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| {{BNU-CHINA/partials/header}} | | {{BNU-CHINA/partials/header}} |
| {{BNU-CHINA/partials/nav | Project=focus}} | | {{BNU-CHINA/partials/nav | Project=focus}} |
| + | {{BNU-CHINA/article/theme | color=#003a6c}} |
| <html> | | <html> |
− | <div class="main-container">
| + | <div class="main-container"> |
− | <div class="container-fluid page-heading"
| + | <div class="container-fluid page-heading" |
− | style="background-image: url(https://static.igem.org/mediawiki/2016/f/f4/T--BNU-China--project.jpg);">
| + | style="background-image: url(https://static.igem.org/mediawiki/2016/f/f4/T--BNU-China--project.jpg);"> |
− | <h3> RESULTS </h3>
| + | <h3> Results </h3> |
− | </div>
| + | </div> |
− | <div>
| + | <div> |
− | <div class="page-story">
| + | <div class="page-story"> |
− | <article id="project" class="col-lg-10 col-lg-offset-1 col-md-12 col-md-offset-0 col-sm-offset-0 col-sm-12">
| + | <article id="project" class="col-lg-10 col-lg-offset-1 col-md-12 col-md-offset-0 col-sm-offset-0 col-sm-12"> |
− | <header class="page-header">
| + | <header class="page-header"> |
− | <h1>Results</h1>
| + | <h1>Results</h1> |
− | <small id="secondary-page-header">Do One Thing</small>
| + | </header> |
− | </header>
| + | |
| | | |
− | <h2>Vector Construction</h2> | + | <h2>Vector Construction</h2> |
− | <h3>Vectors of α-tubulin, β-tubulin, n-luciferase, c-luciferase</h3> | + | <h3>Vectors of α-tubulin, β-tubulin, n-luciferase, c-luciferase</h3> |
− | <p>Gene fragments of α-tubulin、β-tubulin、n-luciferase、c-luciferase were amplified via PCR and verified by electrophoresis(Fig.1). The theoretic gene size of α-tubulin is 1356bp, β-tubulin is 1335bp, n-luciferase is 1248bp, c-luciferase is 459bp, which matched our experimental results.</p> | + | <p>Gene fragments of α-tubulin, β-tubulin, n-luciferase, c-luciferase were amplified via PCR and |
− | <figure class="text-center"> | + | verified |
− | <img src="https://static.igem.org/mediawiki/2016/3/3b/T--BNU-China--Results9.jpg" width="65%">
| + | by electrophoresis(Fig.1). The theoretic gene size of α-tubulin is 1356bp, β-tubulin is 1335bp, |
− | <figcaption>
| + | n-luciferase is 1248bp, c-luciferase is 459bp, which matched our experimental results.</p> |
− | Fig.1 Electrophoresis result of α-tubulin, β-tubulin, n-luciferase, c-luciferase gene fragments </figcaption>
| + | <figure class="text-center"> |
− | </figure>
| + | <img src="https://static.igem.org/mediawiki/2016/3/3b/T--BNU-China--Results9.jpg" width="65%"> |
| + | <figcaption> |
| + | Fig.1 Electrophoresis result of α-tubulin, β-tubulin, n-luciferase, c-luciferase gene fragments |
| + | </figcaption> |
| + | </figure> |
| | | |
− | <p>Gene fragments were ligated to <i>E.coli</i> expression plasmid pET30a(+), after transformation, colony PCR was done to verify the efficiency(Fig.2A and 2B). Meanwhile, the sequencing results further confirmed that we successfully cloned the α-tubulin, β-tubulin, n-luciferase, c-luciferase expression vectors.</p> | + | <p>Gene fragments were ligated to <i>E.coli</i> expression plasmid pET30a(+), after transformation, |
| + | colony PCR was done to verify the efficiency(Fig.2A and 2B). Meanwhile, the sequencing results |
| + | further confirmed that we successfully cloned the α-tubulin, β-tubulin, n-luciferase, c-luciferase |
| + | expression vectors.</p> |
| | | |
− | <figure class="text-center"> | + | <figure class="text-center"> |
− | <img src="https://static.igem.org/mediawiki/2016/c/c0/T--BNU-China--Results5.jpg" width="88%">
| + | <img src="https://static.igem.org/mediawiki/2016/c/c0/T--BNU-China--Results5.jpg" width="88%"> |
− | <figcaption>
| + | <figcaption> |
− | Fig.2 Electrophoresis result of α-tubulin、β-tubulin、n-luciferase、c-luciferase expression vectors
| + | Fig.2 Electrophoresis result of α-tubulin, β-tubulin, n-luciferase, c-luciferase expression |
− | <br/>
| + | vectors |
− | <small style="font-size:80%">(A: electrophoresis result of colony PCR. The arrows show the
| + | <br/> |
− | correct sizes of α-tubulin, n-luciferase and c-luciferase. <br/>B: electrophoresis result of
| + | <small style="font-size:80%">(A: electrophoresis result of colony PCR. The arrows show the |
− | colony PCR. The arrows show the correct size of β-tubulin.
| + | correct sizes of α-tubulin, n-luciferase and c-luciferase. <br/>B: electrophoresis result of |
− | </small>
| + | colony PCR. The arrows show the correct size of β-tubulin. |
− | </figcaption>
| + | </small> |
− | </figure>
| + | </figcaption> |
| + | </figure> |
| | | |
| | | |
− | <h3>Fusion Protein Vectors</h3> | + | <h3>Fusion Protein Vectors</h3> |
− | <h4>By fusion PCR technology</h4> | + | <h4>By fusion PCR technology</h4> |
− | <p> | + | <p> |
− | α-tubulin-YNE, YNE-α-tubulin, α-tubulin-YCE, YCE-α-tubulin, β-tubulin-YNE, YNE-β-tubulin, β-tubulin-YCE, YCE-β-tubulin, α-tubulin-nluc, nluc-α-tubulin, α-tubulin-cluc and cluc-α-tubulin were cloned respectively via fusion PCR. After ligating these fusion gene fragments to pET30a(+) empty vectors, we transformed the target plasmids to Trans5α. When colony PCR was done for screening, we picked correct colonies shown in SDS-PAGE (Fig.3) for plasmid amplification. | + | α-tubulin-YNE, YNE-α-tubulin, α-tubulin-YCE, YCE-α-tubulin, β-tubulin-YNE, YNE-β-tubulin, |
− | </p> | + | β-tubulin-YCE, YCE-β-tubulin, α-tubulin-nluc, nluc-α-tubulin, α-tubulin-cluc and cluc-α-tubulin were |
| + | cloned respectively via fusion PCR. After ligating these fusion gene fragments to pET30a(+) empty |
| + | vectors, we transformed the target plasmids to Trans5α. When colony PCR was done for screening, we |
| + | picked correct colonies shown in electrophoresis gel(Fig.3) for plasmid amplification. |
| + | </p> |
| | | |
| | | |
− | <figure class="text-center">
| + | <figure class="text-center"> |
− | <img src="https://static.igem.org/mediawiki/2016/b/b9/T--BNU-China--Results6.jpg" width="75%">
| + | <img src="https://static.igem.org/mediawiki/2016/b/b9/T--BNU-China--Results6.jpg" width="75%"> |
− | <figcaption>
| + | <figcaption> |
− | Fig.3 Result of colony PCR
| + | Fig.3 Result of colony PCR |
− | <br/>
| + | <br/> |
− | <small style="font-size:80%">Arrows show the correct size of fusion gene fragments:
| + | <small style="font-size:80%">Arrows show the correct size of fusion gene fragments: |
− | α-tubulin-YNE is 1866 bp, α-tubulin-YCE is 1650bp, β-tubulin-YCE is 1629bp,
| + | α-tubulin-YNE is 1866 bp, α-tubulin-YCE is 1650bp, β-tubulin-YCE is 1629bp, |
− | α-tubulin-nluc is 2640bp, α-tubulin-cluc is 1857bp.
