Team:HZAU-China/Labnotes

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             <a href="https://igem.org/Team.cgi?year=2014&team_name=HZAU-China"><img src="https://static.igem.org/mediawiki/2014/b/bb/Hazuteamlogo918.png" alt="HZAU-China" /></a>
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                 <li class="dropdown"><a href="https://2014.igem.org/Team:HZAU-China/Project">Project</a>
                 <li class="dropdown"><a href="https://2014.igem.org/Team:HZAU-China/Project">Project</a>
    <ul>  
    <ul>  
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                         <li><a href="https://2014.igem.org/Team:HZAU-China/Project"><span>-</span>Background</a></li>
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                         <li><a href="https://2014.igem.org/Team:HZAU-China/Design"><span>-</span>Overview</a></li>
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<li><a href="https://2014.igem.org/Team:HZAU-China/Design"><span>-</span>Design overview</a></li>
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                        <li><a href="https://2014.igem.org/Team:HZAU-China/Background"><span>-</span>Background</a></li>
<li><a href="https://2014.igem.org/Team:HZAU-China/Input"><span>-</span>Input module</a></li>
<li><a href="https://2014.igem.org/Team:HZAU-China/Input"><span>-</span>Input module</a></li>
<li><a href="https://2014.igem.org/Team:HZAU-China/Processing"><span>-</span>Processing module</a></li>
<li><a href="https://2014.igem.org/Team:HZAU-China/Processing"><span>-</span>Processing module</a></li>
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<li class="selected dropdown"><a href="https://2014.igem.org/Team:HZAU-China/Review">Wetlab</a>
<li class="selected dropdown"><a href="https://2014.igem.org/Team:HZAU-China/Review">Wetlab</a>
                     <ul>  
                     <ul>  
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                         <li><a href="https://2014.igem.org/Team:HZAU-China/Review"><span>-</span>Overview</a></li>
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                         <li><a href="https://2014.igem.org/Team:HZAU-China/Overview"><span>-</span>Overview</a></li>
<li><a href="https://2014.igem.org/Team:HZAU-China/Construction"><span>-</span>Construction</a></li>
<li><a href="https://2014.igem.org/Team:HZAU-China/Construction"><span>-</span>Construction</a></li>
<li><a href="https://2014.igem.org/Team:HZAU-China/Characterization"><span>-</span>Characterization</a></li>
<li><a href="https://2014.igem.org/Team:HZAU-China/Characterization"><span>-</span>Characterization</a></li>
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                        <li><a href="https://2014.igem.org/Team:HZAU-China/Help"><span>-</span>Help each other</a></li>
                         <li><a href="https://2014.igem.org/Team:HZAU-China/Protocol"><span>-</span>Protocol</a></li>
                         <li><a href="https://2014.igem.org/Team:HZAU-China/Protocol"><span>-</span>Protocol</a></li>
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                         <li><a href="https://2014.igem.org/Team:HZAU-China/Labnotes"><span>-</span>Labnotes</a></li>  
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                         <li><a href="https://2014.igem.org/Team:HZAU-China/Labnotes"><span>-</span>Labnotes</a></li>      
                     </ul>
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                 <li class="dropdown"><a href="https://2014.igem.org/Team:HZAU-China/Modeling">Modeling</a>
                 <li class="dropdown"><a href="https://2014.igem.org/Team:HZAU-China/Modeling">Modeling</a>
                     <ul>  
                     <ul>  
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                         <li><a href="https://2014.igem.org/Team:HZAU-China/Modeling"><span>-</span>Overview</a></li>
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                         <li><a href="https://2014.igem.org/Team:HZAU-China/MOverview"><span>-</span>Overview</a></li>
                         <li><a href="https://2014.igem.org/Team:HZAU-China/Biological"><span>-</span>Biological processes</a></li>
                         <li><a href="https://2014.igem.org/Team:HZAU-China/Biological"><span>-</span>Biological processes</a></li>
                         <li><a href="https://2014.igem.org/Team:HZAU-China/Comparison"><span>-</span>Comparison between different designs</a></li>
                         <li><a href="https://2014.igem.org/Team:HZAU-China/Comparison"><span>-</span>Comparison between different designs</a></li>
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<li><a href="https://2014.igem.org/Team:HZAU-China/Collaboration"><span>-</span>Collaboration</a></li>
<li><a href="https://2014.igem.org/Team:HZAU-China/Collaboration"><span>-</span>Collaboration</a></li>
                         <li><a href="https://2014.igem.org/Team:HZAU-China/Meetup"><span>-</span>Meetup</a></li>
                         <li><a href="https://2014.igem.org/Team:HZAU-China/Meetup"><span>-</span>Meetup</a></li>
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                         <li><a href="https://2014.igem.org/Team:HZAU-China/Humanities"><span>-</span>Humanities and Aesthetics</a></li>
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                         <li><a href="https://2014.igem.org/Team:HZAU-China/Humanities"><span>-</span>Humanities</a></li>
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                         <li><a href="https://2014.igem.org/Team:HZAU-China/Economics"><span>-</span>Economics and Law</a></li>
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                         <li><a href="https://2014.igem.org/Team:HZAU-China/Economics"><span>-</span>Social Sciences</a></li>
                         <li><a href="https://2014.igem.org/Team:HZAU-China/Pubic outreach"><span>-</span>Public outreach</a></li>
                         <li><a href="https://2014.igem.org/Team:HZAU-China/Pubic outreach"><span>-</span>Public outreach</a></li>
                         <li><a href="https://2014.igem.org/Team:HZAU-China/Safety"><span>-</span>Safety</a></li>
                         <li><a href="https://2014.igem.org/Team:HZAU-China/Safety"><span>-</span>Safety</a></li>
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<h3 style="text-align:center;">Molecule labnotes</h3>
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<h3 style="text-align:center;">Molecule Labnotes</h3>
         <p class="highlighttext">Author: Yihui Zheng</p>
         <p class="highlighttext">Author: Yihui Zheng</p>
         <p class="highlighttext">Members: Yihui Zheng, Yajing Zhang, Zhixiang Liu, Xudong Luo, Xiaoxue Guo, Lu Wang, Siqi Li.</p>
         <p class="highlighttext">Members: Yihui Zheng, Yajing Zhang, Zhixiang Liu, Xudong Luo, Xiaoxue Guo, Lu Wang, Siqi Li.</p>
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<p class="highlighttext"><span style="font-weight:bold;">3.Construction of gene circuit two: </span>we constructed this gene circuit by following the method we’ve mentioned above. The only difference from the construction of gene circuit three is that we put the coding sequence of TetR protein behind lox71. By the way, lox66 and lox71 are two recombination sites which are recognized specifically by Cre protein. To our surprise, when we did double-digest to this circuit for verification, we found that the length of product of enzyme-digested was not right and there was an extra stripe that we didn’t know what it was. Results are shown in the Figure 4. According to the results, we had another assumption to explain the phenomenon: there were some recombination systems in DH-5α, resulting in homologous recombination of two strains which had synclastic lox71 site. Definitely, We’ve done a lot of work to verify our assumption which will be explained in the part of characterization.</p>
<p class="highlighttext"><span style="font-weight:bold;">3.Construction of gene circuit two: </span>we constructed this gene circuit by following the method we’ve mentioned above. The only difference from the construction of gene circuit three is that we put the coding sequence of TetR protein behind lox71. By the way, lox66 and lox71 are two recombination sites which are recognized specifically by Cre protein. To our surprise, when we did double-digest to this circuit for verification, we found that the length of product of enzyme-digested was not right and there was an extra stripe that we didn’t know what it was. Results are shown in the Figure 4. According to the results, we had another assumption to explain the phenomenon: there were some recombination systems in DH-5α, resulting in homologous recombination of two strains which had synclastic lox71 site. Definitely, We’ve done a lot of work to verify our assumption which will be explained in the part of characterization.</p>
<img src="https://static.igem.org/mediawiki/2014/0/09/HZAU_LABNOTE_FIGURE_4.png" width="650px" class="img-center"/>
<img src="https://static.igem.org/mediawiki/2014/0/09/HZAU_LABNOTE_FIGURE_4.png" width="650px" class="img-center"/>
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<p class="figuretext">Figure 4. the stripes which arrows point towards are enzyme-digested product of gene circuit two and the rightest stripe in each figure is DL-5000 marker. There were at least three stripes and the length of extra stripe is wrong.</p>
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<p class="figuretext01">Figure 4. the stripes which arrows point towards are enzyme-digested product of gene circuit two and the rightest stripe in each figure is DL-5000 marker. There were at least three stripes and the length of extra stripe is wrong.</p>
</br></br>
</br></br>
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             </ul>
             </ul>
<img src="https://static.igem.org/mediawiki/2014/c/c0/HZAU_LABNOTE_FIGURE11.png" width="810px" class="img-center"/>
<img src="https://static.igem.org/mediawiki/2014/c/c0/HZAU_LABNOTE_FIGURE11.png" width="810px" class="img-center"/>
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<p class="figuretext01">Figure 6. a and b are the visual effects of gene circuit six without AHL and with AHL; c is the quantitative result of these two         samples</p>
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<p class="figuretext">Figure 6. a and b are the visual effects of gene circuit six without AHL and with AHL; c is the quantitative result of these two samples</p>
</br></br>
</br></br>
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<p class="highlighttext"><span style="font-weight:bold;">1.Verification for the first assumption that there were little LacI proteins in DH-5α, and it was not enough to repress P<sub>L</sub>lacI promoter, resulting in the overexpression of cI protein which may restrain the growth of host:</span></p>
<p class="highlighttext"><span style="font-weight:bold;">1.Verification for the first assumption that there were little LacI proteins in DH-5α, and it was not enough to repress P<sub>L</sub>lacI promoter, resulting in the overexpression of cI protein which may restrain the growth of host:</span></p>
<p class="highlighttext">We designed a primer contained <span style="font-style:italic;">Pst</span>I site which could match the middle part of the coding sequence of cI protein, and then, we conducted a PCR by using VF2 primer and the designed primer to get a sequence which can express an inactive cI protein. We put this sequence behind P<sub>L</sub>lacI promoter through the same way mentioned above and the sequencing results showed that we constructed it and it proved our assumption to some extent. Then, we came up with a different way to construct our first repressilator which will be explained in the following.</p>
<p class="highlighttext">We designed a primer contained <span style="font-style:italic;">Pst</span>I site which could match the middle part of the coding sequence of cI protein, and then, we conducted a PCR by using VF2 primer and the designed primer to get a sequence which can express an inactive cI protein. We put this sequence behind P<sub>L</sub>lacI promoter through the same way mentioned above and the sequencing results showed that we constructed it and it proved our assumption to some extent. Then, we came up with a different way to construct our first repressilator which will be explained in the following.</p>
-
<p class="highlighttext"><span style="font-weight:bold;">2.New way to construct our first repressilator: </span>Due to toxicity of cI protein on host bacteria and the expression of cI protein was regulated by P<sub>L</sub>lacI promoter, we came up with a new method that putting the gene circuit three in front of P<sub>L</sub>lacI promoter because circuit three would express LacI protein to repress the activation of P<sub>L</sub>lacI promoter and thus, cI protein would not be overexpressed. So, we digested circuit three by <span style="font-style:italic;">Eco</span>RI enzyme and <span style="font-style:italic;">Spe</span>I enzyme and digested P<sub>L</sub>lacI promoter by <span style="font-style:italic;">Eco</span>RI enzyme and <span style="font-style:italic;">Xba</span>I enzyme, and then do a ligation to get a circuit contained P<sub>L</sub>lacI promoter controlled by LacI protein. After that, we connected the coding sequence of cI protein with the previous product to get a compound contained circuit one and circuit three. Gel electrophoresis spectrum diagram of plasmid is shown in Figure 7.</p>
+
<p class="highlighttext"><span style="font-weight:bold;">2.New way to construct our first repressilator: </span>Due to toxicity of cI protein on host bacteria and the expression of cI protein which was regulated by P<sub>L</sub>lacI promoter, we came up with a new method that putting the gene circuit three in front of P<sub>L</sub>lacI promoter because circuit three would express LacI protein to repress the activation of P<sub>L</sub>lacI promoter and thus, cI protein would not be overexpressed. So, we digested circuit three by <span style="font-style:italic;">Eco</span>RI enzyme and <span style="font-style:italic;">Spe</span>I enzyme and digested P<sub>L</sub>lacI promoter by <span style="font-style:italic;">Eco</span>RI enzyme and <span style="font-style:italic;">Xba</span>I enzyme, and then do a ligation to get a circuit contained P<sub>L</sub>lacI promoter controlled by LacI protein. After that, we connected the coding sequence of cI protein with the previous product to get a compound contained circuit one and circuit three. Gel electrophoresis spectrum diagram of plasmid is shown in Figure 7.</p>
<img src="https://static.igem.org/mediawiki/2014/7/7b/HZAU_LABNOTE_FIGURE_5.png" width="350px" class="img-center"/>
<img src="https://static.igem.org/mediawiki/2014/7/7b/HZAU_LABNOTE_FIGURE_5.png" width="350px" class="img-center"/>
<p class="figuretext">Figure 7. plasmid contained circuit one and circuit three (a,b)</p>
<p class="figuretext">Figure 7. plasmid contained circuit one and circuit three (a,b)</p>
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                 </li>
                 </li>
             </ul>
             </ul>
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<p class="figuretext">Figure 9. a was without any excitation light; b was the phenomenon when we co-transformed the gene circuit seven and eight (with Cre protein); c was the phenomenon when we only transformed the gene circuit seven (without Cre protein) as a comparison.</p>
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<p class="figuretext01">Figure 9. a was without any excitation light; b was the phenomenon when we co-transformed the gene circuit seven and eight (with Cre protein); c was the phenomenon when we only transformed the gene circuit seven (without Cre protein) as a comparison.</p>
<img src="https://static.igem.org/mediawiki/2014/0/09/HZAU_LABNOTE_FIGURE_9.jpg" width="700px" class="img-center"/>
<img src="https://static.igem.org/mediawiki/2014/0/09/HZAU_LABNOTE_FIGURE_9.jpg" width="700px" class="img-center"/>
<p class="figuretext">Figure 10. result of fluorescence intensity</p>
<p class="figuretext">Figure 10. result of fluorescence intensity</p>
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<h3 style="text-align:center;">Cellular Labnotes</h3>
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<h3 style="text-align:center;">Function test labnotes</h3>
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<h6>1. Basic works</h6>
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<h6>1. Input module test</h6>
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<h5>1.1 Confirmation of the unexpected recombination of lox71 site</h5>
+
 
