Team:HZAU-China/Characterization

From 2014.igem.org

(Difference between revisions)
 
(23 intermediate revisions not shown)
Line 143: Line 143:
         <!-- logo start here -->
         <!-- logo start here -->
         <div id="logo-left">
         <div id="logo-left">
-
             <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>
+
             <a href="https://igem.org/Team.cgi?year=2014&team_name=HZAU-China"><img src="https://static.igem.org/mediawiki/2014/4/4c/Hzau-home-lllogo.png" alt="HZAU-China" /></a>
         </div>
         </div>
         <!-- logo end here -->
         <!-- logo end here -->
Line 159: Line 159:
                 <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>  
-
                         <li><a href="https://2014.igem.org/Team:HZAU-China/Project"><span>-</span>Background</a></li>
+
                         <li><a href="https://2014.igem.org/Team:HZAU-China/Design"><span>-</span>Overview</a></li>
-
<li><a href="https://2014.igem.org/Team:HZAU-China/Design"><span>-</span>Design overview</a></li>
+
                        <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>
Line 169: Line 169:
<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>  
-
                         <li><a href="https://2014.igem.org/Team:HZAU-China/Review"><span>-</span>Overview</a></li>
+
                         <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>
 +
                        <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>
-
                         <li><a href="https://2014.igem.org/Team:HZAU-China/Labnotes"><span>-</span>Labnotes</a></li>  
+
                         <li><a href="https://2014.igem.org/Team:HZAU-China/Labnotes"><span>-</span>Labnotes</a></li>        
                     </ul>
                     </ul>
                 </li>
                 </li>
                 <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>  
-
                         <li><a href="https://2014.igem.org/Team:HZAU-China/Modeling"><span>-</span>Overview</a></li>
+
                         <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>
Line 187: Line 188:
<li class="dropdown"><a href="https://2014.igem.org/Team:HZAU-China/Achievements">Achievements</a>
<li class="dropdown"><a href="https://2014.igem.org/Team:HZAU-China/Achievements">Achievements</a>
    <ul>                         
    <ul>                         
-
                         <li><a href="https://2014.igem.org/Team:HZAU-China/Achievements"><span>-</span>Judging</a></li>
+
                         <li><a href="https://2014.igem.org/Team:HZAU-China/Achievements"><span>-</span>Judgement</a></li>
<li><a href="https://2014.igem.org/Team:HZAU-China/Parts"><span>-</span>Parts</a></li>
<li><a href="https://2014.igem.org/Team:HZAU-China/Parts"><span>-</span>Parts</a></li>
                     </ul>
                     </ul>
Line 203: Line 204:
<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>
-
                         <li><a href="https://2014.igem.org/Team:HZAU-China/Humanities"><span>-</span>Humanities and Aesthetics</a></li>
+
                         <li><a href="https://2014.igem.org/Team:HZAU-China/Humanities"><span>-</span>Humanities</a></li>
-
                         <li><a href="https://2014.igem.org/Team:HZAU-China/Economics"><span>-</span>Economics and Law</a></li>
+
                         <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>Pubic 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>
                         <li><a href="https://2014.igem.org/Team:HZAU-China/Social media"><span>-</span>Social media</a></li>   
                         <li><a href="https://2014.igem.org/Team:HZAU-China/Social media"><span>-</span>Social media</a></li>   
Line 230: Line 231:
     <div id="breadcrumb-content">
     <div id="breadcrumb-content">
         <ul>
         <ul>
-
         <li><a href="index.html">Home</a></li>
+
         <li><a href="https://2014.igem.org/Team:HZAU-China">Home</a></li>
-
             <li><a href="#">Wetlab</a></li>
+
             <li>Wetlab</li>
-
    <li><a href="#">Characterization</a></li>
+
    <li>Characterization</li>
         </ul>
         </ul>
     </div>
     </div>
Line 243: Line 244:
         <div class="eleven columns">  
         <div class="eleven columns">  
         <div class="offset-by-one columns">  
         <div class="offset-by-one columns">  
-
<h5>Basic works</h5>
+
<h6>1. Input module test</h6>
-
<p class="highlighttext"><span style="font-weight:bold;">Confirmation of the unexpected recombination of lox71 site</span></p>
+
<h5>1.1 Cre recombinase function test</h5>
-
<img src=""  width="px" class="img-center"/>
+
<p class="highlighttext">To confirm that the Cre recombinase from the registry works properly in <span style="font-style:italic;">E.coli</span>, we co-transformed the Cre recombinase under the control of lacI regulated promoter and the confirmation device we constructed in the basic work. We picked the single colonies for inoculation overnight in Luria Broth containing chloramphenicol, ampicillin and IPTG inducer and observed the culture’s color. The result below showed that only co-transformed bacteria were red(Fig.1). So the Cre recombinase can work in <span style="font-style:italic;">E.coli</span>.</p>
-
<img src=""  width="px" class="img-center"/>
+
<img src="https://static.igem.org/mediawiki/2014/e/e0/Hzau-c-1.png"  width="600px" class="img-center"/>
 +
<p class="figuretext">Figure 1. Cre recombinase work</p>
-