| + | α-tubulin-nluc is 2640bp, α-tubulin-cluc is 1857bp. |
− | </small>
| + | </small> |
− | </figcaption>
| + | </figcaption> |
− | </figure>
| + | </figure> |
| | | |
− | <P>Sequencing results further confirmed that α-tubulin-YNE, YNE-α-tubulin, α-tubulin-YCE, YCE-α-tubulin, β-tubulin-YCE, YCE-β-tubulin, α-tubulin-nluc and cluc-α-tubulin expression vectors were constructed successfully.</P> | + | <P>Sequencing results further confirmed that α-tubulin-YNE, YNE-α-tubulin, α-tubulin-YCE, YCE-α-tubulin, |
| + | β-tubulin-YCE, YCE-β-tubulin, α-tubulin-nluc and cluc-α-tubulin expression vectors were constructed |
| + | successfully.</P> |
| | | |
− | <h4>By Gateway Technology</h4> | + | <h4>By Gateway Technology</h4> |
− | <p>We also tried to construct fusion protein vectors by Gateway Large-scale Cloning technology. We used Invitrogen pENTR/D TOPO to clone β-tubulin into entry vector. Primers were designed based on β-tubulin sequence and PCR was done for verification. Electrophoresis result (Fig.4) showed that β-tubulin was successfully cloned into the entry vector.</p> | + | <p>We also tried to construct fusion protein vectors by Gateway Large-scale Cloning technology. We used |
| + | Invitrogen pENTR/D TOPO to clone β-tubulin into entry vector. Primers were designed based on |
| + | β-tubulin sequence and PCR was done for verification. Electrophoresis result (Fig.4) showed that |
| + | β-tubulin was successfully cloned into the entry vector.</p> |
| | | |
− | <figure class="text-center">
| + | <figure class="text-center"> |
− | <img src="https://static.igem.org/mediawiki/2016/6/63/T--BNU-China--Results14.jpg" width="65%">
| + | <img src="https://static.igem.org/mediawiki/2016/6/63/T--BNU-China--Results14.jpg" width="65%"> |
− | <figcaption>
| + | <figcaption> |
− | Fig.4 PCR verification result of the constructed entry vectors
| + | Fig.4 PCR verification result of the constructed entry vectors |
− | </figcaption>
| + | </figcaption> |
− | </figure>
| + | </figure> |
− | <p>In order to do LR reaction, we used the restriction endonuclease Not I to digest the entry vector. Electrophoresis result (Fig.5) showed that single digestion was efficient.</p> | + | <p>In order to do LR reaction, we used the restriction endonuclease Not I to digest the entry vector. |
− | <figure class="text-center"> | + | Electrophoresis result (Fig.5) showed that single digestion was efficient.</p> |
− | <img src="https://static.igem.org/mediawiki/2016/2/2e/T--BNU-China--Results4.jpg" width="25%">
| + | <figure class="text-center"> |
− | <figcaption>
| + | <img src="https://static.igem.org/mediawiki/2016/2/2e/T--BNU-China--Results4.jpg" width="25%"> |
− | Fig.5 Single endonuclease digestion result of entry vectors
| + | <figcaption> |
− | </figcaption>
| + | Fig.5 Single endonuclease digestion result of entry vectors |
− | </figure>
| + | </figcaption> |
− | <p>Using Invitrogen Gateway LR Clonase II Enzyme Mix, the entry vectors were ligated with pCambia1300-nluc and pCambia1300-cluc respectively. Thus the destination vectors were complete. After transformation and running PCR with β-tubulins primers, electrophoresis result (Fig.6) showed high positive rates, indicating β-tubulins was successfully cloned into the vectors.</p> | + | </figure> |
− | <figure class="text-center"> | + | <p>Using Invitrogen Gateway LR Clonase II Enzyme Mix, the entry vectors were ligated with |
− | <img src="https://static.igem.org/mediawiki/2016/d/d3/T--BNU-China--Results10.jpg" width="90%">
| + | pCambia1300-nluc and pCambia1300-cluc respectively. Thus the destination vectors were complete. |
− | <figcaption>
| + | After transformation and running PCR with β-tubulins primers, electrophoresis result (Fig.6) showed |
− | Fig.6 PCR verification result of the objective vectors
| + | high positive rates, indicating β-tubulins was successfully cloned into the vectors.</p> |
− | </figcaption>
| + | <figure class="text-center"> |
− | </figure>
| + | <img src="https://static.igem.org/mediawiki/2016/d/d3/T--BNU-China--Results10.jpg" width="90%"> |
| + | <figcaption> |
| + | Fig.6 PCR verification result of the objective vectors |
| + | </figcaption> |
| + | </figure> |
| | | |
− | <p>Also, signaling fragments were also need to be tested. By using the reverse primer of β-tubulin and the forward primer of cluc for PCR verification, we found that cluc-β-tubulin fusion protein vector is successfully constructed. Electrophoresis result is shown in Fig.7.</p> | + | <p>Also, signaling fragments were also needed to be tested. By using the reverse primer of β-tubulin and |
| + | the forward primer of cluc for PCR verification, we found that cluc-β-tubulin fusion protein vector |
| + | is successfully constructed. Electrophoresis result is shown in Fig.7.</p> |
| + | <figure class="text-center"> |
| + | <img src="https://static.igem.org/mediawiki/2016/0/07/T--BNU-China--Results11.jpg" width="70%"> |
| + | <figcaption> |
| + | Fig.7 PCR verification result of pCambia-nluc, pCambia-cluc |
| + | <br/> |
| + | <small style="font-size:80%">Arrows show the correct bands</small> |
| + | </figcaption> |
| + | </figure> |
| | | |
| + | <p>In conclusion, we successfully cloned nine fusion protein vectors. α-tubulin-YNE, YNE-α-tubulin, |
| + | α-tubulin-YCE, YCE-α-tubulin, β-tubulin-YCE, YCE-β-tubulin, α-tubulin-nluc, and cluc-α-tubulin were |
| + | ligated to pET30(+) . β-tubulin was cloned to pCambia-cluc plasmid as a form of cluc-β-tubulin |
| + | fusion protein vector.</p> |
| + | <h2>Protein Expression</h2> |
| + | <h3>In TransB(DE3) <i>E.coli</i> expression strain</h3> |
| + | <p>Expression vectors were transformed into <i>E.coli</i> expression strain TransB(DE3). After |
| + | culturing, |
| + | we firstly tested the effect of IPTG inducement. β-tubulin was taken as an example. SDS-PAGE(Fig.8) |
| + | showed that IPTG is very significant in the expressing process.</p> |
| | | |
| + | <figure class="text-center"> |
| + | <img src="https://static.igem.org/mediawiki/2016/7/75/T--BNU-China--Result25.jpg" width="60%"> |
| + | <figcaption> |
| + | Fig.8 SDS-PAGE result of β-tubulin inducement test (left to right: non-induced group, induced |
| + | group), arrow shows the correct molecular weight of target protein(55 kDa)<br/> |
| + | <small style="font-size:80%">the molecular weight of target fusion protein is 74.6kDa. |
| + | arrows show the correct bands. |
| + | </small> |
| + | </figcaption> |
| + | </figure> |
| | | |