 +
<h5>1.1 Cre recombinase function test</h5>
<p class="highlighttext">Author:Lu Wang</p>
<p class="highlighttext">Author:Lu Wang</p>
<p class="highlighttext"><span style="font-weight:bold;">1.1.1 Experimental Background</span></p>
<p class="highlighttext"><span style="font-weight:bold;">1.1.1 Experimental Background</span></p>
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<p class="highlighttext">Cre recombinase is the type I topoisomerase found in phage P1 that can catalyze site-specific recombination between loxP sites. Lox71 is a mutation of loxP site which can also be specifically recognized by Cre recombinase. Our purpose is to confirm that unexpected recombination of lox71 site exits through sequencing result and observation under fluorescence microscope.</p>
+
<p class="highlighttext">Our purpose is to confirm the function of Cre recombinase.</p>
 +
 
<p class="highlighttext"><span style="font-weight:bold;">1.1.2 Materials and equipment</span></p>
<p class="highlighttext"><span style="font-weight:bold;">1.1.2 Materials and equipment</span></p>
<p class="highlighttext"><span style="font-weight:bold;">Materials:</span></br>
<p class="highlighttext"><span style="font-weight:bold;">Materials:</span></br>
-
Part: Lac Promoter; Lox71 site (BBa_K886000); mCherry (BBa_J06504)</br>
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Part:mCherry (BBa_J06504)</br>
-
Strain: Escherichia. Coli (DH5α, DH10b, BL21)</br>
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Strain: Escherichia. Coli (DH5α)</br>
-
Glycerinum, LB broth and plate, assorted antibiotics</br>  
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LB, assorted antibiotics</br></p>
-
<span style="font-weight:bold;">Equipment:</span></br>
+
 
-
Fluorescence microscope</p> 
+
<p class="highlighttext"><span style="font-weight:bold;">1.1.3 Method</span></p>
<p class="highlighttext"><span style="font-weight:bold;">1.1.3 Method</span></p>
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<p class="highlighttext"><span style="font-weight:bold;">Device Assembly</span></br>
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<img src="https://static.igem.org/mediawiki/2014/e/e8/Figure_1._Our_designed_device.png"  width="700px" class="img-center"/>
-
Ligate the Lac promoter with lox71 site, and lox71 site with mCherry using the Bio-brick assembly.</p> 
+
<p class="figuretext">Figure 1. Our designed device</p>
-
<img src=""  width="px" class="img-center"/>
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<p class="highlighttext">Co-transform the pSB1A3 and pSB1C3 which we designed as our Confirmation device (as shown in Figure 1) into Escherichia. Coli (DH5α) competent. Culture it overnight from a fresh single colony of Escherichia. Coli (DH5α) strain in LB containing chloramphenicol, ampicillin and IPTG inducer. And wait for about 8 hours to observe the culture’s color.</p>
-
<p class="figuretext">Fig.1</p>
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-
<img src=""  width="px" class="img-center"/>
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-
<p class="figuretext">Fig.2</p>
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-
<p class="highlighttext">Change the sequence of bio-brick prefix to make it recognized by Pst I restriction endonuclease utilizing a PCR reaction.</p>
+
<p class="highlighttext"><span style="font-weight:bold;">1.1.4 Result and discussion</span></p>
-
<p class="highlighttext">Obtain the PCR product:</p>
+
<p class="highlighttext"><span style="font-weight:bold;">Observation on Group Level</span></p>
-
<img src=""  width="px" class="img-center"/>
+
-
<p class="figuretext">Fig.3</p>
+
-
<p class="highlighttext">Join the Lac promoter-lox71 and lox71-mCherry fragments with double digestion and ligation.
 
-
Transform the confirmation device into DH5α, DH10b, and BL21, and pick the single colonies for inoculation overnight in Luria Broth containing chloramphenicol and IPTG inducer.</p>
 
-
<p class="highlighttext">Observe the culture’s color and take a small amount under the fluorescence microscope to observe the single bacteria. Extract the plasmids from single colonies and sequence the plasmid with general purpose primer.</p>
 
-
<p class="highlighttext">Co-transform the confirmation device and plasmids that code Cre recombinase into DH5α, pick the single colonies for inoculation overnight in Luria Broth containing chloramphenicol, ampicillin and IPTG inducer and observe the culture’s color.</p>
 
-
<p class="highlighttext"><span style="font-weight:bold;">Testing</br>
 
-
(Whether the recombinase with low specificity expressed by <span style="font-style:italic;">E.coli</span> endogenously could catalyze a recombination process between a pair of lox71 sites)</span></p>
 
-
<p class="highlighttext">Transform the confirmation device into DH5α, DH10b, and BL21, and pick the single colonies for inoculation overnight in Luria Broth containing chloramphenicol and IPTG inducer.</p>
 
-
<p class="highlighttext">Observe the culture’s color and take a small amount under the fluorescence microscope to observe the single bacteria. Extract the plasmids from single colonies and sequence the plasmid with general purpose primer.</p>
 
-
<p class="highlighttext"><span style="font-weight:bold;">Confirmation </br>
 
-
(Confirm the plasmids that code Cre recombinase from the registry work properly in <span style="font-style:italic;">E.coli</span>)</span></p>
 
-
<p class="highlighttext">Co-transform the confirmation device and plasmids that code Cre recombinase into DH5α, pick the single colonies for inoculation overnight in Luria Broth containing chloramphenicol, ampicillin and IPTG inducer and observe the culture’s color.</p>
 
-
<img src=""  width="px" class="img-center"/>
 
-
<p class="figuretext">Fig.4</p>
 
-
<img src=""  width="px" class="img-center"/>
 
-
<p class="figuretext">Fig.5</p>
 
-
<p class="highlighttext"><span style="font-weight:bold;">1.1.4 Experimental Results and Discussion</span></p>
 
-
<p class="highlighttext"><span style="font-weight:bold;">Observation on Group Level</span></p>
 
-
<img src=""  width="px" class="img-center"/>
 
-
<img src=""  width="px" class="img-center"/>
 
-
<p class="figuretext">Fig.6</p>
 
-
<p class="highlighttext">The results present that the culture of Negative Control and Group One has become opaque but still keep being pale yellow. The Group Two’s culture has turned red obviously and also opaque.</p>
+
<img src="https://static.igem.org/mediawiki/2014/1/19/Figure_2._Observation_on_Group_Level_%281%29.png"  width="700px" class="img-center"/>
 +
<p class="figuretext01">Figure 2. Observation on Group Level (the left one is Negative Control contained mCherry without promoter,the middle one is Group One only contained confirmation device,the right one is confirmation device with Cre recombinase)</p>
 +
<img src="https://static.igem.org/mediawiki/2014/d/d9/Figure_3._Observation_on_Group_Level_%282%29.png"  width="700px" class="img-center"/>
 +
<p class="figuretext01">Figure 3. Observation on Group Level (the left one is Group One only contained confirmation device,the right one is confirmation device with Cre recombinase)</p>
 +
<p class="highlighttext">The results indicate that the culture of Negative Control and Group One has become opaque but still keep being pale yellow. The Group Two’s culture has turned red obviously and also opaque.</p>
<p class="highlighttext">Therefore, conclusion could be drawn that plasmids which code Cre recombinase in the registry work properly and the confirmation device we assembled is valid.</p>
<p class="highlighttext">Therefore, conclusion could be drawn that plasmids which code Cre recombinase in the registry work properly and the confirmation device we assembled is valid.</p>
-
<img src=""  width="px" class="img-center"/>
 
-
<p class="figuretext">Fig.7</p>
 
-
<p class="highlighttext"><span style="font-weight:bold;">Observation on Individual Level</span></p>
 
-
<p class="highlighttext">The individual cell of DH5α and DH10b were observed through the fluorescence microscope, and the photographs are shown below.</p>
 
-
<img src=""  width="px" class="img-center"/>
 
-
<p class="figuretext">Fig.8 (Strain DH5α, 10*40)</p>
 
-
<img src=""  width="px" class="img-center"/>
 
-
<p class="figuretext">Fig.9 (Strain DH10b, oil immersion lens, 10*100, white light)</p>
 
-
<img src=""  width="px" class="img-center"/>
 
-
<p class="figuretext">Fig.10 (StrainDH10b, oil immersion lens, 10*100, UV-light, red filter)</p>
 
-
<img src=""  width="px" class="img-center"/>
 
-
<p class="figuretext">Fig.11 (Strain DH5α, oil immersion lens, 10*100, white light)</p>
 
-
<img src=""  width="px" class="img-center"/>
 
-
<p class="figuretext">Fig.12 (StrainDH10b, oil immersion lens, 10*100, UV-light, red filter)</p>
 
-
 
-
<p class="highlighttext">The recombinase with low specificity expressed by <span style="font-style:italic;">E.coli</span> endogenously could catalyze a recombination process between a pair of lox71 sites, but with an extremely small probability. The rate of recombination between the lox71 sites in DH10b is lower than that in DH5α.</p>
 
-
<p class="highlighttext"><span style="font-weight:bold;">Sequencing</span></p>
 
-
<p class="highlighttext">We sequenced the plasmids of the positive clones and the result was not bimodal. So far, it could be said that lox71 sites can recombine under the catalysis of the non-specific Recombinase of <span style="font-style:italic;">E.coli</span>, but the probability is so small that neither the construction of plasmids nor the following experiments will be affected.</p>
 
-
<p class="highlighttext"><span style="font-weight:bold;">Discussion </span></p>
 
-
<p class="highlighttext">The recombinase with low specificity expressed by <span style="font-style:italic;">E.coli</span> endogenously could catalyze a recombination process between a pair of lox71 sites, but with an extremely small probability. </p>
 
-
<p class="highlighttext">The rate of recombination between the lox71 sites in DH10b is lower than that in DH5α.</p>
 
-
<p class="highlighttext">Recombination process between a pair of lox71 sites will be catalyzed by <span style="font-style:italic;">E.coli</span> endogenous recombinase, but the probability is small enough that neither the construction of plasmids nor the following experiments will be affected.</p>
 
-
<p class="highlighttext">The probability of unexpected lox71 site-based recombination in DH10b is smaller than that in DH5α.</p>
 
-
<p class="highlighttext">The plasmids that code Cre recombinase from the registry work properly.</p>
 
-
 
+
<h5>1.2 Riboregulator function test</h5>
-
<h5>1.2 Promoter test</h5>
+
<p class="highlighttext">Author:Lu Wang</p>
-
<p class="highlighttext">Author: Yuan Lu</p>
+
<p class="highlighttext"><span style="font-weight:bold;">1.2.1 Experimental Background</span></p>
<p class="highlighttext"><span style="font-weight:bold;">1.2.1 Experimental Background</span></p>
-
<p class="highlighttext">The three devices used in our project are concerned with plenty of functions such as oscillation, feedback and feedforward. Since the difference between frequencies of initiation of the promoters has critical impact on the functions of these regulatory devices, it is important to measure the relative frequencies of initiation between these promoters.</p>
+
<p class="highlighttext">Confirm if the leakage of lac promoter’s expression will be reduced under the control of riboregulator.</p>
-
<p class="highlighttext">Our purpose is to measure the relative initiation frequencies of promoters used in our devices, which are BBa_J23119、BBa_R1051、BBa_R0062、BBa_R0040、BBa_R0063、BBa_R0011.</p>
+
 
