Difference between revisions of "Team:Jilin China/Improve"

 
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<section class="s1">
 
<section class="s1">
 
   <div class="title_con">
 
   <div class="title_con">
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   <p><span>Tools for Curiosity</span><br>Sketching Kits</p>
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   <p>IMPROVE</p>
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  <br />
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   </div>
 
   </div>
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   <div class="title_nav"><h2>Engagement</h2></div>
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  <div style="clear:both;"></div>
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   <div class="title_nav"><h2>Improvement</h2></div>
 
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  </section>
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  <div class="bodycontent" style="padding:0;">
 
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   <!--<section class="s0"></section>-->
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   <ul class="sidenav">
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   <!--ul class="sidenav">
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   <li><a href="#paragraph_0">Preparations</a></li>
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   <li><a href="#paragraph_1">Support</a></li>
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<div>
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    <div>
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       <h3>Sketching Kit – An intuitive tool for curiosity. </h3>
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       <h2>Improvement</h2>
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       <!--div class="paragraph_banner_in sketching"><div></div></div-->
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      <p>This year, we choose Golden Gate assembly especially for construction. (Learn about our <b>Construction</b>? See our <a href="/Team:Jilin_China/Construction">Construction Page</a>) BsaI and BbsI are used as Type IIs restriction endonucleases in our construction device. At the same time, we decided to use sfGFP as our reporter protein due to its faster folded feature and higher fluorescence intensity. However, sfGFP in iGEM part registry (<a href="http://parts.igem.org/Part:BBa_I746916">BBa_I746916</a>) has a BbsI recognition site, which could be affected during Golden Gate assembly. Hence, first coming into our mind was to do a site mutagenesis, BbsI recognition site GAAGAC was altered into GAGGAT while amino acids sequence kept the same. Then we got our sfGFP(BbsI free) part(<a href="http://parts.igem.org/Part:BBa_K2541401">BBa_K2541401</a>), a BbsI-free sfGFP and especially for Golden Gate assembly(<b>figure 1.</b>).  </p>
 +
       <center>
 +
      <img src="https://static.igem.org/mediawiki/parts/7/72/SfGFP_BbsI_free-1.svg" width="50%" /></center>
 +
      <p class="figure">Figure 1. Site mutagenesis for sfGFP BbsI free.</p>
 +
      <p>Because we cannot guarantee the mutated sfGFP has the same fluorescence expression as the previous one, after further study, we found a codon optimized sfGFP<sup>[1,2]</sup> (<b>sfGFP_optimism</b>, <a href="HTTP://parts.igem.org/Part:BBa_K2541400">BBa_K2541400</a>), which removed the BbsI site and had a functional improvement upon an existing superfolder GFP(BBa_I746916).</p>
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       <p></p>
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       <h3>Nucleic Acid Electrophoresis</h3>
−
