Team:HIT-Harbin/Wetlab

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         <a href="https://2014.igem.org/Team:HIT-Harbin/Project" name="top"><span>Project</span></a>
       <ul>
       <ul>
         <li><a href="https://2014.igem.org/Team:HIT-Harbin/Background">Background</a></li>
         <li><a href="https://2014.igem.org/Team:HIT-Harbin/Background">Background</a></li>
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     <div id="wetlab" class="nav1" ><a href="https://2014.igem.org/Team:HIT-Harbin/Wetlab"><span>Wetlab</span></a>
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     <div id="wetlab" class="nav1" ><a href="https://2014.igem.org/Team:HIT-Harbin/Wetlab">Wetlab</a>
       <ul>
       <ul>
         <li><a href="https://2014.igem.org/Team:HIT-Harbin/Protocols">Protocols</a></li>
         <li><a href="https://2014.igem.org/Team:HIT-Harbin/Protocols">Protocols</a></li>
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         <li><a href="https://2014.igem.org/Team:HIT-Harbin/Background">Background</a></li>
         <li><a href="https://2014.igem.org/Team:HIT-Harbin/Background">Background</a></li>
         <li><a href="https://2014.igem.org/Team:HIT-Harbin/Design">Design</a></li>
         <li><a href="https://2014.igem.org/Team:HIT-Harbin/Design">Design</a></li>
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         <li><a href="https://2014.igem.org/Team:HIT-Harbin/FutureWork">Advantages</a></li>
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         <li><a href="https://2014.igem.org/Team:HIT-Harbin/Advantages">Advantages</a></li>
         <li><a href="https://2014.igem.org/Team:HIT-Harbin/Modeling">Modeling</a></li>
         <li><a href="https://2014.igem.org/Team:HIT-Harbin/Modeling">Modeling</a></li>
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         <li><a href="https://2014.igem.org/Team:HIT-Harbin/Attribution">Attribution</a></li>
         <li><a href="https://2014.igem.org/Team:HIT-Harbin/Attribution">Attribution</a></li>
         <li><a href="https://2014.igem.org/Team:HIT-Harbin/Acknowledge">Acknowledge</a></li>
         <li><a href="https://2014.igem.org/Team:HIT-Harbin/Acknowledge">Acknowledge</a></li>
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         <li><a href="https://igem.org/Team.cgi">Profile</a></li>
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         <li><a href="https://igem.org/Team.cgi" target="_blank">Profile</a></li>
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                    <div class="site-description"><h4><span class="colored"><cufon class="cufon cufon-canvas" alt="Project" style="width: 72px; height: 20px;"><canvas width="79" height="27" style="width: 79px; height: 27px; top: -7px; left: 1px;"></canvas><cufontext>Project</cufontext></cufon></span><cufon class="cufon cufon-canvas" alt="/experiments" style="width: 129px; height: 20px;"><canvas width="136" height="27" style="width: 136px; height: 27px; top: -7px; left: 1px;"></canvas><cufontext>/experiments</cufontext></cufon></h4></div>
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<h4 class="hashed"><span><cufon class="cufon cufon-canvas" alt="Experiment " style="width: 111px; height: 20px;"><canvas width="124" height="27" style="width: 124px; height: 27px; top: -7px; left: 1px;"></canvas><cufontext>Experiment </cufontext></cufon><cufon class="cufon cufon-canvas" alt="Schedule" style="width: 85px; height: 20px;"><canvas width="93" height="27" style="width: 93px; height: 27px; top: -7px; left: 1px;"></canvas><cufontext>Schedule</cufontext></cufon></span></h4>
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                          <p></p>JuL. 10~15 Preparation of media, competent cells and experimental reagents
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<p></p>JuL. 16~20 Preparation of parts from iGEM
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<p></p>JuL. 21~31 Respective ligation of strong, intermediate and weak RBS with sub-circuit hrpR/hrpS/tet/RFP
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<p></p>Aug. 1~10 Ligation of terminators
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<p></p>Aug. 11~20 Successful ligation of the four sub-circuits
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<p></p>Aug. 21~28 Combination of sub-circuits: the device
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<p></p>Aug. 29~Sep. 15 Test of the device
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<p></p>Sep. 16~25 Remaining experiments
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<img src="https://static.igem.org/mediawiki/2013/6/6b/HIT-Harbin_team_HIT.png">
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<p>Fig: Agarose electrophoresis for our parts and maker forming "HIT"</p>
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<p>Part A :Plasmid carrying Plac+RBS+hrpR+T</p>
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<p>Part B :Plasmid carrying Ptet+RBS+hrpS+T</p>
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<p>Part C :Plasmid carrying PhrpL+RBS+RFP+T</p>
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<h4 class="hashed"><span><cufon class="cufon cufon-canvas" alt="Ligation " style="width: 78px; height: 20px;"><canvas width="91" height="27" style="width: 91px; height: 27px; top: -7px; left: 1px;"></canvas><cufontext>Ligation </cufontext></cufon><cufon class="cufon cufon-canvas" alt="for " style="width: 36px; height: 20px;"><canvas width="50" height="27" style="width: 50px; height: 27px; top: -7px; left: 1px;"></canvas><cufontext>for </cufontext></cufon><cufon class="cufon cufon-canvas" alt="our " style="width: 38px; height: 20px;"><canvas width="51" height="27" style="width: 51px; height: 27px; top: -7px; left: 1px;"></canvas><cufontext>our </cufontext></cufon><cufon class="cufon cufon-canvas" alt="device" style="width: 60px; height: 20px;"><canvas width="67" height="27" style="width: 67px; height: 27px; top: -7px; left: 1px;"></canvas><cufontext>device</cufontext></cufon></span></h4>
 