-
<p class="highlighttext"><span style="font-weight:bold;"></span></p>
+
<p class="highlighttext"><span style="font-weight:bold;">1.2.2 Materials and equipment</span></p>
-
<p class="highlighttext">1.2.2 Materials and equipment</p>
+
<p class="highlighttext"><span style="font-weight:bold;">Materials:</span></br>
-
<p class="highlighttext"><span style="font-weight:bold;">Materials</span></br>
+
Strain: Escherichia. Coli (DH5α,BL21(DE3))</br>
-
Parts: Promoters (BBa_J23119、BBa_R1051、BBa_R0062、BBa_R0040、BBa_R0063、BBa_R0011), mCherry (BBa_J06504), RBS (BBa_B0034), Terminator (BBa_B0015)</br>
+
LB , ampicillin antibiotic</br>
-
Strain: Escherichia. Coli (DH5α)</br>  
+
<span style="font-weight:bold;">Equipment:</span></br>
-
LB broth and plate, Assorted antibiotics</p>
+
Multifunctional microplate reader</br></p>
-
<p class="highlighttext"><span style="font-weight:bold;">Equipment</span></br>
+
-
Multi-function Enzyme-labeled instrument</p>
+
<p class="highlighttext"><span style="font-weight:bold;">1.2.3 Method</span></p>
<p class="highlighttext"><span style="font-weight:bold;">1.2.3 Method</span></p>
-
<p class="highlighttext">Transformation of DH5α with each of the plasmid necessary in our projects, incubation on a accordingly selective plate for 14 hours or so at 37 and then cultivate the single colony in LB broth for 8 hours at 37 Celsius degree. Extracting plasmid from bacteria and after the sequencing result shows the correct sequence, stored the plasmid at -20 Celsius degree.</p>
+
<img src="https://static.igem.org/mediawiki/2014/e/e3/Figure_4._Designed_Device_%281%29.jpg"  width="400px" class="img-center"/>
-
<p class="highlighttext">Construction of the vectors of different promoters as illustrated by biobrick method.
+
<p class="figuretext">Figure 4. Designed Device (1)</p>
-
Then after the sequencing prove the correction of 6 vectors constructed transformed them and other 6 same vectors but without promoters upstream respectively to the competent cell DH5α in 12 different tubes, and then cultured at 37 Celsius degree. </p>
+
<img src="https://static.igem.org/mediawiki/2014/d/dc/Figure_5._Designed_Device_%282%29.jpg"  width="700px" class="img-center"/>
-
<p class="highlighttext">After culturing for 12 hours measuring the fluorescent degree of 12 different bacteria (6 in which without vector with promoter serve as control) by enzyme-labeled instrument.</p>
+
<p class="figuretext">Figure 5. Designed Device (2)</p>
-
<p class="highlighttext">To determine the effect of inducer, IPTG was added to each of tube in which bacteria contains vector with BBa_R0040 or BBa_R0011 and its according control and then culturing for another 12 hours and then measuring the fluorescent degree by enzyme-labeled instrument.</p>
+
<p class="highlighttext">Firstly, construct these two devices shown in Figure 4 and Figure 5. Respectively transform each of them into Escherichia. Coli strain BL21 (DE3). Pick single colonies and add each of them into 5ml LB liquid with its corresponding antibiotic and incubate it in shaking incubator at 37 ℃,180rpm for 8 hours. Secondly, add 5ul of previous single colony cultural from each tube into 5ml fresh M9 broth. And we divided all the sample tubes into two groups: one was the cells with designed device (1) plasmid and we named it group A. Another was the cells with designed device (2) plasmid and we named it group B. Add 0.1ul IPTG into 12 tubes of both group A and B, while add nothing into the other 12 tubes of both group A and B as control . Incubate them at 37 ℃ for 4 hours. Lastly, measure the fluorescent intensity by the multifunctional microplate reader.</p>
-
<p class="highlighttext"><span style="font-weight:bold;">1.2.4 Experimental Results and Discussion</span></p>
 
-
<img src=""  width="px" class="img-center"/>
 
-
<p class="figuretext">Fig.13 Detection of the part BBa_R0040 after adding inducer</p>
 
-
<img src=""  width="px" class="img-center"/>
 
-
<p class="figuretext">Fig.14 The detection of the relative strength of the promoters</p>
 
-
<p class="highlighttext"><span style="font-weight:bold;">Analysis:</span></p>
 
-
<img src=""  width="px" class="img-center"/>
+
<p class="highlighttext"><span style="font-weight:bold;">1.2.4 Result and discussion</span></p>
-
<p class="figuretext">Fig.15 Detection of the part BBa_R0040 after adding inducer </p>
+
<img src="https://static.igem.org/mediawiki/2014/5/55/Hzau-note-6.jpg"  width="700px" class="img-center"/>
-
<img src=""  width="px" class="img-center"/>
+
<p class="figuretext">Figure 6.The data from the enzyme-labeled instrument.</p>
-
<p class="figuretext">Fig.16 The detection of the relative strength of the promoters</p>
+
<p class="highlighttext"><span style="font-weight:bold;">Experimental analysis:</span></p>
 +
<img src="https://static.igem.org/mediawiki/2014/9/9a/Hzau-note-7.jpg"  width="700px" class="img-center"/>
 +
<p class="figuretext">Figure 7.Experimental analysis based on the data from enzyme-labeled instrument</p>
 +
<p class="highlighttext"><span style="font-weight:bold;">Conclusion:</span></p>
 +
Riboregulators ensure much lower leakage of the promoters. </p>
-
<p class="figuretext">Based on the data from Fig.13, we do the statistical analysis of the detection of the part BBa_R0040 after adding inducer. With analysis diagram shows in Fig.15, the inductive effect of the inducer is not evident in low concentration. However, inductive effect of the inducer is evident when the concentration is up to 10µM. </p>
+
<h5>1.3 Test of input module's tight regulation </h5>
-
<p class="figuretext">Based on the data from Fig.14, we do the statistical analysis. With analysis diagram shows in Fig.16, we can conclude that the rank of relative strength of the promoters from strong to weak is: BBa_R0063、BBa_R0011、BBa_J23119、BBa_R1051、BBa_R0040、BBa_R0062.</p>
+
<p class="highlighttext">Author:Lu Wang</p>
 +
<p class="highlighttext"><span style="font-weight:bold;">1.3.1 Experimental Background</span></p>
 +
<p class="highlighttext">Our purpose is to confirm whether the cre will be expressed or not under the control of riboswitch driven by IPTG.</p>
-
<h5>1.3 pCusC, PpcoA sequencing and promoter test</h5>
+
<p class="highlighttext"><span style="font-weight:bold;">1.3.2 Materials and equipment</span></p>
-
<p class="figuretext">Author: Liu Tian</p>
+
-
<p class="highlighttext"><span style="font-weight:bold;">1.3.1 Experimental Background</span></p>
+
-
<p class="highlighttext">iGEM encourage the communication between teams and help others. So, when we heard HUST-China has something wrong about their promoter, we decided to give a hand. The one problem they had is they tried many times but failed to sequencing those promoters in pET28a, and the other is they put mRFP under the promoter to do a promoter test but didn’t get result. So we got A&B promoters with mRFP from them.</p>
+
-
<p class="highlighttext"><span style="font-weight:bold;">1.3.2 Proposes</span></p>
+
-
<p class="highlighttext">pCusC & PpcoA promoters sequencing</br>pCusC & PpcoA promoters test</p>
+
-
<p class="highlighttext"><span style="font-weight:bold;">1.3.3 Materials and equipment</span></p>
+
<p class="highlighttext"><span style="font-weight:bold;">Materials:</span></br>
<p class="highlighttext"><span style="font-weight:bold;">Materials:</span></br>
-
pCusC & PpcoA promoters with mRFP in pET28a plasmid</br>
+
Vector-pSB1A3, pSB1C3</br>
-
LB medium</br>
+
Strain: Escherichia. Coli (DH5α)</br>
-
Kanamycin</p>
+
LB , assorted antibiotics</br>
-
<p class="highlighttext"><span style="font-weight:bold;">Equipment:</span></p>
+
<span style="font-weight:bold;">Equipment:</span></br>
-
<p class="highlighttext">Multifunctional microplate reader</br>
+
Multifunctional microplate reader</br></p>
-
Shaking incubator </p>
+
-
<p class="highlighttext"><span style="font-weight:bold;">1.3.4 Method</span></p>
+
-
<p class="highlighttext"><span style="font-weight:bold;">Sequencing</span></p>
+
-
<p class="highlighttext">We design the 5’-agatcgggctcgccacttcg-3’ as the reverse primer to sequencing the promoters. And Tsing Ke Biology Technology Company help us synthetize the primer and sequencing those plasmid. You can get the sequencing results of two samples at supplement 1. Analyzed the sample pCusC’s sequencing result, we find that the T7 promoter has not be digested , there is no pCusC in the plasmid, but mRFP has successful ligated. So we can observe the red is the red fluorescent protein expression by the T7 promoter. Therefore, in the subsequent promoter test, we abandoned the pCusC samples. In the PpcoA samples we successfully found PpcoA and mRFP. However, at the end of PpcoA‘ssequencing result, there is more 6bp than HUST’s sequence. We take PpcoA promoter to detection.</p>
+
-
<p class="highlighttext"><span style="font-weight:bold;">Promoter test</span></p>
+
-
<p class="highlighttext">Add 2mL LB culture medium, 2uL Kanamycin (50mg/L) and 20uL bacterial samples to a 5mL centrifuge tube. Shaken overnight in the 37°C shaking incubator and set the rotational speed at 180 rpm/min.</p>
+
-
<p class="highlighttext">Add CuSO4 solution to induce in concentration of 0, 0.02, 0.1 and 1mM. Set three copies for each concentration as repetitions. Cultivate in the 37°C shaking incubator at the rotational speed of 180 rpm/min for 4 hours.</p>
+
-
<p class="highlighttext">Add 200uL bacterial samples from each centrifuge tube to the 96-well plate. Set two copies for each centrifuge tub as repetitions. Meanwhile, add LB culture medium containing and not containing 1.0mM CuSO4 as blank controls.</p>
+
-
<p class="highlighttext">Read the OD600 and fluorescent intensity (with the emission wavelength at 607nM and excitation wavelength at 584nM) using a multifunctional microplate reader.</p>
+
-
<p class="highlighttext">Divided the fluorescent intensity result using the value of OD600. Record the data for compare and analysis. </p>
+
-
<p class="highlighttext"><span style="font-weight:bold;">1.3.5 Result and discussion</span></p>
+
<p class="highlighttext"><span style="font-weight:bold;">1.3.3 Method</span></p>
-
<img src=""  width="px" class="img-center"/>
+
<img src="https://static.igem.org/mediawiki/2014/3/36/Figure._8_input_device.png"  width="770px" class="img-center"/>
-
<p class="figuretext">Fig.17</p>
+
<p class="figuretext"> Figure 8. input device</p>
-
<p class="highlighttext">When we add the 1.0mM Cu to bacterium solution,we observed the color change of the visible to the naked eye. So we try to test the 1.0mM Cu in LB medium. Then we find its fluorescence value and the value of OD600 with only LB samples have a large difference. So we decided to use the 1.0mM Cu in LB medium as the blank samples of the 1.0mM control.We can observe from the figure, when the copper ion concentration is range of 0-0.1mM, with the increase of concentration of copper ion, promoter strength increased. In the copper ion concentration of 1mM, the promoter strength decreased. To this end, we intend to in the copper ion concentration in the range of 0.1-1mM is provided with a plurality of gradient measured next.</p>
+
<img src="https://static.igem.org/mediawiki/2014/3/34/Figure._9_verification_device.png"  width="400px" class="img-center"/>
 +
<p class="figuretext">Figure 9. verification device</p>
-
<h6>2. Input module test</h6>
+
<p class="highlighttext"><span style="font-weight:bold;">Process:</span></p>
-
<h5>2.1 Cre recombinase function test</h5>
+
<p class="highlighttext">1. Co-transform our input device and verification device into BL21(DE3) competent cell and plate it onto a selective plate, incubate it at 37 ℃ for 14 hours.</p>
 +
<p class="highlighttext">2. Pick 3 different single colonies numbered 1, 2, 3 into 3 tubes of LB broth with its corresponding antibiotic and incubate it respectively at 37℃ for 8 hours.</p>
 +
<p class="highlighttext">3. Add 5ul of previous single colony cultural from each tube to two new tubes named A,B respectively with M9 broth in it. Add 0.1ul IPTG to all A tubes (1A, 2A, 3A) while add nothing to all B tubes (1B, 2B, 3B). Incubate them at 37℃ for 4 hours.</p>
 +
<p class="highlighttext">4. Measure the fluorescent intensity by the enzyme-labeled instrument.</p>
 +
 
 +
<p class="highlighttext"><span style="font-weight:bold;">Prediction:</span></p>
 +
<p class="highlighttext">a) If the fluorescent degree in cultural tubes with IPTG showed much less weaker than in the tubes without it, then it indicated that the device had achieved its function for reducing the expression of Cre protein as expected.</p>
 +
<p class="highlighttext">b) If the fluorescent degree in the cultural tubes with IPTG showed almost same or even stronger than in the tubes without it, it meant that the devices had lost its function. However, there may be 3 reasons which are responsible for its failure. The first reason may be the failed function of riboregulator to repress the translation process. Secondly, despite the strong repression dueing to the riboregulator, the leakage expression of Cre protein can still express a little bit Cre protein which may cause the same result of a device without riboregulator. Thirdly, the low production of lacI in BL21(DE3) bacteria is unable to repress the operon efficiently which leads to the high expression of both taRNA and cre mRNA and thus weaken the function of riboregulator.</p>
 +
 
 +
<p class="highlighttext"><span style="font-weight:bold;">1.3.4 Result and discussion</span></p>
 +
<img src="https://static.igem.org/mediawiki/2014/b/b9/Hzau-note-8.jpg"  width="700px" class="img-center"/>
 +
<p class="figuretext">Figure 10. Fluorescent degree (OD<sub>600</sub>)</p>
 +
<img src="https://static.igem.org/mediawiki/2014/f/fa/Hzau-note-9.jpg"  width="700px" class="img-center"/>
 +
<p class="figuretext">Figure 11. The rigour detection of output device</p>
 +
<p class="highlighttext">As shown above, even though the addition of riboregulator does reduce a little of the expression of Cre protein, the difference between control group and experimental group is still very small which means that the riboregulator can hardly exert some critical influence on the control of the expression of cre gene.</p>
 +
 
 +
</br></br>
 +
 
 +
<h6>2. Output module test</h6>
 +
<h5>2.1 The Detection of Aptamer (Qualitative Detection)</h5>
 +
<p class="highlighttext">Author: Yajing Zhang, Siqi Li</p>
<p class="highlighttext"><span style="font-weight:bold;">2.1.1 Experimental Background</span></p>
<p class="highlighttext"><span style="font-weight:bold;">2.1.1 Experimental Background</span></p>
-
<p class="highlighttext">To confirm the function of Cre recombinase</p>
+
<p class="highlighttext">Our purpose is to confirm whether the aptamers is available or not.</p>
 +
 