       <p>For a convenient transportation and distribution, a box to content all necessary tools and materials for pupils was taken into consideration. After several group discussions and contacts with suppliers, we finally decided to use corrugated cases as containers. Each case would contain 2 disposable culture dishes with chloramphenicol add medium, 4 disposable sterile inoculating loop a <a href="/Team:Jilin_China/HP/Supplementary">booklet</a> to introduce microorganisms and an <a href="/Team:Jilin_China/HP/Supplementary">instruction with a drawing template</a>. (links to supplementary)</p>
+
       <p>Free of the BbsI cleavage site was confirmed by nucleic acid electrophoresis(<b>figure 2.</b>). </p>
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       <div class="paragraph_banner_in packaging"><div></div></div>
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       <p class="figure">Figure 2. L1: 1kb DNA marker; L2: BBa_I746916; L3: BBa_I746916+BbsI; L4: BBa_K2541401; L5: BBa_K2541401+BbsI; L6: BBa_K2541400; L7: BBa_K2541400+BbsI; L8: 1kb DNA marker.</p>
−
       <p>With the help of STEM Platform, we prepared a drawing class for pupils as our kit’s first trail. Culture dishes were prepared at the night before the day we set off, all materials were packaged that morning. Then, iGEMers begin their teaching day!</p>
+
       <p></p>
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+
       <h3>Emission and Excitation Spectra</h3>
−
      <h3>Why we choose pupils?</h3>
+
       <p>Next, we got three types of sfGFP emission and excitation spectra. We have set the excitation and emission wavelength range from 400nm to 600nm. For these three types of sfGFP, the excitation wavelength is around 495nm and the emission wavelength is around 510nm. (<b>figure 3.</b>)</p>
−
      <p>In our country, primary students received only basic science knowledge courses and seldom have chances to participate in a hands-on experiment. What’s more, their understanding of the micro world remains superficial. Some of them even hold the opinion that all bacteria are harmful but never have a look of those tiny lives. Letting them get a close look of microorganism in a macroscopic way may help them learn more about cytobiology and a vivid mechanism of molecular biology, which would stimulate their interest in further study. And, pupils are a group of people who are full of curiosity. Hence, we decide to hold a drawing class for them, by using bacterial liquid as pigment. Therefore, masterpieces come into being.</p>
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      <img src="https://static.igem.org/mediawiki/parts/1/1e/SfGFP-1.svg" width="60%"/></center>
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       <h3>Happy Memories</h3>
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      <center><img src="https://static.igem.org/mediawiki/parts/e/e3/SfGFP_BbsI_free.svg" width="60%"/></center>
−
       <p>We came to United Experimental School of AHSJU (Affiliated High School Jilin University) and Livon, where a series of STEM (science, technology, engineering and mathematics) classes was being held to pupils in whole Changchun city. All kids were at place when appointed time has come. Our iGEMers firstly guided them into the micro world. To our surprise, a question asked by our team members triggered a fierce rushing to answer! </p>
+
      <center><img src="https://static.igem.org/mediawiki/parts/5/50/SfGFP_op.svg" width="60%"/></center>
−
      <div class="paragraph_banner_in handsUp"><div></div></div>
+
      <p class="figure">Figure 3. Three types of sfGFP emission and excitation spectra</p>
−
       <p>Therefore, we helped kids equipped well and distribute them our sketching kits, teaching them how to draw with one-to-one instructions. Finally, more than 50 dishes were collected, sealed, and brought back into incubator by our members. Waiting for the blossom of artist works. </p>
+
      <h3>Fluorescence Intensity</h3>
−
 