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                              <p>Fig 1. PCR resuLts of our own parts</p>
 
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                            <p>(1: hrpL; 2: hrpS; 3: hrpR; 4: Ptet +strong RBS+hrpS+T; 5:Ptet+intermediate RBS+hrpS+T; 6: Ptet+weak RBS+hrpS+T; 7: PIPTG +strong RBS+hrpR+T; 8 is not needed;  9: PIPTG +weak RBS+hrpR+T; 10: PhrpL+strong RBS+tetR+T; 11: PhrpL+intermediate RBS+tetR+T; 12: PhrpL+weak RBS+tetR+T; 13: PhrpL+strong RBS+RFP+T; 14: PhrpL+weak RBS+RFP+T; 15: PhrpL+weak RBS+RFP+T)</p>
 
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                            <img src="https://static.igem.org/mediawiki/2013/c/ca/Experiment3.png">
 
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                            <p>Fig 1.EcoR1 and Pst1 double restriction enzyme cleavage for hrpL AND gate(BBa_K1014014) device and B-POM1(BBa_K1014999) </p>
 
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                            <p>1:plasmid carrying BBa_K1014014; 2:double restriction enzyme cleavage for 1; 3:plasmid carrying BBa_K1014999; 4:double restriction enzyme cleavage for 3</p>
 