<p class="highlighttext"><span style="font-weight:bold;">2.1.2 Materials and equipment</span></p>
<p class="highlighttext"><span style="font-weight:bold;">2.1.2 Materials and equipment</span></p>
-
<p class="highlighttext"><span style="font-weight:bold;">Materials:</span>
+
<p class="highlighttext"><span style="font-weight:bold;">Material:</span></br>
-
Part:mCherry (BBa_J06504)</br>
+
DMHBI, M9 broth, Escherichia. Coli(DH5α)</p>
-
Strain: Escherichia. Coli(DH5α, DH10b, BL21)</br>
+
<p class="highlighttext"><span style="font-weight:bold;">Equipment:</span></br>
-
LB broth and plate, assorted antibiotics</p>
+
Fluorescence microscope</p>
-
<p class="highlighttext"><span style="font-weight:bold;">Equipment:</span></br>fluorescence microscope</p>
+
 
<p class="highlighttext"><span style="font-weight:bold;">2.1.3 Method</span></p>
<p class="highlighttext"><span style="font-weight:bold;">2.1.3 Method</span></p>
-
<p class="highlighttext"><span style="font-weight:bold;">Test whether the recombinase with low specificity expressed by <span style="font-style:italic;">E.coli</span> endogenously could catalyze a recombination process between a pair of lox71 sites.</span></p>
+
<p class="highlighttext"><span style="font-weight:bold;">The synthesis of DMHBI</span></p>
-
<p class="highlighttext">Transform the confirmation device into DH5α, DH10b, and BL21, and pick the single colonies for inoculation overnight in Luria Broth containing chloramphenicol and IPTG inducer.</p>
+
<p class="highlighttext">N-Acetylglycine (5.26 g, 0.045mol), anhydrous sodium acetate (3.69 g, 0.045mol), 4-hydroxy-2,3-dimethoxybenzaldehyde (8.19 g, 0.045mol), and acetic anhydride (15 ml) were stirred at 90℃for 2 h. After allowing the reaction to cool to room temperature, cold ethanol (20 ml) was added while still stirring and the reaction was left stirring overnight at 4℃. The resulting crystalline solid was then washed with coldethanol, hot water, hexanesand dried to afford 9.65 g (yield 70%) of 3as a pale yellow solid: H NMR (500 MHz,CDCl_3) δ7.56 (s, 2H), 7.18 (s, 1H), 3.98 (s, 6H), 2.54(s, 3H), 2.39(s, 3H); LC/MS (LC: gradient 20-95% MeCN[0.1% HCO_2 H] over 2.5min, 0.5 ml/min flow rate, MS: ESI^+): retention time, 2.15 min; purity, 95%; 306.36 〖[M+H]〗^+. Then we can get Compound 3.Compound 3 (1.12 g, 0.005mol) was refluxed with 15 ml of ethanol, 1 ml of 40% aqueous methylamine, and 700 mg of potassium carbonate for 4h.  The reaction mixture was removed from heat and upon cooling formed an orange precipitate. The precipitate containing the product was filtered and washed briefly with cold ethanol. The precipitate was then redissolved in a 1:1 mixture of ethyl acetate and 500 mM sodium acetate pH 3.0. The organic layer was separated, dried with anhydrous sodium sulfate and solvent was removed under reduced pressure to yield 717 mg (yield 52%) of DMHBI as an orange solid: 1H NMR (500MHz, DMSO-d_6) δ7.62 (s, 2H), 6.90 (s, 1H), 3.80 (s, 6H), 3.09 (s, 3H), 2.34 (s, 3H). </p>
-
<p class="highlighttext">Observe the culture’s color and take a small amount under the fluorescence microscope to observe the single bacteria. Extract the plasmids from single colonies and sequence the plasmid with general purpose primer.</p>
+
<img src="https://static.igem.org/mediawiki/2014/4/47/Hzau-note-10.png"  width="600px" class="img-center"/>
-
<p class="highlighttext"><span style="font-weight:bold;">Confirm the plasmids that code Cre recombinase from the registry work properly in <span style="font-style:italic;">E.coli</span>.</span></p>
+
<p class="figuretext">Figure 12. The process during synthesis  </p>
-
<p class="highlighttext">Co-transform the confirmation device and plasmids that code Cre recombinase into DH5α, pick the single colonies for inoculation overnight in Luria Broth containing chloramphenicol, ampicillin and IPTG inducer and observe the culture’s color.</p>
+
<p class="highlighttext">Then the DMHBI should be stored in such conditions:</br>
-
<img src=""  width="px" class="img-center"/>
+
Prevent from light and oxygen</br>
-
<p class="figuretext">Fig.18</p>
+
Preserve it in low temperature</p>
-
<img src="" width="px" class="img-center"/>
+
<p class="highlighttext">According to the literature, we will get an orange solid. But in reality, we get something dark brown. After detection, our products are in liquid state and we get 40% in purity of product. In order to get more product in high purity, we invite ShaoQing Zhuang who is a student in Huazhong University of Science and Technology to help us with the synthesis of the DMHBI. And finally we get more than 95% in purity of product.</p>
-
<p class="figuretext">Fig.19</p>
+
<p class="highlighttext">DH5α cells (TransGen Biotech) were transformed with 10μl of plasmid DNA expressing chimeras of the human tRNALys3 scaffold fused to the Spinach aptamer’s sequence. Cells were plated, grown overnight and single colonies were picked for inoculation overnight in Luria Broth containing chloramphenicol. At =0.8, 150μl culture was removed, pelleted and resuspended in 100μl M9 minimal media and cultivate for 1 h at 37 ℃. Cells were washed twice and incubated with 200μM DFHBI in M9 media for 5 min. We tested the spinach RNA aptamer in the Tanon 1600R Gel Imaging System under the 302nm, the control groups are the DMHBI and DMHBI + control RNA. The result is below (Figure 15) and we also observed the spinach RNA aptamer by using the fluorescence microscope, the result is in the Figure 13.</p>
 +
 
<p class="highlighttext"><span style="font-weight:bold;">2.1.4 Result and discussion</span></p>
<p class="highlighttext"><span style="font-weight:bold;">2.1.4 Result and discussion</span></p>
-
<img src=""  width="px" class="img-center"/>
+
<img src="https://static.igem.org/mediawiki/2014/b/b0/Hzau-note-11.png"  width="700px" class="img-center"/>
-
<img src=""  width="px" class="img-center"/>
+
<p class="figuretext">Figure 13. The result of detecting E.coli with promoter  and aptamer by blue exciting light is green point. (10*40, blue filter).</p>
-
<p class="figuretext">Fig.20</p>
+
<img src="https://static.igem.org/mediawiki/2014/4/48/Figure_14._The_picture_of_reference..png"  width="400px" class="img-center"/>
-
<img src=""  width="px" class="img-center"/>
+
<p class="figuretext">Figure 14. The picture of reference.</p>
-
<p class="figuretext">Fig.21</p>
+
<img src="https://static.igem.org/mediawiki/2014/4/4c/Figure_15._The_picture_of_test..jpg"  width="400px" class="img-center"/>
-
<p class="highlighttext">The results present that the culture of Negative Control and Group One has become opaque but still keep being pale yellow. The Group Two’s culture has turned red obviously and also opaque.</p>
+
<p class="figuretext">Figure 15. The picture of test.</p>
-
<p class="highlighttext">Therefore, conclusion could be drawn that plasmids which code Cre recombinase in the registry work properly and the confirmation device we assembled is valid.</p>
+
<p class="highlighttext">Because the brightness of the RNA-fluorophore complex is very low.(According to reference report, it is only decimusly brighter than the GFP ) And the half- life of RNA is just few minutes. So the concentration of RNA-fluorophore complex is at low level in cells. The Figure 15 has shown the difference among three samples.The sample of intermediate is E.coli with P<sub>λ</sub> promoter and aptamer,and it is the brightest. The sample of downmost is E.coli with P<sub>λ</sub> promoter, and it is brighter than the sample of uppermost . The sample of uppermost is control test sample, only with M9 medium and DMHBI ,and it is the darkest.We conclude that the aparter is available.</p>
-
<p class="highlighttext"><span style="font-weight:bold;">Observation on Individual Level</span></p>
+
-
<p class="highlighttext">The individual cell of DH5α and DH10b were observed through the fluorescence microscope, and the photographs are shown below.</p>
+
-
<img src=""  width="px" class="img-center"/>
+
-
<p class="figuretext">Fig.22</p>
+
-
<img src=""  width="px" class="img-center"/>
+
-
<p class="figuretext">Fig.23</p>
+
-
<img src=""  width="px" class="img-center"/>
+
-
<p class="figuretext">Fig.24</p>
+
-
<img src=""  width="px" class="img-center"/>
+
-
<p class="figuretext">Fig.25</p>
+
-
<img src=""  width="px" class="img-center"/>
+
-
<p class="figuretext">Fig.26</p>
+
-
<h5>2.2 Riboregulator function test</h5>
+
<h5>2.2 Fluorescent proteins with LVA tag</h5>
-
<p class="highlighttext">Author:Lu Wang</p>
+
<p class="highlighttext"><span style="font-weight:bold;">2.2.1 Experimental Background</span></p>
<p class="highlighttext"><span style="font-weight:bold;">2.2.1 Experimental Background</span></p>
-
<p class="highlighttext">We want to know under the participation of taRNA and crRNA, whether can reduce leakage of Plac expression.</p>
+
For further use in our project, we are going to test whether fluorescent proteins with LVA tag can degrade rapidly or not.</span></p>
 +
 
<p class="highlighttext"><span style="font-weight:bold;">2.2.2 Materials and equipment</span></p>
<p class="highlighttext"><span style="font-weight:bold;">2.2.2 Materials and equipment</span></p>
-
<p class="highlighttext"><span style="font-weight:bold;">Material:</span></br>
+
<p class="highlighttext"><span style="font-weight:bold;">Material:</span></br>
-
Strain: Escherichia. Coli(DH5α)
+
DMHBI, M9 broth, E.coli (DH5α) </p>
-
LB broth and plate, Assorted antibiotics</p>
+
<p class="highlighttext"><span style="font-weight:bold;">Equipment:</br>
 +
Fluorescence microscope</span></p>
<p class="highlighttext"><span style="font-weight:bold;">2.2.3 Method</span></p>
<p class="highlighttext"><span style="font-weight:bold;">2.2.3 Method</span></p>
-
<img src=""  width="px" class="img-center"/>
+
<img src="https://static.igem.org/mediawiki/2014/b/ba/Figure_16._Output_module_of_plan_2.png"  width="700px" class="img-center"/>
-
<p class="figuretext">Fig.27</p>
+
<p class="figuretext">Figure 16. Output module of plan 2</p>
-
<img src=""  width="px" class="img-center"/>
+
-
<p class="figuretext">Fig.28</p>
+
-
<img src=""  width="px" class="img-center"/>
+
-
<p class="figuretext">Fig.29</p>
+
-
<p class="highlighttext">First, built the three devices and from top to bottom Numbers for 1 2 3, and then, respectively transform the three plasmid into BL21 competent cell.Pick monoclonal colony.Add 5ml LB liquid and incubate at 37 Celsius degree for 8 hours. Secondly, add 5ul of LB liquid cultural medium from each tube to two new tubes named A, B respectively with 5ml M9 broth in it. Add 0.1ul IPTG to all A tubes (1A, 2A, 3A) while add nothing to all B tubes (1B, 2B, 3B). Incubating at 37 Celsius degree for 4 hours. Lastly, measuring the fluorescent degree by utilizing the enzyme-labeled instrument.</p>
+
<p class="highlighttext"><span style="font-weight:bold;">2.2.4 Result and discussion</span></p>
<p class="highlighttext"><span style="font-weight:bold;">2.2.4 Result and discussion</span></p>
 +
<img src="https://static.igem.org/mediawiki/2014/b/bc/Figure_17.png"  width="700px" class="img-center"/>
 +
<p class="figuretext">Figure 17. a: without any excitation light b: mCherry c: CFP</p>
-
<h5>2.3 Tight regulation test to input module</h5>
 
-
<p class="highlighttext">Author: Lu Wang</p>
 
-
<p class="highlighttext"><span style="font-weight:bold;">2.3.1 Experimental Background</span></p>
 
-
<p class="highlighttext">Since the high catalytic capability of the Cre recombinase, a small amount of recombinase can lead to the recombination between the lox sites, combine with the basal expression of the operon in our input device, the lox sites in our others device can easily be recombined in the bacteria before the stimulation by the inducer (IPTG in this case) on the repressor (lacI in this case). To address this problem, we add a riboswitch as a second layer of control upstream to the ORF of the cre to suppress the translation of the mRNA of the reconbinase in absence of the inducer. We wish to tightly control the expression of the Cre recombinase in both transcription and translation. Thus, in our input system, only stimulation by adding inducer can the expression of the recombinase be activated, while the production of the Cre can be extremely low in absence of the inducer. We undertook the experiment to verify the function of this device.</p>
 
-
<p class="highlighttext"><span style="font-weight:bold;">2.3.2 Materials and equipment</span></p>
 
-
<p class="highlighttext"><span style="font-weight:bold;">Material:</span></br>
 
-
Vector-pSB1A3,pSB1C3</br>
 
-
Strain: Escherichia. Coli (DH5α)</br>
 
-
LB broth and plate, Assorted antibiotics</p>
 
-
<p class="highlighttext"><span style="font-weight:bold;">Equipment:</span></br>
 