| + | <p>Then we checked the protein expression predicted website <i>http://www.biotech.ou.edu/</i>. It showed |
| + | that |
| + | our fusion protein would probably expressed as inclusion bodies. We therefore renatured the |
| + | inclusion bodies and verified through SDS-PAGE(Fig.9).</p> |
| + | <figure class="text-center"> |
| + | <img src="https://static.igem.org/mediawiki/2016/a/a0/T--BNU-China--Results7.jpg" width="60%"> |
| + | <figcaption> |
| + | Fig.9 SDS-PAGE of renatured inclusion bodies from α-tubulin-YNE, YNE-α-tubulin |
| + | <br/> |
| + | <small style="font-size:80%">the molecular weight of target fusion protein is 74.6kDa. |
| + | arrows show the correct bands. |
| + | </small> |
| + | </figcaption> |
| + | </figure> |
| + | <p>Also, western-blot(Fig.10) were done to test the protein from supernatant, pellet and renatured |
| + | inclusion body.</p> |
| + | <figure class="text-center" id="ref-1"> |
| + | <img src="https://static.igem.org/mediawiki/2016/6/61/T--BNU-China--result20.jpg" width="80%"> |
| + | <figcaption> |
| + | Fig.10 Western blot result of prokaryotic expression |
| + | <br/> |
| + | <small style="font-size:80%"> Left to right, extracted α-tubulin, expressed empty vector, |
| + | α-tubulin,α-tubulin-YNE fusion protein, α-tubulin-YCE fusion protein, α-tubulin-nluc fusion |
| + | protein. Arrows show the correct bands of target proteins, triangles show the homologous |
| + | tubulin protein(FtsZ,43kDa) from the bacteria. |
| + | </small> |
| + | </figcaption> |
| + | </figure> |
| + | <h3>In Rossatta(DE3) <i>E.coli</i> expression strain</h3> |
| | | |
| + | <p>Rossatta(DE3) is a kind of <i>E.coli</i> strain that can express rare codons and improve the |
| + | expression level of eukaryotic protein. Thus we applied this strain to optimize our protein |
| + | expression.</p> |
| + | <p>SDS-PAGE were done to verify the expression results before(Fig.11) and after(Fig.12) breaking the |
| + | bacteria, and Western blot(Fig.13) was also applied for the further confirmation.</p> |
| + | <figure class="text-center"> |
| + | <img src="https://static.igem.org/mediawiki/2016/3/3c/T--BNU-China--Results19.jpg" width="100%"> |
| + | <figcaption> |
| + | Fig.11 SDS-PAGE of centrifuged cells before ultrasonic breaking. |
| + | <br/> |
| + | <small style="font-size:80%">A: cluc-α-tubulin(74 kDa), α-tubulin-nluc, YCE-β-tubulin(66 |
| + | kDa), β-tubulin-YCE(66 kDa), YCE-α-tubulin(66 kDa), α-tubulin-YCE(66 kDa), expressed |
| + | empty vector. <br/>B: left to right: expressed empty vector, α-tubulin(55 kDa), |
| + | β-tubulin(55 kDa), α-tubulin-YNE(75kDa), YNE-α-tubulin(75kDa).<br/>Arrows show the correct |
| + | bands. |
| + | </small> |
| + | </figcaption> |
| + | </figure> |
| + | <figure class="text-center"> |
| + | <img src="https://static.igem.org/mediawiki/2016/a/a9/T--BNU-China--result21.jpg" width="70%"> |
| + | <figcaption> |
| + | Fig.12 SDS-PAGE of supernatant after ultrasonic breaking the rossatta cells |
| + | <br/> |
| + | <small style="font-size:80%"> Left to right: expressed empty vector, α-tubulin(55 kDa), |
| + | β-tubulin(55 kDa), α-tubulin-YNE(75kDa), YNE-α-tubulin(75kDa), α-tubulin-YCE(66 kDa), |
| + | YCE-α-tubulin(66 kDa), β-tubulin-YCE(66 kDa), YCE-β-tubulin(66 kDa), α-tubulin-nluc, |
| + | cluc-α-tubulin(74 kDa) |
| + | </small> |
| + | </figcaption> |
| + | </figure> |
| | | |
| + | <figure class="text-center"> |
| + | <img src="https://static.igem.org/mediawiki/2016/a/af/T--BNU-China--Result26.jpg" width="70%"> |
| + | <figcaption> |
| + | Fig.13 Western blot of rossatta cells expression. |
| + | <br/> |
| + | <small style="font-size:80%"> A: left to right, protein marker, negative control, α-tubulin in |
| + | pellet(55 kDa), α-tubulin-YNE in pellet(75kDa), α-tubulin-YCE in pellet (66 kDa), |
| + | YCE-α-tubulin in pellet (66 kDa), cluc-α-tubulin in pellet (74 kDa), extracted α-tubulin(55 |
| + | kDa). B: left to right: negative control in pellet, β-tubulin in pellet(55 kDa), |
| + | β-tubulin-YCE in pellet (66 kDa), protein marker, negative control in supernatant, β-tubulin |
| + | in supernatant (55 kDa), β-tubulin-YCE in supernatant (66 kDa), extracted β-tubulin(55 kDa). |
| + | </small> |
| + | </figcaption> |
| + | </figure> |
| + | <p>Based on the results above, we could confirm that α-tubulin, β-tubulin, α-tubulin-YNE, YNE-α-tubulin, |
| + | α-tubulin-YCE, YCE-α-tubulin, β-tubulin-YCE, YCE-β-tubulin, cluc-α-tubulin fusion protein were |
| + | successfully expressed in rossatta cell.</p> |
| + | <p>Particularly, according to figure 7B, the target proteins (β-tubulin and β-tubulin-YCE) can be tested |
| + | out in the supernatant, indicating that they are soluble when expressed in rossatta strain.</p> |
| + | <p>We collaborated with Fujian Agriculture and Forest University and asked them to test the interaction |
| + | between α and β-tubulin. Thus verified the activity of tubulin monomers.</p> |
| | | |
| + | <h2>Fluorescence detection</h2> |
| + | <h3>Protein functional test</h3> |
| + | <p>In order to make sure the protein expressed from prokaryotic cells had their functions. We did a |
| + | functional test at first. Concentrated supernatant of α-tubulin-YNE and α-tubulin-YCE were mixed |
| + | equally and treated with twice as much as the supernatant of β-tubulin. 50μM GTP and 200μM taxol |
| + | were also added for helping aggregation. After incubated in 37℃ for 1 hour,the mixture was tested by |
| + | the absolute recording spectrofluorometer. The result(Fig.14) showed there was a significant |
| + | emission peak in 525nm with the excitation in near 512nm wave length, indicating that YCE and YNE |
| + | protein fragments were combined as a whole YFP and thus verifying their biological functions <i>in |
| + | vitro</i>.</p> |
| + | <p>Also, equally mixed α-tubulin-YNE and β-tubulin-YCE treated with 50μM GTP and 200μM taxol indicated |
| + | the existed α-β tubulin interaction and the YNE-YCE combination.</p> |
| | | |
| + | <figure class="text-center"> |
| + | <img src="https://static.igem.org/mediawiki/2016/c/c3/T--BNU-China--results28.jpg" width="70%"> |
| + | <figcaption> |
| + | Fig.14 Fluorescence intensity - Wave length diagram <br/> |
| + | </figcaption> |
| + | </figure> |
| | | |
| + | <h3>Taxol-concentration based assay</h3> |
| + | <p>After protein functions were confirmed, we further tested the microtubule aggregation level by |
| + | treating serial concentration of taxol samples. Since the excess amount of substrate proteins |
