latest revision as of 22:56, 31 October 2017





Measurement


Background

As a standardized toolkit for site-specific recombination, SMORE complies with the principle of biobrick and synthetic biology, emphasizing on accurate and repeatable measurements of our parts. This is why we have developed simple protocols and specific biobricks to quantify the activity of recombination, in terms of both efficiency and orthogonality.

This protocol involves i) transferring the recombinase generator from pSB1C3 to pSB4K5; ii) transformation of T7 polymerase expressing E. coli strain with recombinase generator in pSB4K5; iii) make competent cells out of the recombinase-carrying E. coli; and iv) transformation of recombinase-carrying E. coli with the appropriate measurement device. The entire experiment takes 7 days.

Protocol

Material needed

  1. Escherichia coli strain for cloning purpose (e.g. TOP10, DH5α)
  2. Any T7 polymerase expressing E. coli strain (e.g. BL21 (DE3))
  3. Biobricks: pSB4K5 plasmid backbone
  4. Biobricks: T7-LacO-regulated Cre/Dre/VCre/SCre/Vika recombinase generators (BBa_K2406080, BBa_K2406081, BBa_K2406082, BBa_K2406083, BBa_K2406084)
  5. Biobricks: Measurement device (BBa_K2406051, BBa_K2406053, BBa_K2406055, BBa_K2406057, BBa_K2406059, BBa_K2406061, BBa_K2406063, BBa_K2406065, BBa_K2406067, BBa_K2406069, BBa_K2406071, BBa_K2406073, BBa_K2406075, BBa_K2406077, BBa_K2406079)
  6. Chloramphenical (stock of 25 mg/mL in EtOH
  7. Kanamycin (stock of 50 mg/mL in dH2O)
  8. IPTG (stock of 0.1 M in dH2O)
  9. LB (Luria Bertani) media and LB agar
  10. MgCl2 (Autoclaved, 0.1 M)
  11. CaCl2 (Autoclaved, 0.1 M)
  12. 100% glycerol
  13. Standard biobrick restriction enzymes – EcoRI and SpeI, and their associated enzyme buffer
  14. T4 DNA ligase and its associated enzyme buffer
  15. Miniprep kit
  16. 50 mL falcon tubes
  17. 1.5 mL eppendorf tubes
  18. Cuvettes
  19. Erlenmeyer flask (250 mL or larger)
  20. Petri dishes
  21. 96-well plates (black ,with flat and transparent bottom)
  22. Pipettes
  23. Spectrophotometer
  24. Plate reader
  25. Water bath and/or heat block
  26. Ice and ice buckets
  27. Dry ice or liquid nitrogen
  28. Microcentrifuge (for eppendorf tubes)
  29. Centrifuge (for falcon tubes)
  30. Incubator at 37ºC


Method

Day 1:

  1. Transform E. coli DH5α with the following inducible recombinase generators:
    1. T7-LacO-Cre (BBa_K2406080)
    2. T7-LacO-Dre (BBa_K2406081)
    3. T7-LacO-Vika (BBa_K2406082)
    4. T7-LacO-VCre (BBa_K2406083)
    5. T7-LacO-SCre (BBa_K2406084)

    Plate on LB agar with chloramphenicol
  2. Transform E. coli DH5α with the following measurement constructs:
    1. Rox-term-Rox (BBa_K2406051)
    2. Vox-term-Vox (BBa_K2406053)
    3. Lox-term-Lox (BBa_K2406055
    4. Vlox-term-Lox (BBa_K2406057)
    5. Vlox-term-Vlox (BBa_K2406059)
    6. Slox-term-Lox (BBa_K2406061)
    7. Slox-term-Vlox (BBa_K2406063)
    8. Slox-term-Slox (BBa_K2406065)
    9. Vox-term-Vlox (BBa_K2406067)
    10. Vox-term-Slox (BBa_K2406069)
    11. Rox-term-Slox (BBa_K2406071)
    12. Rox-term-Vox (BBa_K2406073)
    13. Lox-term-Vox (BBa_K2406075)
    14. Vlox-term-Rox (BBa_K2406077)
    15. Lox-term-Rox (BBa_K2406079)

    Plate on LB agar with chloramphenicol
  3. If the users do not have miniprepped DNA of pSB4K5 backbone, transform E. coli DH5α with it as well, and plate on LB agar with kanamycin.
  4. Incubate all the LB agar plates with transformants overnight at 37ºC.