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<h4 class="hashed"><span><cufon class="cufon cufon-canvas" alt="1.Test " style="width: 58px; height: 20px;"><canvas width="71" height="27" style="width: 71px; height: 27px; top: -7px; left: 1px;"></canvas><cufontext>1.Test </cufontext></cufon><cufon class="cufon cufon-canvas" alt="of " style="width: 25px; height: 20px;"><canvas width="39" height="27" style="width: 39px; height: 27px; top: -7px; left: 1px;"></canvas><cufontext>of </cufontext></cufon><cufon class="cufon cufon-canvas" alt="device" style="width: 60px; height: 20px;"><canvas width="67" height="27" style="width: 67px; height: 27px; top: -7px; left: 1px;"></canvas><cufontext>device</cufontext></cufon></span></h4>
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                    <p>The year,We done the dioxin sensor as the main program of our lab work,they are designed in to 3 following parts.</p>
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                            <p></p>1)Preparation of IPTG solution: add 240mg IPTG powder into 10mL dd H2O. We filtrated the solution to sterilize it and broke it into EP tubes. The concentration is 24mg/mL (100mM/mL). then we stored them in -20℃.
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<p></p>2)To test the four different combination according to the strength of promoters, we made different concentrations of IPTG for the bacteria.
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<div align="center">
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<p> Table 1 IPTG formula</p>
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<img src="https://static.igem.org/mediawiki/2013/a/a9/Experiment4.png">
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<p></p>We grew the IPTG-added culture in 37℃ at 120rpm, overnight. Unfortunately, we haven’t observed the expected color of RFP (Red Fluorescence Protein).
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<img src="https://static.igem.org/mediawiki/2013/c/c2/Experiment5.png">
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      <h5>Control Group</h5>
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<p>Fig 3. No red after night</p>
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<p></p>3)After that, we tested the reporting sub-circuit of our device. We connect the constitutive promoter PLac with RBS+RFP+T, transformed the bacteria and grew it overnight. The red in the results proved that the reporting sub-circuit is working.
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<img class="Project" width="450px" src="https://static.igem.org/mediawiki/2014/d/d2/CG.jpg">
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<p></p>Fig 4.  Constitutive promoter expressing RFP
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<p></p>(Left is 12h, right is 24h)
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                    <p>So as to test the working state of promoter when there is no dioxin, we designed this control circuit showed above. We replaced mdr83-805 by mdr521-805. Its sequence of dioxin receptor was eliminated. When it is stimulated by galactose in the environment, the circuit will express the DNA binding sequence which is independent of dioxin. It is especially for testing whether mdr521-805 can trigger transmembrane transportation. What’s more, Its localization effect as well as zero correction function can be tested.</p>
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      <h5>Experimental Group</h5>
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<img class="Project" width="450px" src="https://static.igem.org/mediawiki/2014/c/c9/EG1.jpg">
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<p></p>4)We sent our whole device to companies for sequencing, but it failed.
 
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<img src="https://static.igem.org/mediawiki/2013/7/71/HIT-Harbin_team.png">
 
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<p>Fig: the happy moment when we saw the culture becoming red for the first time</p>
 
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<h4 class="hashed"><span><cufon class="cufon cufon-canvas" alt="2.Investigating " style="width: 145px; height: 20px;"><canvas width="158" height="27" style="width: 158px; height: 27px; top: -7px; left: 1px;"></canvas><cufontext>2.Investigating </cufontext></cufon><cufon class="cufon cufon-canvas" alt="the " style="width: 36px; height: 20px;"><canvas width="50" height="27" style="width: 50px; height: 27px; top: -7px; left: 1px;"></canvas><cufontext>the </cufontext></cufon><cufon class="cufon cufon-canvas" alt="relationship " style="width: 122px; height: 20px;"><canvas width="135" height="27" style="width: 135px; height: 27px; top: -7px; left: 1px;"></canvas><cufontext>relationship </cufontext></cufon><cufon class="cufon cufon-canvas" alt="between " style="width: 83px; height: 20px;"><canvas width="97" height="27" style="width: 97px; height: 27px; top: -7px; left: 1px;"></canvas><cufontext>between </cufontext></cufon><cufon class="cufon cufon-canvas" alt="the " style="width: 36px; height: 20px;"><canvas width="50" height="27" style="width: 50px; height: 27px; top: -7px; left: 1px;"></canvas><cufontext>the </cufontext></cufon><cufon class="cufon cufon-canvas" alt="concentration " style="width: 141px; height: 20px;"><canvas width="154" height="27" style="width: 154px; height: 27px; top: -7px; left: 1px;"></canvas><cufontext>concentration </cufontext></cufon><cufon class="cufon cufon-canvas" alt="of " style="width: 25px; height: 20px;"><canvas width="39" height="27" style="width: 39px; height: 27px; top: -7px; left: 1px;"></canvas><cufontext>of </cufontext></cufon><cufon class="cufon cufon-canvas" alt="RFP " style="width: 36px; height: 20px;"><canvas width="50" height="27" style="width: 50px; height: 27px; top: -7px; left: 1px;"></canvas><cufontext>RFP </cufontext></cufon><cufon class="cufon cufon-canvas" alt="with " style="width: 47px; height: 20px;"><canvas width="61" height="27" style="width: 61px; height: 27px; top: -7px; left: 1px;"></canvas><cufontext>with </cufontext></cufon><cufon class="cufon cufon-canvas" alt="that " style="width: 46px; height: 20px;"><canvas width="60" height="27" style="width: 60px; height: 27px; top: -7px; left: 1px;"></canvas><cufontext>that </cufontext></cufon><cufon class="cufon cufon-canvas" alt="of " style="width: 25px; height: 20px;"><canvas width="39" height="27" style="width: 39px; height: 27px; top: -7px; left: 1px;"></canvas><cufontext>of </cufontext></cufon><cufon class="cufon cufon-canvas" alt="IPTG" style="width: 37px; height: 20px;"><canvas width="44" height="27" style="width: 44px; height: 27px; top: -7px; left: 1px;"></canvas><cufontext>IPTG</cufontext></cufon></span></h4>
 