<p class="figuretext">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 which can also be specifically recognized by Cre recombinase. But the result of the sequencing in DH5α may suggest that our design cannot be used in <span style="font-style:italic;">E.coli</span> because recombination will happened without cre recombinase. In order to confirm it, we constructed the confirmation device shown in the figure 3 below. Then we transformed the device to kinds of the competent E.coli cells (DH5α,DH10B,BL21(DE3)) we got from different lab.</p>
+
<h5>1.2 Riboregulator function test</h5>
-
<img src=""  width="px" class="img-center"/>
+
<p class="highlighttext">We utilized the riboregulator to reduce the leakage of our input module, so we replaced the coding sequence of Cre protein with the coding sequence of mCherry protein in the input module to detect the efficiency of this riboregulator by fluorescence intensity. We chose the device expressing the mCherry under the control of lac promoter as the positive control. Those devices (Fig. 2) are respectively transformed into BL21(DE3) competent cell. Add IPTG solution to induce in concentration of 0, and 0.1mM. Set 12 copies for each concentration and devices as repetitions. Cultivate in the 37°C shaking incubator at the rotational speed of 180 rpm/min for 4 hours. </p>
-
<p class="figuretext">Fig.3 Confirmation Device</p>
+
<img src="https://static.igem.org/mediawiki/2014/2/27/Hzau-c-2.png"  width="700px" class="img-center"/>
 +
<p class="figuretext">Figure 2. co-transform devices</p>
-
<p class="figuretext">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 fig.4(DH5α) and fig.5(DH10B) below.</p>
+
<p class="highlighttext">The result from the multifunctional microplate revealed that under the non-induced condition, the leakage of the input device with riboregulator is much less than that of the positive control. And the riboregulator can be activated by IPTG. </p>
-
<img src=""  width="px" class="img-center"/>
+
<img src="https://static.igem.org/mediawiki/2014/7/78/Hzau-c-3.jpg"  width="700px" class="img-center"/>
-
<p class="figuretext">Fig.4 unexpected recombination in DH5alpha</p>
+
<p class="figuretext">Figure 3. Riboregulator function test result</p>
-
<img src=""  width="px" class="img-center"/>
+
-
<p class="figuretext">Fig.5 unexpected recombination in DH10B</p>
+
-
<p class="figuretext">At the same time, the samples were sequenced and anything was OK. </p>
+
<h5>1.3 Tight regulation test to input module </h5>
-
<p class="figuretext">In summary, we confirmed the unexpected recombination of lox71 site even if the device is in the stable <span style="font-style:italic;">E.coli</span> 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 <span style="font-style:italic;">E.coli</span>, 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>
+
<p class="highlighttext">We co-transformed the input module which has the Lac promoter and the confirmation device into the BL21(DE3)(Fig. 4). And we incubated 3 different colonies numbered 1, 2, 3 in 3 tubes of LB broth respectively at 37 Celsius degree for 8 hours. After that, we inoculated them into M9 medium and divided them into A group and B group. Then add 0.1ul IPTG to all A group tubes (1A, 2A, 3A) while add nothing to all B group tubes (1B, 2B, 3B). After cultivating the cells in the 37°C shaking incubator at the rotational speed of 180 rpm/min for 4 hours, we measured the fluorescent intensity using the multifunctional microplate reader(Fig. 5).</p>
 +
<img src="https://static.igem.org/mediawiki/2014/a/ad/Hzau-c-4.jpg"  width="700px" class="img-center"/>
 +
<p class="figuretext">Figure 4. the plasmids we co-transformed into BL21(DE3)</p>
 +
<img src="https://static.igem.org/mediawiki/2014/2/24/Hzau-cha-p10.jpg"  width="700px" class="img-center"/>
 +
<p class="figuretext">Figure 5. output device rigour detection</p>
-
<p class="highlighttext"><span style="font-weight:bold;">Promoter test</span></p>
+
<p class="highlighttext">As shown above, even though the addition of riboregulator did reduce a little of the expression of Cre protein, the difference between control group and experimental group was still very small which means that the riboregulator can hardly exert some critical influence on the control of the expression of cre gene.</p>
-
<p class="figuretext">The promoter strength is the one of the most important factor in our processing module. So we tested the inducible promoters we used in our processing module in the DH5α, they are BBa_J23119、BBa_R1051、BBa_R0062、BBa_R0040、BBa_R0063 and we ligated the mCherry with them. We read the OD<sub>600</sub> and fluorescent intensity (with the emission wavelength at 610nM and excitation wavelength at 587nM) using a multifunctional microplate reader. We divided the fluorescent intensity result using the value of OD<sub>600</sub>, and then recorded the data for compare and analysis. The result is below.</p>
+
<p class="highlighttext">In summary, the riboregulator can reduce the leakage of the Cre recombinase. We infer that there are two reasons to cause the input module out of control. The basal expression of Lac promoter is still too high, and the Cre recombinase is efficient. However, if you want to achieve the tight control of Cre recombinase, the conjugation between the bacteria can be considered. Because the recombination has an extremely low probability without the Cre recombinase, depended on the result we got from the lox71 work.</p>
-
<img src=""  width="px" class="img-center"/>
+
        <div class="clear"></div>
-
<p class="figuretext">Fig.6 inducible promoter test result in DH5alpha</p>
+
                <div class="divider"></div>
-
<p class="highlighttext"><span style="font-weight:bold;">Interaction work: pCusC, PpcoA sequencing and promoter test</span></p>
 