+
      <p>For further characterization, we detected the expression intensity of these three types of sfGFP. According to the results(<b>figure 4.</b>), we found out that the expression intensity of sfGFP_optimism is nearly 2.6 as much as the other two types. As a result, we finally choose sfGFP_optimism as our reporter protein.</p>
−
       <div class="paragraph_banner_in teaching"><div></div></div>
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      <center>
−
 
+
      <img src="https://static.igem.org/mediawiki/parts/8/83/Jilin_China-sfGFP-fluorescence-01.svg" width="60%" /></center>
−
       <h3>Online Communication Contributes to Feedback</h3>
+
      <p class="figure">Figure 4. Three Types of sfGFP  expression in <i>E.coli</i>.</p>
−
       <p>After the class, we kept in touch with those kids and their parents, by creating a chatting group online. To our delight, the class successfully draw some kids’ attention, instead of purely having fun in drawing. On the way back to laboratory, they started asking us questions. Some screenshots and translations are listed below. (For privacy, we blurred their nickname and portrait)</p>
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      <span class="figures"><img alt="Screenshots" src="https://static.igem.org/mediawiki/2018/2/20/T--Jilin_China--Engagement--Primary--Screenshot--1.jpeg" /></span>
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      <span class="HP_Quotes">
+
−
        <span class="quotes_portrait">🧒🏻</span>@Rrrrrebekah🍩 Hello teacher. My name is Xinhe Liu. I have 2 questions that I don't understand: 1) why those bacteria have colors? 2) why skin color doesn't change when people got sick and bacteria were produced in the body?<br><br>
+
−
        <span class="quotes_portrait">👩🏻‍🏫</span>@lar As what we have mentioned in class, the phenotype of living bodies (for example, the color of bacteria) is controlled by DNA. However, scientist can transform the DNA. The original version of this kind of bacteria doesn’t have color. But the bacteria we use today were added special kinds of DNAs that can produce color, which means we transform it to make it produce color~<br>
+
−
        <span class="quotes_portrait">👩🏻‍🏫</span>The bacteria that cause illness are not transformed by us, so it won't change skin color.<br><br>
+
−
        <span class="quotes_portrait">🧒🏻</span>Oh! I got it. Thank you teacher. I didn't catch it in class😜</span>
+
−
      <br>It seems that this kid is interested in the color of bacteria, and he misunderstood the relationship between the colored and the harmful kind. Meanwhile, we realized that we taught too fast. The teaching skills needs to be improved. After a while, another kid asked questions:<br><br>
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      <span class="figures"><img alt="Screenshots" src="https://static.igem.org/mediawiki/2018/7/79/T--Jilin_China--Engagement--Primary--Screenshot--2.jpeg" /></span>
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−
      <span class="HP_Quotes">
+
−
        <span class="quotes_portrait">👩🏻‍🏫</span>Feel free to ask any questions at any time!😀<br><br>
+
−
        <span class="quotes_portrait">👦🏻</span>Hello teacher. I'm Haoran Ma. I have a question for you.🙏 Bacteria can fall into two kinds, beneficial bacteria and harmful bacteria. What good could them do to human body? or will it could turn into harmful bacteria suddenly?<br><br>
+
−
        <span class="quotes_portrait">👩🏻‍🏫</span>Beneficial bacteria vary a lot. Take lactobacillus as an example. The lactobacillus in our body can promote intestinal peristalsis, and they can activate immune cells to enhance immunity. The benefits that it could bring is a great many. 😀 And in recent study, scientists have found an amount of different kinds of bacteria have connections to cancer, hypertension and other illness, and their mechanisms are under discovering.<br><br>
+
−
        <span class="quotes_portrait">👦🏻</span>Thanks, teacher👏🏻.<br><br>
+
−
        <span class="quotes_portrait">👩🏻‍🏫</span>For the second question, I think the beneficial won't change into harm under stable conditions. However, if you take it in too much, the beneficial would be harmful, like diarrhea. 💩<br><br>
+
−
        <span class="quotes_portrait">👦🏻</span>😱.<br>
+
−
        <span class="quotes_portrait">👦🏻</span>Thank you for helping.<br><br>
+
−
        <span class="quotes_portrait">👩🏻‍🏫</span>Actually, there're also some neutral bacteria in gut. They help the beneficial when the beneficial become dominated in gut while help the other on the contrary. In a word, microorganisms vary a lot. And gut microbial problems remain to be explored.<br>
+
−
        <span class="quotes_portrait">👩🏻‍🏫</span>Thank you for your questions.😀</span>
+
−
      <br>This kid paid more attention on the effect of bacteria. And his worrying proved the necessity of our class. However, we somehow failed to deliver the right concept to them in class. Our next class needs to be reconsidered.<br>
+
−
      </p>
+
−
      <h3>Fantastic Works</h3>
+
−
      <p>One day passed, we checked dishes. Colonies had formed. Some of them are corresponding to the given template, some are not, while others are too abstract to recognize, but looks pretty. We believe they want to draw their own story! Then, we took dishes pictures and post them to the group. Before destroying them, we put them all together, and removed caps, taking a group photo!</p>
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    <div class="paragraph_banner dishes"><div></div></div>
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     <div>
 