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<p></p>1)Measuring absorbance of RFP
 
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<p></p>We grew bacteria without device and bacteria with our device in same volume until stationary phase. Taking bacteria without device as background, we measured the absorbance of bacteria with our device (the max absorption peak is 504nm).But absorbance in 504nm is higher than 1,which present a bad linear relation between absorbance and concentraton. RFP has absorption in 450nm,and absorbance is between 0.1 and 1(better linear relation).Occasionally, we find a RFP standard curve under 450nm on the web. it was very lucky compared with our failure in testing our device.
 
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Before the mensuration, we diluted the two groups according to table2. We took the mean of two measures as the useful data.
 
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<img src="https://static.igem.org/mediawiki/2013/8/8f/Experiment8.png">
 
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<p>Fig.5 RFP absorbance varying with wave length</p>
 
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<p>Table 2 Dilution of Two groups of bacteria</p>
 
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<img src="https://static.igem.org/mediawiki/2013/2/21/Experiment7.png">
 
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<img src="https://static.igem.org/mediawiki/2013/7/74/Experiment9.png">
 
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<p>Fig 6.  The relationship between RFP concentration and absorbance(OD450)</p>
 
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<p></p>2)The actual relationship between RFP concentration and absorbance
 
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<img src="https://static.igem.org/mediawiki/2013/8/88/Experiment10.png">
 
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<p>Fig 7.  RFP standard curve obtain from the web,<a href="http://www.cellbiolabs.com/sites/default/files/AKR-122-rfp-elisa-kit.pdf">Click here</a>
 
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</p></div>
 
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<p></p>Through the standard curve, we can convert the relative concentration to the absolute concentration, and finally get the relationship between IPTG concentration and RFP concentration.
 
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<p></p>Compared to crushing cells to separate RFP, our method is simpler and easy to practice. Moreover, our relative concentration curve is credible. If the standard curve is reliable, our calculated result of RFP will be precise.
 