-
<p class="figuretext">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="figuretext">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. Analyzed the sequencing result, the P<span style="font-style:italic;">pcoA</span> device is ok but the p<span style="font-style:italic;">CusC</span> is failed to ligate to the p<span style="font-style:italic;">CusC</span> device. (See the detail)</p>
 
-
<p class="figuretext">Then we only tested the P<span style="font-style:italic;">pcoA</span> under different concentration of CuSO4 solution to induce. The result is in Fig.7 below. </p>
 
-
<img src=""  width="px" class="img-center"/>
 
-
<p class="figuretext">Fig.7 PpcoA promoter test</p>
 
-
<p class="figuretext">We used the 1.0mM Cu in LB medium as the blank samples of the 1.0mM control. And we intend to measure the fluorescent intensity in the copper ion concentration of plurality of gradient in the range of 0.1-1mM.</p>
 
-
<h5>Input module test</h5>
 
-
<p class="highlighttext"><span style="font-weight:bold;">Cre recombinase function test</span></p>
 
-
<p class="figuretext">To confirm that the cre recombinase from the registry work properly in E.coli, we co-transformed the cre recombinase under the lacI regulated promoter and the confirmation device we construct in the basic work. We picked the single colonies for inoculation overnight in Luria Broth containing chloramphenicol, ampicillin and IPTG inducer and observed the culture’s color.</p>
 
-
<img src=""  width="px" class="img-center"/>
 
-
<p class="figuretext">Fig. 8 Cre recombinase work</p>
 
-
<p class="figuretext">Only co-transformation bacteria is red so the cre recombinase can work in E.coli.</p>
 