| + | (α-tubulin-YNE, α-tubulin-YCE and β-tubulin) were treated. The aggregation level could be |
| + | represented by the fluorescence intensity. The experimental results (Fig.15 Table 1) showed that |
| + | there is an obvious positive correlation between taxol concentration and fluorescence intensity when |
| + | the substrate proteins are abundant.</p> |
| | | |
| + | <figure class="text-center"> |
| + | <img src="https://static.igem.org/mediawiki/2016/e/ed/T--BNU-China--results-fluoro.png" width="70%"> |
| + | <figcaption> |
| + | Fig.15 Fluorescence intensity - Wave length diagram <br/> |
| + | </figcaption> |
| + | </figure> |
| | | |
| + | <table class="table table-bordered"> |
| + | <caption> |
| + | Table 1 Statistics of serial taxol concentration-integration of fluorescence intensity |
| + | </caption> |
| + | <thead> |
| + | <tr> |
| + | <th style="text-align: center; ">Taxol concentration(μM)</th> |
| + | <th style="text-align: center;">Fluorescence intensity(CPS)</th> |
| + | </tr> |
| + | </thead> |
| + | <tbody> |
| + | <tr> |
| + | <td align="center">0</td> |
| + | <td align="center">31880000</td> |
| + | </tr> |
| + | <tr> |
| + | <td align="center">0.5</td> |
| + | <td align="center">33211000</td> |
| + | </tr> |
| + | <tr> |
| + | <td align="center">5</td> |
| + | <td align="center">33951000</td> |
| + | </tr> |
| + | <tr> |
| + | <td align="center">25</td> |
| + | <td align="center">34267000</td> |
| + | </tr> |
| + | <tr> |
| + | <td align="center">50</td> |
| + | <td align="center">35650000</td> |
| + | </tr> |
| + | <tr> |
| + | <td align="center">100</td> |
| + | <td align="center">35861000</td> |
| + | </tr> |
| + | <tr> |
| + | <td align="center">250</td> |
| + | <td align="center">42479000</td> |
| + | </tr> |
| + | <tr> |
| + | <td align="center">500</td> |
| + | <td align="center">51428000</td> |
| + | </tr> |
| + | </tbody> |
| + | </table> |
| | | |
| + | <p>Further visualized experiment was also carried out. As the aggregated tubulins could be centrifuged |
| + | in room temperature while the tubulin monomers could not. As enough protein substrates |
| + | (α-tubulin-YNE, α-tubulin-YCE and β-tubulin) existed, we centrifuged each experimental group. |
| + | Results(Fig.16) showed obvious difference between each group with serial taxol concentration, further |
| + | indicating that the taxol concentration can be represented by the tubulin aggregation level. |
| + | </p> |
| | | |
| + | <figure class="text-center"> |
| + | <img src="https://static.igem.org/mediawiki/2016/1/16/T--BNU-China--Results100.jpg" width="70%"> |
| + | <figcaption> |
| + | Fig.16 Pellet from each experimental group with serial taxol concentration |
| + | <br/> |
| + | </figcaption> |
| + | </figure> |
| | | |
| + | <p>Based on the statistics above, any random concentration of taxol could be figured out in the following Polynomial interpolation curve(Fig.17).</p> |
| | | |
| + | <figure class="text-center"> |
| + | <img src="https://static.igem.org/mediawiki/2016/c/cc/T-BNU-China--Polynomial_interpolation.png" width="70%"> |
| + | <figcaption> |
| + | Fig.17 Fluoroscence intensity - Taxol concentration polynomial interpolation curve<br/> |
| + | </figcaption> |
| + | </figure> |
| | | |
| + | <h2>Results of tublin extraction <i>in vitro</i></h2> |
| + | <p>After successfully extracting tubulin from porcine brains, we tried to summarize the aggregation |
| + | condition in vitro by using electron microscope.</p> |
| + | <figure class="text-center"> |
| + | <img src="https://static.igem.org/mediawiki/2016/3/34/T--BNU-China--Result27.jpg " width="70%"> |
| + | <figcaption> |
| + | Fig.18 Aggregated microtubule treated with 1μM taxol <br/> |
| + | </figcaption> |
| + | </figure> |
| | | |
| | | |
| + | <p>From pictures taken under the electron microscope(Fig.18), we could see tublin (treated with 1 μM |
| + | taxol) in aggregated form obviously, indicating we have achieved the aggregation process <i>in |
| + | vitro</i>. However, due to the high concentration of our extracted sample, it was hard to tell |
| + | the aggregated length and the quantity of microtubules. Thus we tried to use spectrophotometer to |
| + | measure OD<sub>350</sub> of our experimental samples.</p> |
| + | <h4 style="text-align: center">Table 2 OD<sub>350</sub> of microtubule samples treated with serial |
| + | concentration of taxol</h4> |
| + | <table class="table"> |
| + | <tbody> |
| + | <tr> |
| + | <th style="text-align: center;vertical-align: middle;" rowspan="2" colspan="2">OD<sub>350</sub> |
| + | </th> |
| + | <th style="text-align: center" colspan="5">Experiment Group</th> |
| + | </tr> |
| + | <tr> |
| + | <th style="text-align: center">1</th> |
| + | <th style="text-align: center">2</th> |
| + | <th style="text-align: center">3</th> |
| + | <th style="text-align: center">4</th> |
| + | <th style="text-align: center">5</th> |
| + | </tr> |
| + | <tr> |
| + | <th style="text-align: center; vertical-align: middle; padding: 0; width: 60px;" rowspan="21"> |
| + | <div style="font-size: 20px; transform:rotate(-90deg); |
| + | -ms-transform:rotate(-90deg); /* Internet Explorer */ |
| + | -moz-transform:rotate(-90deg); /* Firefox */ |
| + | -webkit-transform:rotate(-90deg); /* Safari 和 Chrome */ |
| + | -o-transform:rotate(-90deg); /* Opera */">Taxol concentration(μM) |
| + | </div> |
| + | </th> |
| + | <td align="center">0</td> |
| + | <td align="center">0.095</td> |
| + | <td align="center">0.077</td> |
| + | <td align="center"></td> |
| + | <td align="center">0.025</td> |
| + | <td align="center">0.104</td> |
| + | </tr> |
| + | <tr> |
| + | <td align="center">0.001</td> |
| + | <td align="center">0.062</td> |
| + | <td align="center">0.123</td> |
| + | <td align="center">0.119</td> |
| + | <td align="center">0.086</td> |
| + | <td align="center">0.149</td> |
| + | </tr> |
| + | <tr> |
| + | <td align="center">0.01</td> |
| + | <td align="center">0.152</td> |
| + | <td align="center">0.138</td> |
| + | <td align="center">0.129</td> |
| + | <td align="center">0.060</td> |
| + | <td align="center">0.081</td> |
| + | </tr> |
| + | <tr> |
| + | <td align="center">0.1</td> |
| + | <td align="center">0.096</td> |
| + | <td align="center">0.106</td> |
| + | <td align="center">0.123</td> |
| + | <td align="center">0.082</td> |
| + | <td align="center">0.134</td> |
| + | </tr> |
| + | <tr> |
| + | <td align="center">1</td> |
| + | <td align="center">0.148</td> |