-
Multifunctional microplate reader</p>
 
-
<p class="highlighttext"><span style="font-weight:bold;">2.3.3 Method</span></p>
+
</br></br>
-
<p class="highlighttext">Co-transformation of bl21 with input device and verification device and plate the transformation onto a selective plate, incubate at 37 Celsius degree for 14hours or so.</p>
+
<h6>3. Processing module test</h6>
-
<p class="highlighttext">Incubate 3 different colonies numbered 1, 2, 3 in 3 tubes of LB broth respectively at 37 Celsius degree for 8 hours.</p>
+
<p class="highlighttext">Author :Yajing Zhang, Yihui Zheng</p>
-
<p class="highlighttext">Add 5ul of LB liquid cultural medium from each tube to two new tubes named A, B respectively with M9 broth in it. Add 0.1ul IPTG to all A tubes (1A, 2A, 3A) while add nothing to all B tubes (1B, 2B, 3B). Incubating at 37 Celsius degree for 4 hours.</p>
+
<p class="highlighttext"><span style="font-weight:bold;">3.1 Experimental Background</span></p>
-
<p class="highlighttext">Measuring the fluorescent degree by utilizing the enzyme-labeled instrument.</p>
+
To test our processing module.</span></p>
-
<p class="highlighttext"><span style="font-weight:bold;">Prediction and analysis:</span></p>
+
-
<p class="highlighttext">If the fluorescent degree in the cultural tube with IPTG showed much less intensity than in the tube without it, then it indicated that the device had achieved its function for reducing cre translation as expected.</p>
+
-
<p class="highlighttext">If the fluorescent degree in the cultural tube with IPTG showed almost same or even larger intensity than in the tube without it, it meant the devices had failed its purpose. While 3 reasons may be responsible for its failure. The first reason may be the failed function of riboswitch to repress the translation process. Second, despite the strong repression of translation due to the riboswitch, the tremendous catalytic activity of cre complements the low concentration of cre in bacteria. Third, the low production of lacI in bl21 bacteria is unable to repress the operon efficiently which leads to the high expression of both taRNA and cre mRNA and thus undermine the function of riboswitch.</p>
+
-
<p class="highlighttext"><span style="font-weight:bold;">2.3.4 Result and discussion</span></p>
+
<p class="highlighttext"><span style="font-weight:bold;">3.2 Materials and equipment</span></p>
-
<img src="" width="px" class="img-center"/>
+
<p class="highlighttext"><span style="font-weight:bold;">Material:</span></br>
-
<p class="figuretext">Fig.30</p>
+
Parts: BBa_K805016, BBa_R0062, BBa_R0063, BBa_K516022, BBa_I0462, BBa_I718016, BBa_K886000, BBa_I714075, BBa_B0015, BBa_J61047</br>
-
<img src=""  width="px" class="img-center"/>
+
Strain: Escherichia. Coli (BL21 (DE3))</br>
-
<p class="figuretext">Fig.31</p>
+
LB , assorted antibiotics</p>
-
<p class="highlighttext">As showed above, even though the addition of riboswitch does reduce a little of the production of Cre protein, the level of reduction is too slight and can hardly exert some critical influence on the experiment.</p>
+
<p class="highlighttext"><span style="font-weight:bold;">Equipment:</span></br>
 +
Perkin-Elmer plate reader</p>
 +
<p class="highlighttext"><span style="font-weight:bold;">3.3 Method</span></p>
 +
<img src="https://static.igem.org/mediawiki/2014/3/3d/HZAU_LABNOTE_FIGURE2B.jpg"  width="700px" class="img-center"/>
 +
<p class="figuretext">Figure 18. The testing of processing device.</p>
 +
<p class="highlighttext">The processing plasmid and input plasmid were co-transformed into the Escherichia. Coli (BL21 (DE3)) strain as experimental group and only the processing plasmid was transformed into the Escherichia. Coli (BL21 (DE3)) as control.</br>
 +
<p class="highlighttext">Single colonies were used to inoculate 5 mL LB media supplemented with 50ug/ mL ampicillin , 50ug/mL kanamycin and grown overnight at 37°C.And the control was used to inoculate 5 mL LB media supplemented with 50ug/mL kanamycin.</p>
 +
<p class="highlighttext">The next morning, overnight cultures were inoculated with 5 mL fresh LB cultures supplemented with antibiotics at 1:100 dilution. Cultures were grown at the desired temperature in constant temperature shaker following 3 h or 5h of growth (Both the experimental group and control were set two contrats further to test if the inducer, IPTG for experimental group and AHL for control group, would make influnece. ), then cells were plated in triplicate in a 96-well BD-Falcon plate. The optical density at 600 nm and fluorescence (X excitation = 475 nm and X emission = 515 nm) were measured using a Perkin-Elmer plate reader every 15 or 20 minutes.</p>
 +
<p class="highlighttext">When cells grow to mid-stage, one of the experiment groups was induced with 100uM IPTG, the optical density at 600 nm and fluorescence (X excitation = 475 nm and X emission = 515 nm) were measured using a Perkin-Elmer plate reader every 15 or 20 minutes after 3 hours ,at the same time cells were grown at 30°C. (Figure 17)</p>
-
<h6>3. Output module test</h6>
+
<p class="highlighttext"><span style="font-weight:bold;">3.4 Result and discussion</span></p>
-
<h5>3.1 The Detection of Aptamer (Qualitative Detection)</h5>
+
<img src="https://static.igem.org/mediawiki/2014/d/d8/Figure_19.png"  width="700px" class="img-center"/>
-
<p class="highlighttext">Author: Yajing Zhang, Siqi Li</p>
+
<p class="figuretext">Figure 19. The value of fluorescence intensity changes over time, the time begins with all the E.coli growing to mid-log.</p>
-
<p class="highlighttext"><span style="font-weight:bold;">3.1.1 Experimental Background</span></p>
+
<p class="highlighttext">The result indicated the processing device without Cre recombinase was in high fluorescence and the processing device with Cre recombinase was in low fluorescence, it works as expected. And whether it was induced by IPTG had no difference, the reason was mentioned in the tight regulation test for input module, because of the leakage of Cre recombinase. We also found the experimental groups grew better than the control with only processing plasmid. (Figure 18) Maybe the positive feedback caused more loading, but the negative feedback didn't. So we chose the data when all groups of the cells grow to mid-log phase.</p>
-
<p class="highlighttext">Detect the aptamers whether available individually. In the case of the entire project is out of the entire project.</p>
+
<img src="https://static.igem.org/mediawiki/2014/e/e7/Figure_20.png"  width="700px" class="img-center"/>
 +
<p class="figuretext">Figure 20. the growth curve of the E.coli , a: the control. b, the control with 100nM AHL. c: the experimental group without IPTG inducer. d: the experimental group with 100uM IPTG inducer.  e :the experimental group with 100uM IPTG inducer after growing to mid-log. f: the control growing to stable phase.</p>
-
<p class="highlighttext"><span style="font-weight:bold;">3.1.2 Materials and equipment</span></p>
+
</br></br>
-
<p class="highlighttext"><span style="font-weight:bold;">Material:</span></br>
+
-
DMHBI, M9 broth, E.coli(DH5α) </p>
+
-
<p class="highlighttext"><span style="font-weight:bold;">Equipment:</span></br>
+
-
fluorescence microscope</p>
+
-
<p class="highlighttext"><span style="font-weight:bold;">3.1.3 Method</span></p>
+
<h6>4. Other work</h6>
-
<p class="highlighttext"><span style="font-weight:bold;">The synthesis of DMHBI</span></p>
+
<p class="highlighttext"><span style="font-weight:bold;">4.1.Interaction work(pCusC, PpcoA sequencing and promoter test)</span></p>
-
<p class="highlighttext">N-Acetylglycine (5.26 g, 0.045mol), anhydrous sodium acetate (3.69 g, 0.045mol), 4-hydroxy-2,3-dimethoxybenzaldehyde (8.19 g, 0.045mol), and acetic anhydride (15 ml) were stirred at 90℃for 2 h. After allowing the reaction to cool to room temperature, cold ethanol (20 ml) was added while still stirring and the reaction was left stirring overnight at 4℃. The resulting crystalline solid was then washed with coldethanol, hot water, hexanesand dried to afford 9.65 g (yield 70%) of 3as a pale yellow solid: H NMR (500 MHz,CDCl_3) δ7.56 (s, 2H), 7.18 (s, 1H), 3.98 (s, 6H), 2.54(s, 3H), 2.39(s, 3H); LC/MS (LC: gradient 20-95% MeCN [0.1% HCO_2 H] over 2.5min, 0.5 ml/min flow rate, MS: ESI^+): retention time, 2.15 min; purity, 95%; 306.36 〖[M+H]〗^+. Then we can get Compound 3.Compound 3 (1.12 g, 0.005mol) was refluxed with 15 ml of ethanol, 1 ml of 40% aqueous methylamine, and 700 mg of potassium carbonate for 4h.  The reaction mixture was removed from heat and upon cooling formed an orange precipitate. The precipitate containing the product was filtered and washed briefly with cold ethanol. The precipitate was then redissolved in a 1:1 mixture of ethyl acetate and 500 mM sodium acetate pH 3.0. The organic layer was separated, dried with anhydrous sodium sulfate and solvent was removed under reduced pressure to yield 717 mg (yield 52%) of DMHBI as an orange solid: 1H NMR (500MHz, DMSO-d_6) δ7.62 (s, 2H), 6.90 (s, 1H), 3.80 (s, 6H), 3.09 (s, 3H), 2.34 (s, 3H). The process during synthesis has been shown in Fig.28.</p>
+
<p class="highlighttext">Author:Liu Tian </p>
-
<img src=""  width="px" class="img-center"/>
+
<p class="highlighttext"><span style="font-weight:bold;">4.1.1 Experimental Background</span></p>
-
<p class="figuretext">Fig.32</p>
+
<p class="highlighttext">The iGEM encourages us to communicate and help with each other. So, when we heard HUST-China has something wrong with their promoter, we decided to give a hand. The one problem they met was the failure in sequencing their promoters in pET28a, and another was that they put mRFP under the promoter to do a promoter test but without result. So we got A&B promoters with mRFP from them.</p>
-
<p class="highlighttext">Then the DMHBI should be stored in such condition:</br>
+
-
Avoid light vacuum</br>
+
-
Preserve it in low temperature</br>
+
-
Isolate it from oxygen</p>
+
-
<p class="highlighttext">According to the literature, we will get an orange solid. But in reality, we get something dark brown. After detection, our products are in liquid state and we get 40% in purity of product. In order to get more product in high purity, we invite ShaoQing Zhuang who is a student in Huazhong University of Science and Technology to help us with the synthesis of the DMHBI. And finally we get more than 95% in purity of product.</p>
+
-
<p class="highlighttext">Inoculation and training <span style="font-style:italic;">E.coli</span> all the night. When the =0.8, take out and centrifugal 1.5ml bacterial suspension(6000rpm,1min), and resuspend it with 1ml M9 medium. Then training it 1h in the shakers(37℃, 170rpm). Remove the background of bacterial. Resuspend it with 1ml M9 medium, and add 1μl DMHBI(MW=276). After being dissolved, the concentration of DMHBI is 200mM, And the concentration of what we needed is 200μΜ. Mix and training it 10min in the shakers(37℃, 170rpm). Detect it by fluorescence microscope.</p>
+
-
<p class="highlighttext"><span style="font-weight:bold;">3.1.4 Result and discussion</span></p>
+
<p class="highlighttext"><span style="font-weight:bold;">4.1.2 Proposes</span></p>
-
<p class="highlighttext"><span style="font-weight:bold;">The result of detect E.coli with promoter  and aptamer by blue exciting light is green point. And the result of detect E.coli with promoter  and aptamer by purple exciting light is blue point. Hence, the result is same with the reference.</span></p>
+
<p class="highlighttext">pCusC & PpcoA promoters sequencing</br>
-
<img src=""  width="px" class="img-center"/>
+
pCusC & PpcoA promoters test</p>
-
<p class="figuretext">Fig33. The result of detect <span style="font-style:italic;">E.coli</span> with promoter and aptamer by blue exciting light is green point. (10*40, blue filter).</p>
+
-
<p class="highlighttext">On the one hand, the display of fluorescence microscope is a plane picture. Hence, there are different picture, and we got different fluorescence point, with different focal length. The light, sent from aptamer, is similar to the light sent from GFP, so the light’s penetrating power is weak. Hence, not any bacterial to fluoresce, and we can not see anyone to fluoresce, and we can not see a piece of light. We can see fewer solitary light point.</p>
+
-
<p class="highlighttext">On the other hand, the glowing effect depends on the incubation time of <span style="font-style:italic;">E.coli</span> with DMHBI, and it could be found in the result of the detection of the same day and the detection of the next day. But there are another problem with the incubation time extend. The bad growth state of E.coli will affect the observation.</p>
+
-
<p class="highlighttext">We want take a photo that distinct and have dense fluorescence point. But, there are no ideal picture.We absent <span style="font-style:italic;">E.coli</span> with promoter and no DMHBI. And we do the comparison of other sample in the gel image system ,we can see the result as follow. The sample of intermediate is <span style="font-style:italic;">E.coli</span> with promoter  and aptamer,and it is the brightest. The sample of downmost is <span style="font-style:italic;">E.coli</span> with promoter , and it is brighter than the sample of uppermost . The sample of uppermost is control test sample, only with M9 medium and DMHBI ,and it is the darkest.</p>
+
-
<img src=""  width="px" class="img-center"/>
+
-
<p class="figuretext">Fig.34  The picture of reference.</p>
+
-
<img src=""  width="px" class="img-center"/>
+
-
<p class="figuretext">Fig.35 The picture of test.</p>
+
-
<h5>3.2 Fluorescent proteins with LVA tag</h5>
+
<p class="highlighttext"><span style="font-weight:bold;">4.1.3 Materials and equipment</span></p>
-
<p class="highlighttext"><span style="font-weight:bold;">3.2.1 Experimental Background</span></p>
+
<p class="highlighttext"><span style="font-weight:bold;">Materials</span></br>
-
<p class="highlighttext"><span style="font-weight:bold;">Testing whether Fluorescent proteins with LVA tag can degrade rapidly or not for further use in our project.</span></p>
+
pCusC & PpcoA promoters with mRFP in pET28a plasmid</br>
 +
LB medium</br>
 +
Kanamycin</p>
 +
<p class="highlighttext"><span style="font-weight:bold;">Equipment</span></br>
 +
Multifunctional microplate reader</br>
 +
Shaking incubator </p>
-
<p class="highlighttext"><span style="font-weight:bold;">3.2.2 Materials and equipment</span></p>
+
<p class="highlighttext"><span style="font-weight:bold;">4.1.4 Method</span></br>
-
<p class="highlighttext"><span style="font-weight:bold;">Material:</span></br>
+
<p class="highlighttext"><span style="font-weight:bold;">Sequencing</span></p>
-
DMHBI, M9 broth, E.coli (DH5α) </p>
+
<p class="highlighttext">We designed the 5’-agatcgggctcgccacttcg-3’ as the reverse primer to sequence the promoters. And Tsing Ke Biology Technology Company helped us to synthesize the primer and sequence those plasmid. (You can get the sequencing results of two samples at supplement 1.) After analyzing the sequencing result for sample pCusC , we found that the T7 promoter had't been digested, and no pCusC sequence in the plasmid. But mRFP had been successfully ligated. So we can observed the red fluorescence due to the expression of mRFP driven by T7 promoter. Therefore, in the subsequent promoter test, we abandoned samples of the pCusC. As for the PpcoA, we successfully found PpcoA and mRFP. However, at the end of PpcoA's sequencing result, there is 6 more base-pairs than HUST’s sequence. We took PpcoA promoter to detection.</p>
-
<p class="highlighttext"><span style="font-weight:bold;">Equipment:</br>
+
<p class="highlighttext"><span style="font-weight:bold;">Promoter test</span></p>
-
fluorescence microscope</span></p>
+
<p class="highlighttext">Add 2mL LB culture medium, 2uL Kanamycin (50mg/L) and 20uL bacterial samples to a 5mL centrifuge tube. Shaken overnight in the 37°C shaking incubator and set the rotational speed at 180 rpm/min.</p>
 +
<p class="highlighttext"> Add CuSO4 solution to induce in concentration of 0, 0.02, 0.1 and 1mM. Set three repetitions for each concentration. Cultivate in the 37°C shaking incubator at the rotational speed of 180 rpm/min for 4 hours.</p>
 +
<p class="highlighttext">Add 200uL bacterial samples from each centrifuge tube to the 96-well plate. Set two copies for each centrifuge tub. Meanwhile, add LB culture medium containing 1.0mM CuSO4 or not as blank controls.</p>
 +
<p class="highlighttext">Read the OD<sub>600</sub> and fluorescent intensity (with the emission wavelength at 607nM and excitation wavelength at 584nM) using a multifunctional microplate reader.</p>
 +
<p class="highlighttext">Divided the fluorescent intensity result using the value of OD<sub>600</sub>. Record the data for compare and analysis. </p>
-
<p class="highlighttext"><span style="font-weight:bold;">3.2.3 Method</span></p>
+
<p class="highlighttext"><span style="font-weight:bold;">4.1.5 Result and discussion</span></p>
-
<img src=""  width="px" class="img-center"/>
+
<img src="https://static.igem.org/mediawiki/2014/9/9a/Figure_21..jpg"  width="700px" class="img-center"/>
-
<p class="figuretext">Fig.36 output module of plan 2</p>
+
<p class="figuretext">Figure 21. pCoa Promoter Test</p>
 +
<p class="highlighttext">When we added the 1.0mM Cu to bacterium solution,we observed the color change of the visible to the naked eye. So we tried to test the 1.0mM copper ion in LB medium. Then we found its fluorescence value and the value of OD<sub>600</sub> with only LB medium samples differred greatly. So we decided to use the 1.0mM copper ion in LB medium as the blank samples of the 1.0mM control. The result shown in figure presents that promoter strength increases with the rising of copper ion concentration below 0.1mM, and then drops down once above 0.1mM.</p>
-
<p class="highlighttext"><span style="font-weight:bold;">3.2.4 Result and discussion</span></p>
 