-
<p class="highlighttext"><span style="font-weight:bold;">Riboregulator function test</p>
+
<h6>2. Output module test</h6>
-
<p class="figuretext">In order to test the riboregulator, we replace the cre recombinase with mCherry.</p>
+
<p class="highlighttext">We design our output module in two plans. One is the fluorescent protein with the fast degradation tag. And the other is the RNA aptamer.</p>
-
<p class="highlighttext"><span style="font-weight:bold;">Tight regulation test to input module </span></p>
+
<p class="highlighttext"><span style="font-weight:bold;">Plan 1: RNA aptamer</span></p>
-
<p class="figuretext">We co-transformation the input module which has the Lac promoter and the confirmation device into the BL21(DE3). And we incubated 3 different colonies numbered 1, 2, 3 in 3 tubes of LB broth respectively at 37 Celsius degree for 8 hours. After that, we inoculated them into M9 medium and divided them into A group and B group. Then add 0.1ul IPTG to all A tubes (1A, 2A, 3A) while add nothing to all B tubes (1B, 2B, 3B). Cultivate in the 37°C shaking incubator at the rotational speed of 180 rpm/min for 4 hours. Then we used the multifunctional microplate reader to get the result.</p>
+
<p class="highlighttext">We tried our best to synthesis the fluorophore (DMHBI) during the summer. At last, it was synthesized but its purity cannot meet the experimental requirement. (Fig. 6) Finally, we turned to the senior in HUST and he helped us to synthesis it.</p>
-
<img src=""  width="px" class="img-center"/>
+
<img src="https://static.igem.org/mediawiki/2014/d/df/Hzau-c-6.jpg"  width="400px" class="img-center"/>
-
<p class="figuretext">Fig.9 the plasmid we co-transformation</p>
+
<p class="figuretext">Figure 6. Processes during synthesis</p>
-
<img src=""  width="px" class="img-center"/>
+
-
<p class="figuretext">Fig. 10 output device rigour detection</p>
+
-
<p class="figuretext">As showed above, even though the addition of ribore 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="figuretext">In summary, the Riboregulator can reduce the leakage of the cre recombinase. We infer there is two reasons may cause the input module out of control. The one is there is too much leakage of the Lac promoter, and the other is the cre recombinase is high efficient. However, if you want to achieve the tight control of cre recombinase, the conjugation between the bacteria can be considered. Depended on the result we got from the basic work, recombination has an extremely low probability without the cre recombinase.</p>
+
-
<h5>Output module test</h5>
+
<p class="highlighttext">We prepared the sample as mentioned in previous paper(Paige <span style="font-style:italic;">et al.</span>, 2011). Then 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 7) and we also observed the spinach RNA aptamer by the fluorescence microscope, the result is in the Fig. 8.</p>
-
<p class="figuretext">We design our output module in two plans. One is the fluorescent protein with the fast degradation tag. And the other is the RNA aptamer.</p>
+
<img src="https://static.igem.org/mediawiki/2014/6/6c/HZAU-China_2014_tl_RNA_apdater_1.png"  width="700px" class="img-center"/>
-
<p class="figuretext"></p>
+
<p class="figuretext">Figure 7. photographed under illumination with 302 nm of light.</p>
-
<div class="clear"></div>
+
<img src="https://static.igem.org/mediawiki/2014/2/28/Hzau-c-8.png"  width="600px" class="img-center"/>
-
                <div class="divider"></div>                    
+
<p class="figuretext">Figure 8. photographed by the fluorescence microscope (400X)</p>
 +
 
 +
<p class="highlighttext"><span style="font-weight:bold;">Plan 2 fluorescent proteins with LVA tag</span></p>
 +
<p class="highlighttext">We observed fluorescence created by the output device (Fig.9) from the fluorescence microscope. The results indicated that the output device worked as expected (Fig. 10). </p>
 +
<img src="https://static.igem.org/mediawiki/2014/3/38/Hzau-c-15.png"  width="700px" class="img-center"/>
 +
<p class="figuretext">Figure 9. output module of plan 2</p>
 +
<img src="https://static.igem.org/mediawiki/2014/e/e6/Hzau-c-10.png"  width="750px" class="img-center"/>
 +
<p class="figuretext">Figure 10. photographed by the fluorescence microscope (400X) a: phase; b: mCherry; c: CFP</p>
 +
 