| + | <td align="center">0.140</td> |
| + | <td align="center">0.149</td> |
| + | <td align="center">0.061</td> |
| + | <td align="center">0.092</td> |
| + | </tr> |
| + | <tr> |
| + | <td align="center">2</td> |
| + | <td align="center">0.047</td> |
| + | <td align="center">0.093</td> |
| + | <td align="center">0.108</td> |
| + | <td align="center">0.052</td> |
| + | <td align="center">0.080</td> |
| + | </tr> |
| + | <tr> |
| + | <td align="center">3</td> |
| + | <td align="center"></td> |
| + | <td align="center"></td> |
| + | <td align="center"></td> |
| + | <td align="center">0.068</td> |
| + | <td align="center">0.091</td> |
| + | </tr> |
| + | <tr> |
| + | <td align="center">4</td> |
| + | <td align="center"></td> |
| + | <td align="center"></td> |
| + | <td align="center"></td> |
| + | <td align="center">0.035</td> |
| + | <td align="center">0.050</td> |
| + | </tr> |
| + | <tr> |
| + | <td align="center">5</td> |
| + | <td align="center">0.020</td> |
| + | <td align="center">0.059</td> |
| + | <td align="center">0.140</td> |
| + | <td align="center">0.078</td> |
| + | <td align="center">0.100</td> |
| + | </tr> |
| + | <tr> |
| + | <td align="center">6</td> |
| + | <td align="center"></td> |
| + | <td align="center"></td> |
| + | <td align="center"></td> |
| + | <td align="center">0.079</td> |
| + | <td align="center">0.112</td> |
| + | </tr> |
| + | <tr> |
| + | <td align="center">7</td> |
| + | <td align="center"></td> |
| + | <td align="center"></td> |
| + | <td align="center"></td> |
| + | <td align="center">0.076</td> |
| + | <td align="center">0.076</td> |
| + | </tr> |
| + | <tr> |
| + | <td align="center">8</td> |
| + | <td align="center"></td> |
| + | <td align="center"></td> |
| + | <td align="center"></td> |
| + | <td align="center">0.050</td> |
| + | <td align="center">0.067</td> |
| + | </tr> |
| + | <tr> |
| + | <td align="center">9</td> |
| + | <td align="center"></td> |
| + | <td align="center"></td> |
| + | <td align="center"></td> |
| + | <td align="center">0.079</td> |
| + | <td align="center">0.107</td> |
| + | </tr> |
| + | <tr> |
| + | <td align="center">10</td> |
| + | <td align="center">0.053</td> |
| + | <td align="center">0.111</td> |
| + | <td align="center">0.185</td> |
| + | <td align="center">0.086</td> |
| + | <td align="center">0.100</td> |
| + | </tr> |
| + | <tr> |
| + | <td align="center">12.5</td> |
| + | <td align="center"></td> |
| + | <td align="center"></td> |
| + | <td align="center"></td> |
| + | <td align="center">0.077</td> |
| + | <td align="center">0.065</td> |
| + | </tr> |
| + | <tr> |
| + | <td align="center">20</td> |
| + | <td align="center">0.028</td> |
| + | <td align="center">0.099</td> |
| + | <td align="center">0.108</td> |
| + | <td align="center"></td> |
| + | <td align="center"></td> |
| + | </tr> |
| + | <tr> |
| + | <td align="center">25</td> |
| + | <td align="center"></td> |
| + | <td align="center"></td> |
| + | <td align="center"></td> |
| + | <td align="center">0.076</td> |
| + | <td align="center">0.097</td> |
| + | </tr> |
| + | <tr> |
| + | <td align="center">30</td> |
| + | <td align="center">0.093</td> |
| + | <td align="center">0.164</td> |
| + | <td align="center">0.154</td> |
| + | <td align="center"></td> |
| + | <td align="center"></td> |
| + | </tr> |
| + | <tr> |
| + | <td align="center">50</td> |
| + | <td align="center">0.043</td> |
| + | <td align="center">0.162</td> |
| + | <td align="center">0.096</td> |
| + | <td align="center">0.074</td> |
| + | <td align="center">0.090</td> |
| + | </tr> |
| + | <tr> |
| + | <td align="center">70</td> |
| + | <td align="center">0.113</td> |
| + | <td align="center">0.188</td> |
| + | <td align="center">0.156</td> |
| + | <td align="center"></td> |
| + | <td align="center"></td> |
| + | </tr> |
| + | <tr> |
| + | <td align="center">100</td> |
| + | <td align="center"></td> |
| + | <td align="center"></td> |
| + | <td align="center"></td> |
| + | <td align="center">0.072</td> |
| + | <td align="center">0.088</td> |
| + | </tr> |
| + | </tbody> |
| + | </table> |
| | | |
| | | |
| + | <p>From the results shown in table 2, we found that there was no obvious relationship between OD |
| + | statistics and taxol concentration. The reason may be the machine issue. Due to the wave length for |
| + | measuring OD is 350nm, which is between the ultraviolet light and visible light, there is a high |
| + | requirement for instruments and always leads to a huge deviation. As the high technologic |
| + | instruments could not be owned by every laboratory in different areas, our fusion proteins which can |
| + | detect the relatively accurate concentration of anti-microtubule drugs will have a broad application |
| + | prospect.</p> |
| | | |
| | | |
− | | + | </article> |
− | | + | |
− | | + | |
− | | + | |
− | | + | |
− | | + | |
− | <h2>The expression of α-tubulin, β-tubulin, n-luciferase, c-luciferase</h2>
| + | |
− | <h3>Vector construction</h3>
| + | |
− | | + | |
− | | + | |
− | <p>Gene fragments of α-tubulin, β-tubulin, n-luciferase, c-luciferase were amplified via PCR and
| + | |
− | verified by electrophoresis (Fig.1). The theoretic gene size of α-tubulin is 1356bp, β-tubulin is
| + | |
− | 1335bp, n-luciferase is 1248bp, c-luciferase is 459bp, which matched our experimental results.</p>
| + | |
− | <h3>Protein expression</h3>
| + | |
− | <p>Gene fragments were ligated to <i>E.coli</i> expression plasmid pET30a(+), after transformation, colony PCR
| + | |
− | was done to verify the efficiency (Fig.2A and Fig.2B). Meanwhile, the sequencing results further
| + | |
− | confirmed that we successfully constructed the α-tubulin, β-tubulin, n-luciferase, c-luciferase
| + | |
− | expression vectors. </p>
| + | |
− | | + | |
− |
| + | |
− | <p>Expression vectors were transformed to <i>E.coli</i> expression strain TransB(DE3). After culturing
| + | |
− | and inducing with IPTG, bacteria were lysed and SDS-PAGE(Fig.3) / western-blot (Fig.4) were done to
| + | |
− | test the protein from supernatant, pellet and renatured inclusion body.</p>
| + | |
− | <figure class="text-center">
| + | |
− | <img src="https://static.igem.org/mediawiki/2016/7/7d/T--BNU-China--Results13.jpg" width="65%">
| + | |
− | <figcaption>
| + | |
− | Fig.3 SDS-PAGE result of β-tubulin
| + | |
− | <br/>
| + | |
− | <small style="font-size:80%">(left to right: non-induced group, expressed empty vector, induced group) arrow
| + | |
− | shows the correct molecular weight of target protein(55 kDa).