-
<img src=""  width="px" class="img-center"/>
 
-
<p class="figuretext">Fig.37</p>
 
-
<h6>4. Processing module test</h6>
+
<p class="highlighttext"><span style="font-weight:bold;">4.2 Promoter test</span></p>
 +
<p class="highlighttext">Author: Yuan Lu</p>
 +
<p class="highlighttext"><span style="font-weight:bold;">4.2.1 Experimental Background</span></p>
 +
<p class="highlighttext">The three devices used in our project are concerned with plenty of functions, such as repessilator, feedback and feedforward. Since the difference between frequencies of initiation of the promoters has critical impact on the functions of these processing module, it is important to measure the relative frequencies of initiation between these promoters.</p>
 +
<p class="highlighttext">Our purpose is to measure the relative initiation frequencies of promoters used in our devices, which are BBa_J23119、BBa_R1051、BBa_R0062、BBa_R0040、BBa_R0063 and BBa_R0011.</p>
-
<p class="highlighttext"><span style="font-weight:bold;"></span></p>
+
<p class="highlighttext"><span style="font-weight:bold;">4.2.2 Materials and equipment</span></p>
 +
<p class="highlighttext"><span style="font-weight:bold;">Materials</span></br>
 +
Parts:  Promoters (BBa_J23119、BBa_R1051、BBa_R0062、BBa_R0040、BBa_R0063、BBa_R0011), mCherry (BBa_J06504), RBS (BBa_B0034), Terminator (BBa_B0015)</br>
 +
Strain: Escherichia Coli (DH5α)</br>
 +
LB , assorted antibiotics</p>
 +
<p class="highlighttext"><span style="font-weight:bold;">Equipment</span></br>
 +
Multi-function Enzyme-labeled instrument</p>
 +
 
 +
<p class="highlighttext"><span style="font-weight:bold;">4.2.3 Method</span></br>
 +
<p class="highlighttext">Using BioBrick method, we constructed our detection device by ligating each of the six promoters (LuxpR, LuxpL, BBa_J23119, pcI, plac, ptet) to mCherry (RFP). After transformation, we set up overnight culture from single colonies of Escherichia. Coli (DH5α) strain in LB medium. After 8 hours, we measured the fluorescent degree of each of the samples by using enzyme-labeled instrument. </p>
<p class="highlighttext"></p>
<p class="highlighttext"></p>
-
<img src=""  width="px" class="img-center"/>
+
<p class="highlighttext"></p>
-
<p class="figuretext"></p>
+
 
 +
<p class="highlighttext"><span style="font-weight:bold;">4.2.4 Experimental Results and Discussion</span></p>
 +
<img src="https://static.igem.org/mediawiki/2014/e/eb/Figure_23._The_detection_of_the_relative_strength_of_the_promoters.png"  width="700px" class="img-center"/>
 +
<p class="figuretext">Figure 22. The detection of the relative strength of the promoters</p>
 +
 
 +
 
 +
<p class="highlighttext"><span style="font-weight:bold;">Analysis:</span></p>
 +
<img src="https://static.igem.org/mediawiki/2014/3/31/Hzau-note-23.png"  width="700px" class="img-center"/>
 +
 
 +
<p class="figuretext">Figure 23. The detection of the relative strength of the promoters</p>
 +
<p class="highlighttext">Based on the data from Figure 22, we do the statistical analysis. With analysis diagram shows in Figure 23,  we can conclude that the rank of relative strength of the promoters from strong to weak is: BBa_R0063、BBa_R0011、BBa_J23119、BBa_R1051、BBa_R0040、BBa_R0062.</p>
 +
 
 +
<p class="highlighttext"><span style="font-weight:bold;">4.3 Extra characterization of Part</span></p>
 +
<p class="highlighttext">Author:Lu Wang</p>
 +
<p class="highlighttext"><span style="font-weight:bold;">4.3.1 Experimental Background</span></p>
 +
<p class="highlighttext">Cre recombinase is the type I topoisomerase found in phage P1 that can catalyze site-specific recombination between loxP sites. Lox71 is a mutation of loxP site which can also be specifically recognized by Cre recombinase. Our purpose is to confirm that unexpected recombination of lox71 site exits through sequencing result and observation under fluorescence microscope.</p>
 +
 
 +
<p class="highlighttext"><span style="font-weight:bold;">4.3.2 Materials and equipment</span></p>
 +
<p class="highlighttext"><span style="font-weight:bold;">Materials</span></br>
 +
Part: Lac Promoter; Lox71 site (BBa_K886000); mCherry (BBa_J06504)</br>
 +
Strain: Escherichia. Coli(DH5α, DH10b, BL21(DE3))</br>
 +
Glycerinum, LB , assorted antibiotics</p>
 +
<p class="highlighttext"><span style="font-weight:bold;">Equipment</span></br>
 +
Fluorescence microscope</p>
 +
 
 +
<p class="highlighttext"><span style="font-weight:bold;">4.3.3 Method</span></p>
 +
<p class="highlighttext"><span style="font-weight:bold;">Device Assembly</span></p>
 +
<p class="highlighttext">Ligate the Lac promoter with lox71 site and lox71 site with mCherry by using the BioBrick assembly.</p>
 +
<img src="https://static.igem.org/mediawiki/2014/1/17/Figure_26._Confirmation_device.png"  width="700px" class="img-center"/>
 +
<p class="figuretext">Figure 24. Confirmation device</p>
 +
<p class="highlighttext">Join the Lac promoter-lox71 and lox71-mCherry fragments with double digestion and ligation.
 +
Transform the confirmation device into Escherichia. Coli ( DH5α, DH10B), and set up overnight culture from fresh single colonies of Escherichia. Coli( DH5α) strain in Luria Broth containing chloramphenicol and IPTG inducer at 37℃ ,180 rpm.</p>
 +
<p class="highlighttext">Observe the culture’s color and take a small amount under the fluorescence microscope to observe the single bacteria. Extract the plasmids from single colonies and sequence the plasmid with general purpose primer.</p>
 +
<p class="highlighttext">Co-transform the confirmation device and plasmids that code Cre recombinase into Escherichia. Coli(DH5α), set up overnight culture from fresh single colonies of Escherichia. Coli( DH5α) strain in Luria Broth containing chloramphenicol and IPTG inducer at 37℃ ,180 rpm.</p>
 +
 
 +
<p class="highlighttext"><span style="font-weight:bold;">4.3.4 Experimental Results and Discussion</span></p>
 +
<p class="highlighttext">After cultivate with IPTG in the 37°C shaking incubator at the rotational speed of 180 rpm/min for 24 hours, we used the fluorescence microscope to observe bacteria and you can see result at Figure 25(DH5α) and Figure 26(DH10B)below.</p>
 +
<img src="https://static.igem.org/mediawiki/2014/5/54/Figure_27._Unexpected_recombination_in_DH5%CE%B1.png"  width="600px" class="img-center"/>
 +
<p class="figuretext">Figure 25. unexpected recombination in DH5α</p>
 +
<img src="https://static.igem.org/mediawiki/2014/8/81/Figure._28_Unexpected_recombination_in_DH10B.png"  width="600px" class="img-center"/>
 +
<p class="figuretext">Figure 26. Unexpected recombination in DH10B</p>
 +
<p class="highlighttext">At the same time, the samples were sequenced and anything was OK. In summary, we confirmed the unexpected recombination of lox71 site even if the device is in the stable E.coli strains such as the DH5α and DH10B. But the probability of its occurrence is extremely low. And it doesn’t affect the sequencing result in a short time. The tetR protein might be poisonous to the E.coli, when the promoter is deleted by lox71 site, the bacteria will get the survival advantage and quickly occupy the dominant position in LB medium. Therefore, we recommend that you save the plasmid rather than bacteria when you use the cre/lox system.</p>
 +
<div class="clear"></div>
 +
                <div class="divider"></div> 
 +
 
<h5>Reference:</h5>
<h5>Reference:</h5>

Latest revision as of 03:48, 18 October 2014

<!DOCTYPE html> 2014HZAU-China

Labnotes

Molecule Labnotes

Author: Yihui Zheng

Members: Yihui Zheng, Yajing Zhang, Zhixiang Liu, Xudong Luo, Xiaoxue Guo, Lu Wang, Siqi Li.

Introduction:

We have eight gene circuits to construct (as shown in the Figure 1), and after finishing these circuits, we will integrate some of them into one plasmid to form two repressilators and two detection devices (as shown in the Figure 2a and Figure 2b).

Figure 1. eight gene circuits

Figure 2a. first rewirable circuit and its detection device

Figure 2b. second rewirable circuit and its detection device (quorum sensing)



May

Content: we found the parts we needed in the Plate Kits and did a lot of transformations to get the plasmid for subsequent experiments.

Parts list: BBa_J06602 BBa_E0420 BBa_K886000 BBa_R1051 BBa_P0412 BBa_R0040 BBa_P0451 BBa_R0011 BBa_R0051 BBa_I714075 BBa_J61047 BBa_R0063 BBa_K516022 BBa_K805016 BBa_J37032 BBa_K081019 BBa_K773003 BBa_I13602 BBa_R0061 BBa_R0062 BBa_R0063 BBa_C0060 BBa_B0015 BBa_I718016 BBa_I0462 BBa_P0440 BBa_E0022 BBa_J01010



June

Content: eight gene circuits’ construction

Construction details:

1.Construction of gene circuit one: we used standard method of biobrick connection to put the coding sequence of cI protein behind PLlacI promoter. But we failed to construct it because of some unknown reasons. The sequence results showed that there is either a pretty long and unknown segment which insert into the coding sequence of cI protein or deficiency of the sequence of PLlacI promoter(as shown in the Figure 3). So, we had an assumption that there were little LacI proteins in DH-5α, and it was not much enough to repress PLlacI promoter, resulting in the overexpression of cI protein which may restrain the growth of host.