     <div>
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       <div class="HP_Reflections"><h3>Time to Conclude</h3>
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       <h2>Reference</h2>
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      <p style="display:none;"></p>
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      <ul>
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       <p>This is our first time to be real teachers. We have to admit that we need to improve a lot: teaching skills, patience, communication skills with children, difficulty and pace of the courses… We hope that we can have more experiences in the future EPE activities, especially when talking with children and students. Then more people would be willing to learn about synthetic biology.</p>
+
       <li>[1]Segall-Shapiro, Thomas H., Eduardo D. Sontag, and Christopher A. Voigt. "Engineered promoters enable constant gene expression at any copy number in bacteria." Nature biotechnology 36.4 (2018): 352.</li>
−
       <p>Of course, during the activity with the children, we were really surprised by their enthusiasm, curiosity and earnest. Although it would be a little hard for them to use inoculating loop or this is their first time to know what is <i>E.coli</i>, they truly have pure interest in science and nature. As an iGEMer, we definitely support them and guide them to the scientific world. It must be an unforgettable experience in their childhood.</p>
+
       <li>[2]Overkamp, Wout, et al. "Benchmarking various GFP variants in Bacillus subtilis, Streptococcus pneumoniae and Lactococcus lactis for live cell imaging." Applied and Environmental Microbiology (2013): AEM-02033.</li>
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Latest revision as of 22:02, 17 October 2018

IMPROVE

Improvement

  • Improvement

    This year, we choose Golden Gate assembly especially for construction. (Learn about our Construction? See our Construction Page) BsaI and BbsI are used as Type IIs restriction endonucleases in our construction device. At the same time, we decided to use sfGFP as our reporter protein due to its faster folded feature and higher fluorescence intensity. However, sfGFP in iGEM part registry (BBa_I746916) has a BbsI recognition site, which could be affected during Golden Gate assembly. Hence, first coming into our mind was to do a site mutagenesis, BbsI recognition site GAAGAC was altered into GAGGAT while amino acids sequence kept the same. Then we got our sfGFP(BbsI free) part(BBa_K2541401), a BbsI-free sfGFP and especially for Golden Gate assembly(figure 1.).

    Figure 1. Site mutagenesis for sfGFP BbsI free.

    Because we cannot guarantee the mutated sfGFP has the same fluorescence expression as the previous one, after further study, we found a codon optimized sfGFP[1,2] (sfGFP_optimism, BBa_K2541400), which removed the BbsI site and had a functional improvement upon an existing superfolder GFP(BBa_I746916).

    Nucleic Acid Electrophoresis

    Free of the BbsI cleavage site was confirmed by nucleic acid electrophoresis(figure 2.).

    Figure 2. L1: 1kb DNA marker; L2: BBa_I746916; L3: BBa_I746916+BbsI; L4: BBa_K2541401; L5: BBa_K2541401+BbsI; L6: BBa_K2541400; L7: BBa_K2541400+BbsI; L8: 1kb DNA marker.

    Emission and Excitation Spectra

    Next, we got three types of sfGFP emission and excitation spectra. We have set the excitation and emission wavelength range from 400nm to 600nm. For these three types of sfGFP, the excitation wavelength is around 495nm and the emission wavelength is around 510nm. (figure 3.)

    Figure 3. Three types of sfGFP emission and excitation spectra

    Fluorescence Intensity

    For further characterization, we detected the expression intensity of these three types of sfGFP. According to the results(figure 4.), we found out that the expression intensity of sfGFP_optimism is nearly 2.6 as much as the other two types. As a result, we finally choose sfGFP_optimism as our reporter protein.

    Figure 4. Three Types of sfGFP expression in E.coli.

  • Reference

    • [1]Segall-Shapiro, Thomas H., Eduardo D. Sontag, and Christopher A. Voigt. "Engineered promoters enable constant gene expression at any copy number in bacteria." Nature biotechnology 36.4 (2018): 352.
    • [2]Overkamp, Wout, et al. "Benchmarking various GFP variants in Bacillus subtilis, Streptococcus pneumoniae and Lactococcus lactis for live cell imaging." Applied and Environmental Microbiology (2013): AEM-02033.