| + | |
− | </small>
| + | |
− | </figcaption>
| + | |
− | </figure>
| + | |
− | | + | |
− | <figure class="text-center" id="ref-1">
| + | |
− | <img src="https://static.igem.org/mediawiki/2016/6/61/T--BNU-China--result20.jpg" width="80%">
| + | |
− | <figcaption>
| + | |
− | Fig.4 Western blot result of prokaryotic expression
| + | |
− | <br/>
| + | |
− | <small style="font-size:80%">(left to right, extracted α-tubulin, expressed empty vector,
| + | |
− | α-tubulin, α-tubulin-YNE fusion protein, α-tubulin-YCE fusion protein, α-tubulin-nluc fusion
| + | |
− | protein) arrows show the correct bands of target proteins, triangles show the homologous
| + | |
− | tubulin protein(FtsZ,43kDa) from the bacteria.
| + | |
− | </small>
| + | |
− | </figcaption>
| + | |
− | </figure>
| + | |
− | | + | |
− | <p>Apart from this, we also transformed plasmids to Rossatta(DE3) which can express rare codons and
| + | |
− | improve the expression level of eukaryotic protein.</p>
| + | |
− | <p id="ref-2">Before breaking the bacteria via ultrasonic waves, SDS-PAGE (Fig.5) was established to
| + | |
− | verify the results.</p>
| + | |
− | | + | |
− | <figure class="text-center">
| + | |
− | <img src="https://static.igem.org/mediawiki/2016/a/a0/T--BNU-China--Results16.jpg" width="65%">
| + | |
− | <figcaption>
| + | |
− | Fig.5 SDS-PAGE of rossatta cells before ultrasonic breaking
| + | |
− | <br/>
| + | |
− | <small style="font-size:80%">(left to right: protein marker, expressed empty vector,
| + | |
− | α-tubulin(55 kDa), β-tubulin(55 kDa), α-tubulin-YNE(75kDa), YNE-α-tubulin(75 kDa) ) arrows
| + | |
− | show the correct band.
| + | |
− | </small>
| + | |
− | </figcaption>
| + | |
− | </figure>
| + | |
− | | + | |
− | <p id="ref-3">After breaking the bacteria, SDS-PAGE (Fig.6) was also established to verify the
| + | |
− | results.</p>
| + | |
− | | + | |
− | <figure class="text-center">
| + | |
− | <img src="https://static.igem.org/mediawiki/2016/a/a9/T--BNU-China--result21.jpg" width="70%">
| + | |
− | <figcaption>
| + | |
− | Fig.6 SDS-PAGE of supernatant after ultrasonic breaking the rossatta cells
| + | |
− | <br/>
| + | |
− | <small style="font-size:80%">left to right: expressed empty vector, α-tubulin(55 kDa),
| + | |
− | β-tubulin(55 kDa), α-tubulin-YNE(75kDa), YNE-α-tubulin(75kDa), α-tubulin-YCE(66 kDa),
| + | |
− | YCE-α-tubulin(66 kDa), β-tubulin-YCE(66 kDa), YCE-β-tubulin(66 kDa), α-tubulin-nluc,
| + | |
− | cluc-α-tubulin(74 kDa).
| + | |
− | </small>
| + | |
− | </figcaption>
| + | |
− | </figure>
| + | |
− | | + | |
− | <p id="ref-4">Western blot(Fig.7) was also applied for the further confirmation.</p>
| + | |
− | | + | |
− | <figure class="text-center">
| + | |
− | <img src="https://static.igem.org/mediawiki/2016/6/67/T--BNU-China--result22.jpg" width="70%">
| + | |
− | <figcaption>
| + | |
− | Fig.7 Western blot of rossatta cells expression
| + | |
− | <br/>
| + | |
− | <small style="font-size:80%">A: left to right, protein marker, negative control, α-tubulin(55
| + | |
− | kDa), α-tubulin-YNE(75kDa), α-tubulin-YCE(66 kDa), YCE-α-tubulin(66 kDa), cluc-α-tubulin(74
| + | |
− | kDa), extracted α-tubulin(55 kDa).<br/> B: left to right: negative control in pellet,
| + | |
− | β-tubulin in pellet(55 kDa), β-tubulin-YCE in pellet (66 kDa), protein marker, negative
| + | |
− | control in supernatant, β-tubulin in supernatant(55 kDa), β-tubulin-YCE in supernatant(66
| + | |
− | kDa), extracted β-tubulin(55 kDa).
| + | |
− | </small>
| + | |
− | </figcaption>
| + | |
− | </figure>
| + | |
− | | + | |
− | <p>According to Fig.7B, the target protein can be tested out in the supernatant, indicating that they
| + | |
− | are soluble when expressed in rossatta strain.</p>
| + | |
− | <p>Based on the results above, we can confirm that α-tubulin and β-tubulin were successfully expressed
| + | |
− | in cell.</p>
| + | |
− | <p>We collaborated with Fujian Agriculture and Forest University and asked them to test the interaction
| + | |
− | between α and β-tubulin. Thus verified the activity of tubulin monomers.</p>
| + | |
− |
| + | |
− | | + | |
− | <h2> The expression of fusion protein</h2>
| + | |
− | <h3> Fusion PCR</h3>
| + | |
− | <p>α-tubulin-YNE, YNE-α-tubulin, α-tubulin-YCE, YCE-α-tubulin, β-tubulin-YNE, YNE-β-tubulin,
| + | |
− | β-tubulin-YCE, YCE-β-tubulin, α-tubulin-nluc, nluc-α-tubulin, α-tubulin-cluc, cluc-α-tubulin
| + | |
− | were constructed respectively via fusion PCR. After ligating these fusion gene fragments to
| + | |
− | pET30a(+) with restriction enzyme, we transformed the target plasmids to Trans5α. When colony
| + | |
− | PCR was done, we picked correct colony shown in electrophoresis(Fig.8) for plasmid amplification.</p>
| + | |
− | <p>Sequencing results showed that α-tubulin-YNE, YNE-α-tubulin, α-tubulin-YCE, YCE-α-tubulin,
| + | |
− | β-tubulin-YCE, YCE-β-tubulin, α-tubulin-nluc, cluc-α-tubulin expression vectors were constructed
| + | |
− | successfully.</p>
| + | |
− | <p>We transformed the expression plasmids to <i>E.coli</i> expression strain TranB(DE3). The protein
| + | |
− | expression predicted website http://www.biotech.ou.edu/ showed that our fusion protein would
| + | |
− | probably expressed as inclusion bodies. We therefore renatured the inclusion bodies and verified
| + | |
− | through SDS-PAGE(Fig.9).</p>
| + | |
− | | + | |
− | <figure class="text-center">
| + | |
− | <img src="https://static.igem.org/mediawiki/2016/a/a0/T--BNU-China--Results7.jpg" width="60%">
| + | |
− | <figcaption>
| + | |
− | Fig.9 SDS-PAGE of renatured inclusion bodies from α-tubulin-YNE, YNE-α-tubulin
| + | |
− | <br/>
| + | |
− | <small style="font-size:80%">the molecular weight of target fusion protein is 74.6kDa.