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                    <p>In order to make yeast be capable of detecting dioxins in the environment, we designed the circuit above. TEF is the constitutive promoter which can activate downstream sequences to express dual domain protein, namely, lexA-DBD/mDR83-80. Comparing with other amplifiers, lexA can rapidly and efficiently induce the expression of downstream gene. At the meantime, we designed and applied the operator-lexop which possesses eight LEXAdbd binding sites, thus the catalytic effect of lexA operator is further improved. mDR83-805 can express mdr83-805（mouse dioxin receptor）. And after it combining intracellular dissociative dioxin, it will pass through karyotheca and combine with lexA operator to switch up downstream promoters and then activate the expression of green fluorescent protein downstream. Then we can know by observation whether there is dioxin in the environment or not.</p>
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<p>Considering the amount of dioxin in the environment is in micro level or even trace level, when the concentration of dioxin decreases, those who can successfully combine with fusion protein lexadbd/mdr83-805 and then nucleic DNA will be less. Thus, we added a memory system to amplify the signal intensity by positive feedback regulation.</p>
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<p>When there is a trace of dioxin, with the assistance of native intercellular hsp90 and arnt, the combination of fusion protein lexadbd/mdr83-805 and dioxin will activate the expression of florescent protein. Lex-A-DBD/mDR521-805 depicted in the picture is also dual domain protein but without binding site of dioxin hence cannot combine with dioxin. However, it can still pass through the nuclear membrane and combine with DNA to activate the expression of GFP. Such signal of the trace of dioxin can lead to the expression of a lot of GFP by amplification.</p>
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<h2>Reference:</h2>
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<p>1.Ajo-Franklin C M, Drubin D A, Eskin J A, et al. Rational design of memory in eukaryotic cells[J]. Genes & development, 2007, 21(18): 2271-2276.
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<p>2.Whitelaw M L, McGuire J, Picard D, et al. Heat shock protein hsp90 regulates dioxin receptor function in vivo[J]. Proceedings of the National Academy of Sciences, 1995, 92(10): 4437-4441.
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Latest revision as of 01:58, 18 October 2014

The year,We done the dioxin sensor as the main program of our lab work,they are designed in to 3 following parts.

Control Group

So as to test the working state of promoter when there is no dioxin, we designed this control circuit showed above. We replaced mdr83-805 by mdr521-805. Its sequence of dioxin receptor was eliminated. When it is stimulated by galactose in the environment, the circuit will express the DNA binding sequence which is independent of dioxin. It is especially for testing whether mdr521-805 can trigger transmembrane transportation. What’s more, Its localization effect as well as zero correction function can be tested.

Experimental Group

In order to make yeast be capable of detecting dioxins in the environment, we designed the circuit above. TEF is the constitutive promoter which can activate downstream sequences to express dual domain protein, namely, lexA-DBD/mDR83-80. Comparing with other amplifiers, lexA can rapidly and efficiently induce the expression of downstream gene. At the meantime, we designed and applied the operator-lexop which possesses eight LEXAdbd binding sites, thus the catalytic effect of lexA operator is further improved. mDR83-805 can express mdr83-805（mouse dioxin receptor）. And after it combining intracellular dissociative dioxin, it will pass through karyotheca and combine with lexA operator to switch up downstream promoters and then activate the expression of green fluorescent protein downstream. Then we can know by observation whether there is dioxin in the environment or not.

Considering the amount of dioxin in the environment is in micro level or even trace level, when the concentration of dioxin decreases, those who can successfully combine with fusion protein lexadbd/mdr83-805 and then nucleic DNA will be less. Thus, we added a memory system to amplify the signal intensity by positive feedback regulation.

When there is a trace of dioxin, with the assistance of native intercellular hsp90 and arnt, the combination of fusion protein lexadbd/mdr83-805 and dioxin will activate the expression of florescent protein. Lex-A-DBD/mDR521-805 depicted in the picture is also dual domain protein but without binding site of dioxin hence cannot combine with dioxin. However, it can still pass through the nuclear membrane and combine with DNA to activate the expression of GFP. Such signal of the trace of dioxin can lead to the expression of a lot of GFP by amplification.

Reference:

1.Ajo-Franklin C M, Drubin D A, Eskin J A, et al. Rational design of memory in eukaryotic cells[J]. Genes & development, 2007, 21(18): 2271-2276.

2.Whitelaw M L, McGuire J, Picard D, et al. Heat shock protein hsp90 regulates dioxin receptor function in vivo[J]. Proceedings of the National Academy of Sciences, 1995, 92(10): 4437-4441.

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