 +
 
 +
<h6>Processing module test</h6>
 +
<p class="highlighttext">We completed the device which is related to quorum sensing to perform the change from positive feedback to negative feedback. The processing & output module and input module (Fig. 11) were co-transformed into the DH5α strain as experimental group and only the processing & output module was transformed into the DH5α strain as control.</p>
 +
<img src="https://static.igem.org/mediawiki/2014/4/4a/Hzau-c-11.png"  width="700px" class="img-center"/>
 +
<p class="figuretext">Figure 11. the plasmids we co-transformed into DH5α</p>
 +
 
 +
<p class="highlighttext">Cultivate in the 37°C shaking incubator at the rotational speed of 180 rpm/min overnight. Add or not add the inducer to the experiment group and control group as the Fig. 12 (a: the control. b, the control with 100nM AHL. c: the experiment group without IPTG inducer. d: the experiment group with 100uM IPTG inducer.  e :the experiment group with 100uM IPTG inducer after growing to mid-log. f: the control growing to stable phase.). Shaker for 3 h or 5h 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. And the result of growth curve is shown in the Fig. 12.</p>
 +
<img src="https://static.igem.org/mediawiki/2014/b/bb/Hzau-c-14.jpg"  width="700px" class="img-center"/>
 +
<p class="figuretext">Figure 12. the growth curve of the <span style="font-style:italic;">E.coli</span> </p>
 +
 
 +
<p class="highlighttext">The experiment indicated whether it was induced by IPTG had no difference, the reason was mentioned in the tight regulation test to input module. And we also found the experiment groups grow better than the control with only processing plasmid  (Fig. 12). It may be that the positive feedback circle causes more over-loading, the negative feedback on the contrast. So we chose the data to analyze when all groups of the <span style="font-style:italic;">E.coli</span> grow to mid-log, the result is below. (Fig. 13).</p>
 +
<img src="https://static.igem.org/mediawiki/2014/5/5a/Hzau-c-13.png"  width="700px" class="img-center"/>
 +
<p class="figuretext">Figure 13. Fluorescence result of design 2</p>
 +
 
 +
<p class="highlighttext">The fluorescence intensity of the group without Cre is much higher than the group with Cre. So it suggested that the circuits in experimental group have already been rewired to perform the negative feedback function.</p>
 +
 
 +
<div class="clear"></div>
 +
<div class="divider"></div>  
 +
 
 +
<h5>Reference</h5>
 +
<p class="highlighttext">PPaige J S, Wu K Y, Jaffrey S R. RNA mimics of green fluorescent protein[J]. Science, 2011, 333(6042): 642-646.</p>
 +
 
 +
 
 +
                   
                 </div>
                 </div>
                 </div>
                 </div>

Latest revision as of 03:49, 18 October 2014

<!DOCTYPE html> 2014HZAU-China

Characterization

1. Input module test
1.1 Cre recombinase function test

To confirm that the Cre recombinase from the registry works properly in E.coli, we co-transformed the Cre recombinase under the control of lacI regulated promoter and the confirmation device we constructed in the basic work. We picked the single colonies for inoculation overnight in Luria Broth containing chloramphenicol, ampicillin and IPTG inducer and observed the culture’s color. The result below showed that only co-transformed bacteria were red(Fig.1). So the Cre recombinase can work in E.coli.

Figure 1. Cre recombinase work

1.2 Riboregulator function test

We utilized the riboregulator to reduce the leakage of our input module, so we replaced the coding sequence of Cre protein with the coding sequence of mCherry protein in the input module to detect the efficiency of this riboregulator by fluorescence intensity. We chose the device expressing the mCherry under the control of lac promoter as the positive control. Those devices (Fig. 2) are respectively transformed into BL21(DE3) competent cell. Add IPTG solution to induce in concentration of 0, and 0.1mM. Set 12 copies for each concentration and devices as repetitions. Cultivate in the 37°C shaking incubator at the rotational speed of 180 rpm/min for 4 hours.