| + | |
− | arrows show the correct bands.
| + | |
− | </small>
| + | |
− | </figcaption>
| + | |
− | </figure>
| + | |
− | | + | |
− | <p>Also, western-blot (<a href="https://2016.igem.org/Team:BNU-China/Proof#ref-1">Fig.4</a>) were
| + | |
− | done to test the protein from supernatant, pellet and renatured inclusion body.</p>
| + | |
− | <p>Apart from these, we also expressed our target protein through Rossatta(DE3) strain and used
| + | |
− | SDS-PAGE(Fig.10) to verify the expression.</p>
| + | |
− | | + | |
− | <figure class="text-center">
| + | |
− | <img src="https://static.igem.org/mediawiki/2016/3/3c/T--BNU-China--Results19.jpg" width="100%">
| + | |
− | <figcaption>
| + | |
− | Fig.10 SDS-PAGE of rossatta cells before ultrasonic breaking
| + | |
− | <br/>
| + | |
− | <small style="font-size:80%">A: cluc-α-tubulin(74 kDa), α-tubulin-nluc, YCE-β-tubulin(66
| + | |
− | kDa), β-tubulin-YCE(66 kDa), YCE-α-tubulin(66 kDa), α-tubulin-YCE(66 kDa), expressed
| + | |
− | empty vector. <br/>B: left to right: expressed empty vector, α-tubulin(55 kDa),
| + | |
− | β-tubulin(55 kDa), α-tubulin-YNE(75kDa), YNE-α-tubulin(75kDa).
| + | |
− | </small>
| + | |
− | </figcaption>
| + | |
− | </figure>
| + | |
− | | + | |
− | <p>SDS-PAGE were done to verify the expression results before(<a
| + | |
− | href="https://2016.igem.org/Team:BNU-China/Proof#ref-2">Fig.5</a>) and after(<a
| + | |
− | href="https://2016.igem.org/Team:BNU-China/Proof#ref-3">Fig.6</a>) breaking the bacteria, and
| + | |
− | Western blot(<a href="https://2016.igem.org/Team:BNU-China/Proof#ref-4">Fig.7</a>) was also
| + | |
− | applied for the further confirmation.</p>
| + | |
− | <p>Based on the results above, we can confirm that α-tubulin-YNE, YNE-α-tubulin, α-tubulin-YCE,
| + | |
− | YCE-α-tubulin, β-tubulin-YCE, YCE-β-tubulin, cluc-α-tubulin fusion protein were successfully
| + | |
− | expressed in cell.</p>
| + | |
− | | + | |
− | | + | |
− | <h3>Gateway</h3>
| + | |
− | <p>In our experiment, we also try to construct fusion protein vectors with Gateway Large-scale
| + | |
− | Cloning technology. We used Invitrogen pENTR/D TOPO to clone β-tubulin into entry vector.
| + | |
− | Designing primer based on β-tubulin sequence and running PCR procedure. From this picture, the
| + | |
− | band of β-tubulin was correct.</p>
| + | |
− | | + | |
− | <p>In order to do LR reaction, we used the restriction endonuclease Not I to digest the entry
| + | |
− | vector. The bands of linear vector and the origin vector suggested that the digestion was
| + | |
− | efficiency.</p>
| + | |
− |
| + | |
− | <p>Using Invitrogen Gateway LR Clonase II Enzyme Mix, the entry vector can be ligate with
| + | |
− | pCambia1300-nluc and pCambia1300-cluc respectively. So the destination vectors were complete.
| + | |
− | After transformation, running PCR with β-tubulin's primers, the bands show high positive rates
| + | |
− | as showed in Fig.13</p>
| + | |
− | | + | |
− |
| + | |
− | <p>Extracting plasmid, the product gal bands show that cluc-β-tubulin is correct. Running PCR using
| + | |
− | the reverse primer of β-tubulin and the forward primer of cluc, the correct band existed.</p>
| + | |
− | | + | |
− | <figure class="text-center">
| + | |
− | <img src="https://static.igem.org/mediawiki/2016/0/07/T--BNU-China--Results11.jpg" width="70%">
| + | |
− | <figcaption>
| + | |
− | Fig.14 PCR verification result of pCambia-nluc, pCambia-cluc
| + | |
− | <br/>
| + | |
− | <small style="font-size:80%">arrows show the correct bands</small>
| + | |
− | </figcaption>
| + | |
− | </figure>
| + | |
− | | + | |
− | <h2>Results of tublin extraction <i>in vitro</i></h2>
| + | |
− | <p>After successfully extracting tubulin from porcine brains, we tried to summarize the aggregation
| + | |
− | condition<i>in vitro</i> by using electron microscope.</p>
| + | |
− | | + | |
− | <figure class="text-center">
| + | |
− | <img src="https://static.igem.org/mediawiki/2016/c/c0/T--BNU-China--Results5.jpg" width="88%">
| + | |
− | <figcaption>
| + | |
− | Fig.15 Aggregated microtubule treated with 1μM taxol
| + | |
− | <br/>
| + | |
− |
| + | |
− | </figcaption>
| + | |
− | </figure>
| + | |
− | <P>From pictures taken under the electron microscope(Fig.15), we
| + | |
− | could see tubulin (treated with 1μM taxol) in aggregated form obviously, indicating we
| + | |
− | have achieved the aggregation process <i>in vitro</i>. However, due to the high
| + | |
− | concentration of our extracted sample, it was hard to tell the aggregated length and the
| + | |
− | quantity of microtubules. Thus we tried to use spectrophotometer to measure OD350 of
| + | |
− | our experimental samples.</P>
| + | |
− | <h4 style="text-align: center">Table 1 OD350 of microtubule samples treated with serial concentration of taxol</h4>
| + | |
− | <table class="table">
| + | |
− | <tbody>
| + | |
− | <tr>
| + | |
− | <th style="text-align: center">Taxol concentraion(μM)</th>
| + | |
− | <th style="text-align: center">1</th>
| + | |
− | <th style="text-align: center">2</th>
| + | |
− | <th style="text-align: center">3</th>
| + | |
− | <th style="text-align: center">4</th>
| + | |
− | <th style="text-align: center">5</th>
| + | |
− | </tr>
| + | |
− | <tr>
| + | |
− | <td align="center">0</td>
| + | |
− | <td align="center">0.095</td>
| + | |
− | <td align="center">0.077</td>
| + | |
− | <td align="center"></td>
| + | |
− | <td align="center">0.025</td>
| + | |
− | <td align="center">0.104</td>
| + | |
− | </tr>
| + | |
− | <tr>
| + | |
− | <td align="center">0.001</td>
| + | |
− | <td align="center">0.062</td>
| + | |
− | <td align="center">0.123</td>
| + | |
− | <td align="center">0.119</td>
| + | |
− | <td align="center">0.086</td>
| + | |
− | <td align="center">0.149</td>
| + | |
− | </tr>
| + | |
− | <tr>
| + | |
− | <td align="center">0.01</td>
| + | |
− | <td align="center">0.152</td>
| + | |
− | <td align="center">0.138</td>
| + | |