Figure 3. diagram of sequence results

2.Construction of gene circuit three: Firstly, we used standard method of biobrick connection to put the coding sequence of LacI protein behind lox66 because the length of lox66 was 34bp and it was too small to purify from the enzyme-digested product and then put it in front of LacI protein. Due to the same reason, we put the product of previous step behind PLtet01 promoter to finish this gene circuit. Compared to construction of other gene circuits, it seemed that this gene circuit was the simplest job we’ve made since that we only tried once and took almost eight days to finish this work.

3.Construction of gene circuit two: we constructed this gene circuit by following the method we’ve mentioned above. The only difference from the construction of gene circuit three is that we put the coding sequence of TetR protein behind lox71. By the way, lox66 and lox71 are two recombination sites which are recognized specifically by Cre protein. To our surprise, when we did double-digest to this circuit for verification, we found that the length of product of enzyme-digested was not right and there was an extra stripe that we didn’t know what it was. Results are shown in the Figure 4. According to the results, we had another assumption to explain the phenomenon: there were some recombination systems in DH-5α, resulting in homologous recombination of two strains which had synclastic lox71 site. Definitely, We’ve done a lot of work to verify our assumption which will be explained in the part of characterization.

Figure 4. the stripes which arrows point towards are enzyme-digested product of gene circuit two and the rightest stripe in each figure is DL-5000 marker. There were at least three stripes and the length of extra stripe is wrong.



July

Content: eight gene circuits’ construction

1.Construction of gene circuit four: we used standard method of biobrick connection to put the coding sequence of mCherry protein behind Pλ promoter. And when we planned to construct the part CFP expression controlled by PLlacI promoter, we didn’t find CFP with LVA tag in the Plate Kits, so we decided to get this part through PCR which means that we added a LVA tag behind the coding sequence of CFP. And then, we used standard method of biobrick to connect PLlacI promoter with the coding sequence of CFP to finish the whole circuit. Figure 5 will show the visual effects of mCherry protein expression and CFP expression under the fluorescent microscope.

Figure 5. a was without any excitation light; b and c were mCherry protein expression and CFP expression under the fluorescent microscope

2.Construction of gene circuit five: Because of the complex stem-loop structure of RNA aptamer, it was very hard to get this part by overlapping PCR. So, we found a company to help us to synthetize the sequence of RNA aptamer, and we standardized RNA aptamer and also we connected it with the promoter (lambda cI regulated). To test whether it works or not, we synthetized corresponding fluorophore called DHMBI, and saw the figure under the fluorescent microscope as shown in the output module.

3.Construction of gene circuit six and eight: Construction of gene circuit six and eight: These two gene circuits are familiar in their structure and then, we constructed them simultaneously. Firstly, we divided the circuit into two parts: riboregulator and Cre protein with double terminators. When it came to the synthesis of riboregulator, we designed four specific primers which had almost 20 overlapping sequence and conducted twice PCR to get our riboregulator (more details are shown in our protocol and the input module). Then, we connected both of them by the standard method of biobrick connection. Figure 6a and 6b are the visual effects of gene circuit six without AHL and gene circuit with AHL under the fluorescent microscope, and Figure 6c is the quantitative result of these two samples and we can find that there is a obvious difference between gene circuit six without AHL and with AHL which is accord to our expectation.

Figure 6. a and b are the visual effects of gene circuit six without AHL and with AHL; c is the quantitative result of these two samples



August

Content: we verified our first assumption that the overexpression of cI protein may restrain the growth of host which cause the failure of construction of gene circuit one, so, we designed another method to construct it and finally made it. Then, we integrated three circuits into one plasmid to form our repressilator.

1.Verification for the first assumption that there were little LacI proteins in DH-5α, and it was not enough to repress PLlacI promoter, resulting in the overexpression of cI protein which may restrain the growth of host:

We designed a primer contained PstI site which could match the middle part of the coding sequence of cI protein, and then, we conducted a PCR by using VF2 primer and the designed primer to get a sequence which can express an inactive cI protein. We put this sequence behind PLlacI promoter through the same way mentioned above and the sequencing results showed that we constructed it and it proved our assumption to some extent. Then, we came up with a different way to construct our first repressilator which will be explained in the following.

2.New way to construct our first repressilator: Due to toxicity of cI protein on host bacteria and the expression of cI protein which was regulated by PLlacI promoter, we came up with a new method that putting the gene circuit three in front of PLlacI promoter because circuit three would express LacI protein to repress the activation of PLlacI promoter and thus, cI protein would not be overexpressed. So, we digested circuit three by EcoRI enzyme and SpeI enzyme and digested PLlacI promoter by EcoRI enzyme and XbaI enzyme, and then do a ligation to get a circuit contained PLlacI promoter controlled by LacI protein. After that, we connected the coding sequence of cI protein with the previous product to get a compound contained circuit one and circuit three. Gel electrophoresis spectrum diagram of plasmid is shown in Figure 7.

Figure 7. plasmid contained circuit one and circuit three (a,b)



September

Content: we constructed the first repressilator and change the vector of detection device from high copy to low copy.

1.The construction of first repressilator: The first repressilator contained three gene circuits: circuit one, two and three, and at that moment, we connected circuit one with circuit three, lacking of circuit two. We tried our best to construct it and due to the special condition that we needed to connect this two part reversely and change the vector of repressilator from high copy to low copy, we designed a new method to connect two parts reversely that we digested circuit one and three by EcoRI enzyme and PstI enzyme, meanwhile, we digested circuit two by EcoRI enzyme and XbaI enzyme and target vector by XbaI enzyme and PstI enzyme, leading to the result that there was a common EcoRI site for ligation between circuit two and the compound contained circuit one and three. We’ve tried at least five times in this construction and we tried to separate this procedure into two steps: connection first and then change the vector, but all tries failed. Surprisingly, in the sixth trial, we may design an optimal system of ligation including the proportion between target DNA and vector, we constructed this repressilator successfully. Gel electrophoresis spectrum diagram of plasmid is shown in Figure 8

Figure 8. plasmid of repressilator

2.Changing the vector of detection device from high copy to low copy: we should change the vectors of two detection devices including the compound contained circuit four and circuit six and the gene circuit eight, and at the same time, we changed the vector of RNA aptamer generator which played the same function as fluorescent protein. The indicator to judge whether it had changed successfully or not was the resistance gene: the vector of high copy had chloramphenicol-resistance gene and the vector of low copy had Ampicillin-resistance gene.



October

Content: we constructed our seventh gene circuit about quorum sensing and tested the intensity of GFP through fluorescent microscope and microplate reader.

Construction of gene circuit seven: We divided this circuit into four parts: AiiA protein generator, LuxI protein generator, LuxR protein generator and GFP generator. Both the parts GFP expression controlled by LuxR promoter and luxR protein generator under regulation were found in Plate Kits. As for another two parts, we did them the same thing as the construction of gene circuit three. And then, we did the same thing as the construction of our first repressilator to connect three segments to form our seventh gene circuit. To judge whether it was connected correctly and successfully, we co-transformed our gene circuit seven and eight (with Cre protein) and transformed only the gene circuit seven (without Cre protein) as a comparison, the results shown in the Figure 9 and Figure 10 were exactly accord to our expectation which indicated that we constructed it successfully.

Figure 9. a was without any excitation light; b was the phenomenon when we co-transformed the gene circuit seven and eight (with Cre protein); c was the phenomenon when we only transformed the gene circuit seven (without Cre protein) as a comparison.

Figure 10. result of fluorescence intensity

Function test labnotes

1. Input module test
1.1 Cre recombinase function test

Author:Lu Wang

1.1.1 Experimental Background

Our purpose is to confirm the function of Cre recombinase.

1.1.2 Materials and equipment

Materials:
Part:mCherry (BBa_J06504)
Strain: Escherichia. Coli (DH5α)
LB, assorted antibiotics

1.1.3 Method

Figure 1. Our designed device

Co-transform the pSB1A3 and pSB1C3 which we designed as our Confirmation device (as shown in Figure 1) into Escherichia. Coli (DH5α) competent. Culture it overnight from a fresh single colony of Escherichia. Coli (DH5α) strain in LB containing chloramphenicol, ampicillin and IPTG inducer. And wait for about 8 hours to observe the culture’s color.

1.1.4 Result and discussion

Observation on Group Level

Figure 2. Observation on Group Level (the left one is Negative Control contained mCherry without promoter,the middle one is Group One only contained confirmation device,the right one is confirmation device with Cre recombinase)

Figure 3. Observation on Group Level (the left one is Group One only contained confirmation device,the right one is confirmation device with Cre recombinase)

The results indicate that the culture of Negative Control and Group One has become opaque but still keep being pale yellow. The Group Two’s culture has turned red obviously and also opaque.

Therefore, conclusion could be drawn that plasmids which code Cre recombinase in the registry work properly and the confirmation device we assembled is valid.

1.2 Riboregulator function test

Author:Lu Wang

1.2.1 Experimental Background

Confirm if the leakage of lac promoter’s expression will be reduced under the control of riboregulator.

1.2.2 Materials and equipment

Materials:
Strain: Escherichia. Coli (DH5α,BL21(DE3))
LB , ampicillin antibiotic
Equipment:
Multifunctional microplate reader

1.2.3 Method

Figure 4. Designed Device (1)

Figure 5. Designed Device (2)

Firstly, construct these two devices shown in Figure 4 and Figure 5. Respectively transform each of them into Escherichia. Coli strain BL21 (DE3). Pick single colonies and add each of them into 5ml LB liquid with its corresponding antibiotic and incubate it in shaking incubator at 37 ℃,180rpm for 8 hours. Secondly, add 5ul of previous single colony cultural from each tube into 5ml fresh M9 broth. And we divided all the sample tubes into two groups: one was the cells with designed device (1) plasmid and we named it group A. Another was the cells with designed device (2) plasmid and we named it group B. Add 0.1ul IPTG into 12 tubes of both group A and B, while add nothing into the other 12 tubes of both group A and B as control . Incubate them at 37 ℃ for 4 hours. Lastly, measure the fluorescent intensity by the multifunctional microplate reader.

1.2.4 Result and discussion

Figure 6.The data from the enzyme-labeled instrument.

Experimental analysis:

Figure 7.Experimental analysis based on the data from enzyme-labeled instrument

Conclusion:

Riboregulators ensure much lower leakage of the promoters.

1.3 Test of input module's tight regulation

Author:Lu Wang

1.3.1 Experimental Background

Our purpose is to confirm whether the cre will be expressed or not under the control of riboswitch driven by IPTG.

1.3.2 Materials and equipment

Materials:
Vector-pSB1A3, pSB1C3
Strain: Escherichia. Coli (DH5α)
LB , assorted antibiotics
Equipment:
Multifunctional microplate reader

1.3.3 Method

Figure 8. input device

Figure 9. verification device

Process:

1. Co-transform our input device and verification device into BL21(DE3) competent cell and plate it onto a selective plate, incubate it at 37 ℃ for 14 hours.

2. Pick 3 different single colonies numbered 1, 2, 3 into 3 tubes of LB broth with its corresponding antibiotic and incubate it respectively at 37℃ for 8 hours.

3. Add 5ul of previous single colony cultural from each tube to two new tubes named A,B respectively with M9 broth in it. Add 0.1ul IPTG to all A tubes (1A, 2A, 3A) while add nothing to all B tubes (1B, 2B, 3B). Incubate them at 37℃ for 4 hours.

4. Measure the fluorescent intensity by the enzyme-labeled instrument.

Prediction:

a) If the fluorescent degree in cultural tubes with IPTG showed much less weaker than in the tubes without it, then it indicated that the device had achieved its function for reducing the expression of Cre protein as expected.

b) If the fluorescent degree in the cultural tubes with IPTG showed almost same or even stronger than in the tubes without it, it meant that the devices had lost its function. However, there may be 3 reasons which are responsible for its failure. The first reason may be the failed function of riboregulator to repress the translation process. Secondly, despite the strong repression dueing to the riboregulator, the leakage expression of Cre protein can still express a little bit Cre protein which may cause the same result of a device without riboregulator. Thirdly, the low production of lacI in BL21(DE3) bacteria is unable to repress the operon efficiently which leads to the high expression of both taRNA and cre mRNA and thus weaken the function of riboregulator.

1.3.4 Result and discussion

Figure 10. Fluorescent degree (OD600)

Figure 11. The rigour detection of output device

As shown above, even though the addition of riboregulator does reduce a little of the expression of Cre protein, the difference between control group and experimental group is still very small which means that the riboregulator can hardly exert some critical influence on the control of the expression of cre gene.



2. Output module test
2.1 The Detection of Aptamer (Qualitative Detection)

Author: Yajing Zhang, Siqi Li

2.1.1 Experimental Background

Our purpose is to confirm whether the aptamers is available or not.