Day 2:

  1. Inoculate the twenty transformed parts (pSB4K5 too if done) with 5 mL LB broth with chloramphenicol. Shake at 220 rpm, 37ºC overnight (16 – 18 hours)


Day 3:

  1. Miniprep the twenty parts (pSB4K5 too if done) using the miniprep kit.
  2. Reserve the fifteen measurement devices for later use. For the inducible recombinase generator, mix its miniprep with the miniprep of pSB4K5, and perform restriction digest and heat inactivation with EcoRI and SpeI according to the recommendation by supplier.
  3. Add T4 DNA ligase and its buffer to the heat-inactivated reaction mix (make sure that the T4 DNA ligase is not incompatible with the previous buffer). Incubate according to the supplier’s recommendation.
  4. Transform the ligated products (a total of five, one for each recombinases) into E. coli BL21 (DE3). Plate on LB agar with kanamycin.


Day 4:

  1. Pick out a white colony from each of the five transformations done yesterday. Inoculate the white colony in 10 mL of LB broth + kanamycin overnight (220 rpm, 37ºC).


Day 5:

  1. Inoculate 100 mL of LB broth + kanamycin with 1 mL of overnight culture. Shake at 220 rpm, 37ºC for approximately 2 hours, or until the OD600 reaches 0.3 – 0.6 (absorbance at 600 nm).
  2. Transfer the culture to two 50mL falcon tubes and leave on ice for 30 minutes.
  3. Transfer at least 150 mL of 0.1M MgCl2 and 150 mL of 0.1M CaCl2 onto ice.
  4. Prepare 20 mL of CaCl2/Glycerol solution by mixing 17mL of 0.1M CaCl2 with 3mL of 100% glycerol.
  5. Centrifuge at 4000x g for 5 minutes at 4ºC.
  6. Pour out the supernatant and resuspend the pellet gently with 25 mL ice cold 0.1M MgCl2.
  7. Incubate on ice for 30 minutes.
  8. Centrifuge at 4000x g for 5 minutes at 4ºC.
  9. Pour out the supernatant and resuspend the pellet gently with 25 mL ice cold 0.1M CaCl2.
  10. Incubate on ice for 30 minutes.
  11. Centrifuge at 4000x g for 5 minutes at 4ºC
  12. Resuspend the pellet gently with 1.25 mL of CaCl2/Glycerol solution prepared earlier.
  13. Aliquot 100 μL of cell resuspension into eppendorf tubes.
  14. Flash freeze the aliquots on dry ice or liquid nitrogen.
  15. Store the recombinase-expressing competent cells in -80ºC freezer.


Day 6:

  1. Thaw the recombinase-expressing competent cells on ice. You will need five tubes for each recombinase.
  2. Transform the measurement devices into the competent cells according to this table:
    Competent cells Measurement devices to transform
    Cre
    1. Lox-term-Lox (BBa_K2406055)
    2. Vlox-term-Lox (BBa_K2406057)
    3. Slox-term-Lox (BBa_K2406061)
    4. Lox-term-Vox (BBa_K2406075)
    5. Lox-term-Rox (BBa_K2406079)
    Dre
    1. Rox-term-Rox (BBa_K2406051)
    2. Rox-term-Slox (BBa_K2406071)
    3. Rox-term-Vox (BBa_K2406073)
    4. Vlox-term-Rox (BBa_K2406077)
    5. Lox-term-Rox (BBa_K2406079)
    Vika
    1. Vox-term-Vox (BBa_K2406053)
    2. Vox-term-Vlox (BBa_K2406067)
    3. Vox-term-Slox (BBa_K2406069)
    4. Rox-term-Slox (BBa_K2406071)
    5. Lox-term-Vox (BBa_K2406075)
    VCre
    1. Vlox-term-Lox (BBa_K2406057)
    2. Slox-term-Vlox (BBa_K2406063)
    3. Vox-term-Vlox (BBa_K2406067)
    4. Vlox-term-Rox (BBa_K2406077)
    5. Vlox-term-Vlox (BBa_K2406059)
    SCre
    1. Slox-term-Slox (BBa_K2406065)
    2. Slox-term-Lox (BBa_K2406061)
    3. Slox-term-Vlox (BBa_K2406063)
    4. Vox-term-Slox (BBa_K2406069)
    5. Rox-term-Slox (BBa_K2406071)