− | <td align="center">0.129</td>
| + | |
− | <td align="center">0.060</td>
| + | |
− | <td align="center">0.081</td>
| + | |
− | </tr>
| + | |
− | <tr>
| + | |
− | <td align="center">0.1</td>
| + | |
− | <td align="center">0.096</td>
| + | |
− | <td align="center">0.106</td>
| + | |
− | <td align="center">0.123</td>
| + | |
− | <td align="center">0.082</td>
| + | |
− | <td align="center">0.134</td>
| + | |
− | </tr>
| + | |
− | <tr>
| + | |
− | <td align="center">1</td>
| + | |
− | <td align="center">0.148</td>
| + | |
− | <td align="center">0.140</td>
| + | |
− | <td align="center">0.149</td>
| + | |
− | <td align="center">0.061</td>
| + | |
− | <td align="center">0.092</td>
| + | |
− | </tr>
| + | |
− | <tr>
| + | |
− | <td align="center">2</td>
| + | |
− | <td align="center">0.047</td>
| + | |
− | <td align="center">0.093</td>
| + | |
− | <td align="center">0.108</td>
| + | |
− | <td align="center">0.052</td>
| + | |
− | <td align="center">0.080</td>
| + | |
− | </tr>
| + | |
− | <tr>
| + | |
− | <td align="center">3</td>
| + | |
− | <td align="center"></td>
| + | |
− | <td align="center"></td>
| + | |
− | <td align="center"></td>
| + | |
− | <td align="center">0.068</td>
| + | |
− | <td align="center">0.091</td>
| + | |
− | </tr>
| + | |
− | <tr>
| + | |
− | <td align="center">4</td>
| + | |
− | <td align="center"></td>
| + | |
− | <td align="center"></td>
| + | |
− | <td align="center"></td>
| + | |
− | <td align="center">0.035</td>
| + | |
− | <td align="center">0.050</td>
| + | |
− | </tr>
| + | |
− | <tr>
| + | |
− | <td align="center">5</td>
| + | |
− | <td align="center">0.020</td>
| + | |
− | <td align="center">0.059</td>
| + | |
− | <td align="center">0.140</td>
| + | |
− | <td align="center">0.078</td>
| + | |
− | <td align="center">0.100</td>
| + | |
− | </tr>
| + | |
− | <tr>
| + | |
− | <td align="center">6</td>
| + | |
− | <td align="center"></td>
| + | |
− | <td align="center"></td>
| + | |
− | <td align="center"></td>
| + | |
− | <td align="center">0.079</td>
| + | |
− | <td align="center">0.112</td>
| + | |
− | </tr>
| + | |
− | <tr>
| + | |
− | <td align="center">7</td>
| + | |
− | <td align="center"></td>
| + | |
− | <td align="center"></td>
| + | |
− | <td align="center"></td>
| + | |
− | <td align="center">0.076</td>
| + | |
− | <td align="center">0.076</td>
| + | |
− | </tr>
| + | |
− | <tr>
| + | |
− | <td align="center">8</td>
| + | |
− | <td align="center"></td>
| + | |
− | <td align="center"></td>
| + | |
− | <td align="center"></td>
| + | |
− | <td align="center">0.050</td>
| + | |
− | <td align="center">0.067</td>
| + | |
− | </tr>
| + | |
− | <tr>
| + | |
− | <td align="center">9</td>
| + | |
− | <td align="center"></td>
| + | |
− | <td align="center"></td>
| + | |
− | <td align="center"></td>
| + | |
− | <td align="center">0.079</td>
| + | |
− | <td align="center">0.107</td>
| + | |
− | </tr>
| + | |
− | <tr>
| + | |
− | <td align="center">10</td>
| + | |
− | <td align="center">0.053</td>
| + | |
− | <td align="center">0.111</td>
| + | |
− | <td align="center">0.185</td>
| + | |
− | <td align="center">0.086</td>
| + | |
− | <td align="center">0.100</td>
| + | |
− | </tr>
| + | |
− | <tr>
| + | |
− | <td align="center">12.5</td>
| + | |
− | <td align="center"></td>
| + | |
− | <td align="center"></td>
| + | |
− | <td align="center"></td>
| + | |
− | <td align="center">0.077</td>
| + | |
− | <td align="center">0.065</td>
| + | |
− | </tr>
| + | |
− | <tr>
| + | |
− | <td align="center">20</td>
| + | |
− | <td align="center">0.028</td>
| + | |
− | <td align="center">0.099</td>
| + | |
− | <td align="center">0.108</td>
| + | |
− | <td align="center"></td>
| + | |
− | <td align="center"></td>
| + | |
− | </tr>
| + | |
− | <tr>
| + | |
− | <td align="center">25</td>
| + | |
− | <td align="center"></td>
| + | |
− | <td align="center"></td>
| + | |
− | <td align="center"></td>
| + | |
− | <td align="center">0.076</td>
| + | |
− | <td align="center">0.097</td>
| + | |
− | </tr>
| + | |
− | <tr>
| + | |
− | <td align="center">30</td>
| + | |
− | <td align="center">0.093</td>
| + | |
− | <td align="center">0.164</td>
| + | |
− | <td align="center">0.154</td>
| + | |
− | <td align="center"></td>
| + | |
− | <td align="center"></td>
| + | |
− | </tr>
| + | |
− | <tr>
| + | |
− | <td align="center">50</td>
| + | |
− | <td align="center">0.043</td>
| + | |
− | <td align="center">0.162</td>
| + | |
− | <td align="center">0.096</td>
| + | |
− | <td align="center">0.074</td>
| + | |
− | <td align="center">0.090</td>
| + | |
− | </tr>
| + | |
− | <tr>
| + | |
− | <td align="center">70</td>
| + | |
− | <td align="center">0.113</td>
| + | |
− | <td align="center">0.188</td>
| + | |
− | <td align="center">0.156</td>
| + | |
− | <td align="center"></td>
| + | |
− | <td align="center"></td>
| + | |
− | </tr>
| + | |
− | <tr>
| + | |
− | <td align="center">100</td>
| + | |
− | <td align="center"></td>
| + | |
− | <td align="center"></td>
| + | |
− | <td align="center"></td>
| + | |
− | <td align="center">0.072</td>
| + | |
− | <td align="center">0.088</td>
| + | |
− | </tr>
| + | |
− | </tbody>
| + | |
− | </table>
| + | |
− | <p>From the results shown in Table 1, we found that there was no obvious relationship between OD
| + | |
− | statistics and taxol concentration. The reason may be the machine issue. Due to the wave length
| + | |
− | for measuring OD is 350nm, which is between the ultraviolet light and visible light, there is a
| + | |
− | high requirement for instruments and always leads to a huge deviation. As the high technologic
| + | |
− | instruments could not be owned by every laboratory in different areas, our fusion proteins which
| + | |
− | can detect the relatively accurate concentration of anti-microtubule drugs will have a broad
| + | |
− | application prospect.</p>
| + | |
− | | + | |
− | </article>
| + | |
− | </div>
| + | |
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