Figure 2. co-transform devices

The result from the multifunctional microplate revealed that under the non-induced condition, the leakage of the input device with riboregulator is much less than that of the positive control. And the riboregulator can be activated by IPTG.

Figure 3. Riboregulator function test result

1.3 Tight regulation test to input module

We co-transformed the input module which has the Lac promoter and the confirmation device into the BL21(DE3)(Fig. 4). And we incubated 3 different colonies numbered 1, 2, 3 in 3 tubes of LB broth respectively at 37 Celsius degree for 8 hours. After that, we inoculated them into M9 medium and divided them into A group and B group. Then add 0.1ul IPTG to all A group tubes (1A, 2A, 3A) while add nothing to all B group tubes (1B, 2B, 3B). After cultivating the cells in the 37°C shaking incubator at the rotational speed of 180 rpm/min for 4 hours, we measured the fluorescent intensity using the multifunctional microplate reader(Fig. 5).

Figure 4. the plasmids we co-transformed into BL21(DE3)

Figure 5. output device rigour detection

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

In summary, the riboregulator can reduce the leakage of the Cre recombinase. We infer that there are two reasons to cause the input module out of control. The basal expression of Lac promoter is still too high, and the Cre recombinase is efficient. However, if you want to achieve the tight control of Cre recombinase, the conjugation between the bacteria can be considered. Because the recombination has an extremely low probability without the Cre recombinase, depended on the result we got from the lox71 work.

2. Output module test

We design our output module in two plans. One is the fluorescent protein with the fast degradation tag. And the other is the RNA aptamer.

Plan 1: RNA aptamer

We tried our best to synthesis the fluorophore (DMHBI) during the summer. At last, it was synthesized but its purity cannot meet the experimental requirement. (Fig. 6) Finally, we turned to the senior in HUST and he helped us to synthesis it.

Figure 6. Processes during synthesis

We prepared the sample as mentioned in previous paper(Paige et al., 2011). Then 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 7) and we also observed the spinach RNA aptamer by the fluorescence microscope, the result is in the Fig. 8.

Figure 7. photographed under illumination with 302 nm of light.

Figure 8. photographed by the fluorescence microscope (400X)

Plan 2 fluorescent proteins with LVA tag

We observed fluorescence created by the output device (Fig.9) from the fluorescence microscope. The results indicated that the output device worked as expected (Fig. 10).

Figure 9. output module of plan 2

Figure 10. photographed by the fluorescence microscope (400X) a: phase; b: mCherry; c: CFP

Processing module test

We completed the device which is related to quorum sensing to perform the change from positive feedback to negative feedback. The processing & output module and input module (Fig. 11) were co-transformed into the DH5α strain as experimental group and only the processing & output module was transformed into the DH5α strain as control.

Figure 11. the plasmids we co-transformed into DH5α

Cultivate in the 37°C shaking incubator at the rotational speed of 180 rpm/min overnight. Add or not add the inducer to the experiment group and control group as the Fig. 12 (a: the control. b, the control with 100nM AHL. c: the experiment group without IPTG inducer. d: the experiment group with 100uM IPTG inducer. e :the experiment group with 100uM IPTG inducer after growing to mid-log. f: the control growing to stable phase.). Shaker for 3 h or 5h 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. And the result of growth curve is shown in the Fig. 12.

Figure 12. the growth curve of the E.coli

The experiment indicated whether it was induced by IPTG had no difference, the reason was mentioned in the tight regulation test to input module. And we also found the experiment groups grow better than the control with only processing plasmid (Fig. 12). It may be that the positive feedback circle causes more over-loading, the negative feedback on the contrast. So we chose the data to analyze when all groups of the E.coli grow to mid-log, the result is below. (Fig. 13).

Figure 13. Fluorescence result of design 2

The fluorescence intensity of the group without Cre is much higher than the group with Cre. So it suggested that the circuits in experimental group have already been rewired to perform the negative feedback function.

Reference

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

Contacts
  • No.1, Shizishan Street, Hongshan District
    Wuhan, Hubei Province
    430070 P.R.China
  • Wechat : hzauigem
  • QQ Group : 313297095
  • YouTube : hzauigem