Day 7:

  1. Inoculate a colony from each of the transformation into 5 mL of LB broth + chloramphenicol and kanamycin in the morning.
    *With only 96 wells in each plate, it is recommended to split the 25 transformations into several experiments, inoculate only a portion of the transformants each time.
  2. When the OD600 reaches 0.3 – 0.6, dilute the cultures with LB broth + chloramphenicol and kanamycin until all the cultures have similar OD600, between 0.1 – 0.2.
  3. Aliquot 100 μL of the diluted cultures into the 96-well plates. Reserve a column for loading the blank (LB Broth with chloramphenicol and kanamycin, without any E. coli). To half of the cultures, add 0.5 μL of 0.1M IPTG to reach a final concentration of about 500 μM.
  4. Seal the 96-well plate and put into the plate reader. Measure the RFP fluorescence intensity every 30 minutes over 48 hours with the following conditions:
    • Excitation: 584 nm
    • Emission: 610 nm
    • Shaking frequency: 300 rpm
    • Shaking mode: double orbital
    • Incubation temperature: 37ºC


Results

Results are summarized below. We have obtained quantitative results regarding the cross-reactivity of our four recombinases: Dre, Vika, VCre, and SCre. Our devices detected some unexpected cross-reactivity. For example, VCre and SCre were shown to recognize one another’s target site. Below is a “heat map” of every recombinases activity with respect to every measurement device tested. Note grey corresponds to constructs that were not tested due to time constraints. Also included is a graph demonstrating observed fluorescence output over time for every single measurement taken by our team.






Heat map showing describing the orthogonality of different recombinase systems in E. coli. Percentage of orthogonality is normalised with Rox-Rox in Dre being 100%.






Discussion

The results show that are measurement constructs function and are able to detect cross-reactivity between two recombinases. Indeed, observed sensitivity of our devices was so high that we demonstrated significant cross-reactivity between SCre and VCre, two recombinases originally reported as orthogonal [1]. Furthermore, we demonstrate that there is minimal cross-reactivity between Dre [2], Vika [3], and Cre, indicating that these recombinases can be used to catalyse distinct recombination events within one cell.

Our measurement protocol is also very repeatable. Any team wishing to carry out their own measurement using our parts could simply order the relevant parts from the registry and follow our above protocol. Also, the principle behind these measurement devices is useful for other projects. For example, if another team wanted to utilize Gin and Tn3 invertases [4][5] in one cell, they would be wise to first test if the two systems were orthogonal. They could achieve this by following our design plan and our wet-lab protocol. Our project would simplify the process of designing constructs through our web-based support tool: our oligo designer. Our recombinase target sites could be tricky to design because they contain repetitive sequences that cannot be chemically synthesized.

In conclusion, we have developed a robust tool for assessing recombinase activity. Our measurement devices provided invaluable insight into our own project by distinguishing which of our recombinases were orthogonal and which would cross-react. The devices we have produced are valuable to the iGEM community as a whole due to our detailed protocol, submission of standardized measurement devices, generalizable platform, and web-based support for designing elements of the devices.

References

[1] Suzuki E. and Nakayama, M. 2011. “VCre/VloxP and SCre/SloxP: new site-specific recombination systems for genome engineering.” Nucleic Acids Research 39(8):e49.

[2] Anastassiadis, K., Fu, J., Patsch, C., Hu, S., Weidlich, S., Duerschke, K., Buchholz, F., Edenhofer, F., and Stewart A.F. 2009. “Dre recombinase, like Cre, is a highly efficient site-specific recombinase in E. coli, mammalian cells and mice.” Disease Models and Mechanisms: Sep-Oct; 2(9-10):508-515

[3] Karimova, M., Abi-Ghanem, J., Berger, N., Surendranath, V., Pisabarro, M.T., Buchholz, F. 2013 “Vika/vox, a novel efficient and specific Cre/loxP-like site-specific recombination system”. Nucleic Acids Research 41(2):e37.

[4] Maeser, S. and Kahmann, R. 1991 “The Gin recombinase of phage Mu can catalyse site-specific recombination in plant protoplasts.” Molecular Genetics and Genomics 230(1-2):170-176

[5] Olorunniji, F.J. and Stark, W.M. 2010 “Catalysis of site-specific recombination by Tn3 resolvase”. Biochemical Society Transactions 38(2):417-421