2.1.2 Materials and equipment

Material:
DMHBI, M9 broth, Escherichia. Coli(DH5α)

Equipment:
Fluorescence microscope

2.1.3 Method

The synthesis of DMHBI

N-Acetylglycine (5.26 g, 0.045mol), anhydrous sodium acetate (3.69 g, 0.045mol), 4-hydroxy-2,3-dimethoxybenzaldehyde (8.19 g, 0.045mol), and acetic anhydride (15 ml) were stirred at 90℃for 2 h. After allowing the reaction to cool to room temperature, cold ethanol (20 ml) was added while still stirring and the reaction was left stirring overnight at 4℃. The resulting crystalline solid was then washed with coldethanol, hot water, hexanesand dried to afford 9.65 g (yield 70%) of 3as a pale yellow solid: H NMR (500 MHz,CDCl_3) δ7.56 (s, 2H), 7.18 (s, 1H), 3.98 (s, 6H), 2.54(s, 3H), 2.39(s, 3H); LC/MS (LC: gradient 20-95% MeCN[0.1% HCO_2 H] over 2.5min, 0.5 ml/min flow rate, MS: ESI^+): retention time, 2.15 min; purity, 95%; 306.36 〖[M+H]〗^+. Then we can get Compound 3.Compound 3 (1.12 g, 0.005mol) was refluxed with 15 ml of ethanol, 1 ml of 40% aqueous methylamine, and 700 mg of potassium carbonate for 4h. The reaction mixture was removed from heat and upon cooling formed an orange precipitate. The precipitate containing the product was filtered and washed briefly with cold ethanol. The precipitate was then redissolved in a 1:1 mixture of ethyl acetate and 500 mM sodium acetate pH 3.0. The organic layer was separated, dried with anhydrous sodium sulfate and solvent was removed under reduced pressure to yield 717 mg (yield 52%) of DMHBI as an orange solid: 1H NMR (500MHz, DMSO-d_6) δ7.62 (s, 2H), 6.90 (s, 1H), 3.80 (s, 6H), 3.09 (s, 3H), 2.34 (s, 3H).

Figure 12. The process during synthesis

Then the DMHBI should be stored in such conditions:
Prevent from light and oxygen
Preserve it in low temperature

According to the literature, we will get an orange solid. But in reality, we get something dark brown. After detection, our products are in liquid state and we get 40% in purity of product. In order to get more product in high purity, we invite ShaoQing Zhuang who is a student in Huazhong University of Science and Technology to help us with the synthesis of the DMHBI. And finally we get more than 95% in purity of product.

DH5α cells (TransGen Biotech) were transformed with 10μl of plasmid DNA expressing chimeras of the human tRNALys3 scaffold fused to the Spinach aptamer’s sequence. Cells were plated, grown overnight and single colonies were picked for inoculation overnight in Luria Broth containing chloramphenicol. At =0.8, 150μl culture was removed, pelleted and resuspended in 100μl M9 minimal media and cultivate for 1 h at 37 ℃. Cells were washed twice and incubated with 200μM DFHBI in M9 media for 5 min. We tested the spinach RNA aptamer in the Tanon 1600R Gel Imaging System under the 302nm, the control groups are the DMHBI and DMHBI + control RNA. The result is below (Figure 15) and we also observed the spinach RNA aptamer by using the fluorescence microscope, the result is in the Figure 13.

2.1.4 Result and discussion

Figure 13. The result of detecting E.coli with promoter and aptamer by blue exciting light is green point. (10*40, blue filter).

Figure 14. The picture of reference.

Figure 15. The picture of test.

Because the brightness of the RNA-fluorophore complex is very low.(According to reference report, it is only decimusly brighter than the GFP ) And the half- life of RNA is just few minutes. So the concentration of RNA-fluorophore complex is at low level in cells. The Figure 15 has shown the difference among three samples.The sample of intermediate is E.coli with Pλ promoter and aptamer,and it is the brightest. The sample of downmost is E.coli with Pλ promoter, and it is brighter than the sample of uppermost . The sample of uppermost is control test sample, only with M9 medium and DMHBI ,and it is the darkest.We conclude that the aparter is available.

2.2 Fluorescent proteins with LVA tag

2.2.1 Experimental Background

For further use in our project, we are going to test whether fluorescent proteins with LVA tag can degrade rapidly or not.

2.2.2 Materials and equipment

Material:
DMHBI, M9 broth, E.coli (DH5α)

Equipment:
Fluorescence microscope

2.2.3 Method

Figure 16. Output module of plan 2

2.2.4 Result and discussion

Figure 17. a: without any excitation light b: mCherry c: CFP



3. Processing module test

Author :Yajing Zhang, Yihui Zheng

3.1 Experimental Background

To test our processing module.

3.2 Materials and equipment

Material:
Parts: BBa_K805016, BBa_R0062, BBa_R0063, BBa_K516022, BBa_I0462, BBa_I718016, BBa_K886000, BBa_I714075, BBa_B0015, BBa_J61047
Strain: Escherichia. Coli (BL21 (DE3))
LB , assorted antibiotics

Equipment:
Perkin-Elmer plate reader

3.3 Method

Figure 18. The testing of processing device.

The processing plasmid and input plasmid were co-transformed into the Escherichia. Coli (BL21 (DE3)) strain as experimental group and only the processing plasmid was transformed into the Escherichia. Coli (BL21 (DE3)) as control.

Single colonies were used to inoculate 5 mL LB media supplemented with 50ug/ mL ampicillin , 50ug/mL kanamycin and grown overnight at 37°C.And the control was used to inoculate 5 mL LB media supplemented with 50ug/mL kanamycin.

The next morning, overnight cultures were inoculated with 5 mL fresh LB cultures supplemented with antibiotics at 1:100 dilution. Cultures were grown at the desired temperature in constant temperature shaker following 3 h or 5h of growth (Both the experimental group and control were set two contrats further to test if the inducer, IPTG for experimental group and AHL for control group, would make influnece. ), then cells were plated in triplicate in a 96-well BD-Falcon plate. The optical density at 600 nm and fluorescence (X excitation = 475 nm and X emission = 515 nm) were measured using a Perkin-Elmer plate reader every 15 or 20 minutes.

When cells grow to mid-stage, one of the experiment groups was induced with 100uM IPTG, the optical density at 600 nm and fluorescence (X excitation = 475 nm and X emission = 515 nm) were measured using a Perkin-Elmer plate reader every 15 or 20 minutes after 3 hours ,at the same time cells were grown at 30°C. (Figure 17)

3.4 Result and discussion

Figure 19. The value of fluorescence intensity changes over time, the time begins with all the E.coli growing to mid-log.

The result indicated the processing device without Cre recombinase was in high fluorescence and the processing device with Cre recombinase was in low fluorescence, it works as expected. And whether it was induced by IPTG had no difference, the reason was mentioned in the tight regulation test for input module, because of the leakage of Cre recombinase. We also found the experimental groups grew better than the control with only processing plasmid. (Figure 18) Maybe the positive feedback caused more loading, but the negative feedback didn't. So we chose the data when all groups of the cells grow to mid-log phase.

Figure 20. the growth curve of the E.coli , a: the control. b, the control with 100nM AHL. c: the experimental group without IPTG inducer. d: the experimental group with 100uM IPTG inducer. e :the experimental group with 100uM IPTG inducer after growing to mid-log. f: the control growing to stable phase.



4. Other work

4.1.Interaction work(pCusC, PpcoA sequencing and promoter test)

Author:Liu Tian

4.1.1 Experimental Background

The iGEM encourages us to communicate and help with each other. So, when we heard HUST-China has something wrong with their promoter, we decided to give a hand. The one problem they met was the failure in sequencing their promoters in pET28a, and another was that they put mRFP under the promoter to do a promoter test but without result. So we got A&B promoters with mRFP from them.

4.1.2 Proposes

pCusC & PpcoA promoters sequencing
pCusC & PpcoA promoters test

4.1.3 Materials and equipment

Materials
pCusC & PpcoA promoters with mRFP in pET28a plasmid
LB medium
Kanamycin

Equipment
Multifunctional microplate reader
Shaking incubator

4.1.4 Method

Sequencing

We designed the 5’-agatcgggctcgccacttcg-3’ as the reverse primer to sequence the promoters. And Tsing Ke Biology Technology Company helped us to synthesize the primer and sequence those plasmid. (You can get the sequencing results of two samples at supplement 1.) After analyzing the sequencing result for sample pCusC , we found that the T7 promoter had't been digested, and no pCusC sequence in the plasmid. But mRFP had been successfully ligated. So we can observed the red fluorescence due to the expression of mRFP driven by T7 promoter. Therefore, in the subsequent promoter test, we abandoned samples of the pCusC. As for the PpcoA, we successfully found PpcoA and mRFP. However, at the end of PpcoA's sequencing result, there is 6 more base-pairs than HUST’s sequence. We took PpcoA promoter to detection.

Promoter test

Add 2mL LB culture medium, 2uL Kanamycin (50mg/L) and 20uL bacterial samples to a 5mL centrifuge tube. Shaken overnight in the 37°C shaking incubator and set the rotational speed at 180 rpm/min.

Add CuSO4 solution to induce in concentration of 0, 0.02, 0.1 and 1mM. Set three repetitions for each concentration. Cultivate in the 37°C shaking incubator at the rotational speed of 180 rpm/min for 4 hours.

Add 200uL bacterial samples from each centrifuge tube to the 96-well plate. Set two copies for each centrifuge tub. Meanwhile, add LB culture medium containing 1.0mM CuSO4 or not as blank controls.

Read the OD600 and fluorescent intensity (with the emission wavelength at 607nM and excitation wavelength at 584nM) using a multifunctional microplate reader.

Divided the fluorescent intensity result using the value of OD600. Record the data for compare and analysis.

4.1.5 Result and discussion

Figure 21. pCoa Promoter Test

When we added the 1.0mM Cu to bacterium solution,we observed the color change of the visible to the naked eye. So we tried to test the 1.0mM copper ion in LB medium. Then we found its fluorescence value and the value of OD600 with only LB medium samples differred greatly. So we decided to use the 1.0mM copper ion in LB medium as the blank samples of the 1.0mM control. The result shown in figure presents that promoter strength increases with the rising of copper ion concentration below 0.1mM, and then drops down once above 0.1mM.

4.2 Promoter test

Author: Yuan Lu

4.2.1 Experimental Background

The three devices used in our project are concerned with plenty of functions, such as repessilator, feedback and feedforward. Since the difference between frequencies of initiation of the promoters has critical impact on the functions of these processing module, it is important to measure the relative frequencies of initiation between these promoters.

Our purpose is to measure the relative initiation frequencies of promoters used in our devices, which are BBa_J23119、BBa_R1051、BBa_R0062、BBa_R0040、BBa_R0063 and BBa_R0011.

4.2.2 Materials and equipment

Materials
Parts: Promoters (BBa_J23119、BBa_R1051、BBa_R0062、BBa_R0040、BBa_R0063、BBa_R0011), mCherry (BBa_J06504), RBS (BBa_B0034), Terminator (BBa_B0015)
Strain: Escherichia Coli (DH5α)
LB , assorted antibiotics

Equipment
Multi-function Enzyme-labeled instrument

4.2.3 Method

Using BioBrick method, we constructed our detection device by ligating each of the six promoters (LuxpR, LuxpL, BBa_J23119, pcI, plac, ptet) to mCherry (RFP). After transformation, we set up overnight culture from single colonies of Escherichia. Coli (DH5α) strain in LB medium. After 8 hours, we measured the fluorescent degree of each of the samples by using enzyme-labeled instrument.

4.2.4 Experimental Results and Discussion

Figure 22. The detection of the relative strength of the promoters

Analysis:

Figure 23. The detection of the relative strength of the promoters

Based on the data from Figure 22, we do the statistical analysis. With analysis diagram shows in Figure 23, we can conclude that the rank of relative strength of the promoters from strong to weak is: BBa_R0063、BBa_R0011、BBa_J23119、BBa_R1051、BBa_R0040、BBa_R0062.

4.3 Extra characterization of Part

Author:Lu Wang

4.3.1 Experimental Background

Cre recombinase is the type I topoisomerase found in phage P1 that can catalyze site-specific recombination between loxP sites. Lox71 is a mutation of loxP site which can also be specifically recognized by Cre recombinase. Our purpose is to confirm that unexpected recombination of lox71 site exits through sequencing result and observation under fluorescence microscope.

4.3.2 Materials and equipment

Materials
Part: Lac Promoter; Lox71 site (BBa_K886000); mCherry (BBa_J06504)
Strain: Escherichia. Coli(DH5α, DH10b, BL21(DE3))
Glycerinum, LB , assorted antibiotics

Equipment
Fluorescence microscope

4.3.3 Method

Device Assembly

Ligate the Lac promoter with lox71 site and lox71 site with mCherry by using the BioBrick assembly.

Figure 24. Confirmation device

Join the Lac promoter-lox71 and lox71-mCherry fragments with double digestion and ligation. Transform the confirmation device into Escherichia. Coli ( DH5α, DH10B), and set up overnight culture from fresh single colonies of Escherichia. Coli( DH5α) strain in Luria Broth containing chloramphenicol and IPTG inducer at 37℃ ,180 rpm.

Observe the culture’s color and take a small amount under the fluorescence microscope to observe the single bacteria. Extract the plasmids from single colonies and sequence the plasmid with general purpose primer.

Co-transform the confirmation device and plasmids that code Cre recombinase into Escherichia. Coli(DH5α), set up overnight culture from fresh single colonies of Escherichia. Coli( DH5α) strain in Luria Broth containing chloramphenicol and IPTG inducer at 37℃ ,180 rpm.

4.3.4 Experimental Results and Discussion

After cultivate with IPTG in the 37°C shaking incubator at the rotational speed of 180 rpm/min for 24 hours, we used the fluorescence microscope to observe bacteria and you can see result at Figure 25(DH5α) and Figure 26(DH10B)below.

Figure 25. unexpected recombination in DH5α

Figure 26. Unexpected recombination in DH10B

At the same time, the samples were sequenced and anything was OK. In summary, we confirmed the unexpected recombination of lox71 site even if the device is in the stable E.coli strains such as the DH5α and DH10B. But the probability of its occurrence is extremely low. And it doesn’t affect the sequencing result in a short time. The tetR protein might be poisonous to the E.coli, when the promoter is deleted by lox71 site, the bacteria will get the survival advantage and quickly occupy the dominant position in LB medium. Therefore, we recommend that you save the plasmid rather than bacteria when you use the cre/lox system.

Reference:

Paige J S, Wu K Y, Jaffrey S R. RNA mimics of green fluorescent protein[J]. Science, 2011, 333(6042): 642-646.

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