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− | #contain #detail h4{ | + | |
+ | #contain #detail h4 { | ||
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synthetic biology tools to produce the expected results. 30 new BioBricks were designed for our | synthetic biology tools to produce the expected results. 30 new BioBricks were designed for our | ||
project (See | project (See | ||
− | the Parts and Design details in <a href="https://2021.igem.org/Team:CPU_CHINA/partsOverview">Parts </a> and <a href="https://2021.igem.org/Team:CPU_CHINA/Design">Design</a>.</span></p> | + | the Parts and Design details in <a |
+ | href="https://2021.igem.org/Team:CPU_CHINA/partsOverview"><strong> Parts </strong></a> and | ||
+ | <a href="https://2021.igem.org/Team:CPU_CHINA/Design"><strong> Design</strong></a>.)</span></p> | ||
<p><span>We have done a lot of amazing work to make sure they can realize the goals we set. Moreover, it | <p><span>We have done a lot of amazing work to make sure they can realize the goals we set. Moreover, it | ||
is worth | is worth | ||
− | mentioning that BBa_K3853011, BBa_K3853010 and | + | mentioning that <strong><a href="http://parts.igem.org/Part:BBa_K3853011">BBa_K3853011</a>, <a |
+ | href="http://parts.igem.org/Part:BBa_K3853010">BBa_K3853010</a></strong> and <strong><a | ||
+ | href="http://parts.igem.org/Part:BBa_K3853008">BBa_K3853008</a></strong> are the | ||
+ | representatives among them. | ||
These three | These three | ||
BioBricks work well to perform their functions and occupations as expected. All the related data | BioBricks work well to perform their functions and occupations as expected. All the related data | ||
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<div class="section" id="section2"> | <div class="section" id="section2"> | ||
<h2 id='engineering-success'><span>ENGINEERING SUCCESS</span></h2> | <h2 id='engineering-success'><span>ENGINEERING SUCCESS</span></h2> | ||
− | < | + | <h3 id='1-dcas9-spycatcher-bbak3853011'><span>1. dCas9-SpyCatcher (<a |
+ | href="http://parts.igem.org/Part:BBa_K3853011">BBa_K3853011</a>)</span></h3> | ||
<p><span>In our 2021 project, we fused deactivated CRISPR-associated protein 9 (dCas9) with SpyCatcher, | <p><span>In our 2021 project, we fused deactivated CRISPR-associated protein 9 (dCas9) with SpyCatcher, | ||
turning it | turning it | ||
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expressed and | expressed and | ||
purified to test its function. </span></p> | purified to test its function. </span></p> | ||
− | < | + | <h4><span> </span><strong><span>1-1. Agarose Gel Electrophoresis</span></strong></h4> |
<p><strong><span>Method: </span></strong><span>We used PCR to obtain three homologous recombination | <p><strong><span>Method: </span></strong><span>We used PCR to obtain three homologous recombination | ||
fragments | fragments | ||
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construction of | construction of | ||
expression plasmid. The successfully recombined plasmid was transformed into | expression plasmid. The successfully recombined plasmid was transformed into | ||
− | </span><em><span>E.coli</span></em><span>, and the monoclonal colonies with positive transformation | + | </span><em><span>E.coli </span></em>BL21(DE3)<span>, and the monoclonal colonies with positive |
+ | transformation | ||
results were | results were | ||
selected for subsequent sequencing verification.</span></p> | selected for subsequent sequencing verification.</span></p> | ||
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successfully obtained by PCR, and the result was shown in </span><strong><span>Fig. | successfully obtained by PCR, and the result was shown in </span><strong><span>Fig. | ||
1</span></strong><span>. | 1</span></strong><span>. | ||
− | The sequencing results of the </span><em><span>E.coli</span></em><span> monoclonal colonies | + | The sequencing results of the </span><em><span>E.coli</span></em> BL21(DE3)<span> monoclonal |
− | (</span><strong><a href="https://static.igem.org/mediawiki/2021/b/b9/T--CPU_CHINA--dCas9-SpyCatcher_sequencing_results.zip">File 1</a></strong><span>) showed a successful transformation.</span> | + | colonies |
+ | (</span><strong><a | ||
+ | href="https://static.igem.org/mediawiki/2021/b/b9/T--CPU_CHINA--dCas9-SpyCatcher_sequencing_results.zip">File | ||
+ | 1</a></strong><span>) showed a successful transformation.</span> | ||
</p> | </p> | ||
− | + | <img src="https://static.igem.org/mediawiki/2021/d/dd/T--CPU_CHINA--Engineering_success_fig_1.jpg"> | |
− | + | ||
<p class="imgdescribe"><strong><span>Fig. 1 Agarose gel electrophoresis of PCR | <p class="imgdescribe"><strong><span>Fig. 1 Agarose gel electrophoresis of PCR | ||
− | products.</span></strong><span> </span><em><span>Vector | + | products.</span></strong><span> </span><em><span class="reference">Vector |
− | refers to plasmid pET-28a. G3S*4 refers to quadruple Ser/Gly link</span></em></p> | + | refers to plasmid pET-28a. G3S*4 refers to quadruple Ser/Gly link.</span></em></p> |
− | < | + | <h4><span> </span><strong><span>1-2. SDS-PAGE</span></strong></h4> |
<p><strong><span>Method:</span></strong><span> We used Ni-NTA affinity column to purify | <p><strong><span>Method:</span></strong><span> We used Ni-NTA affinity column to purify | ||
dCas9-SpyCatcher. </span> | dCas9-SpyCatcher. </span> | ||
</p> | </p> | ||
<p><strong><span>Result:</span></strong><span> Target bands could be observed at the position of about | <p><strong><span>Result:</span></strong><span> Target bands could be observed at the position of about | ||
− | 176.2 kDa, | + | 176.2 kDa (<strong><span>Fig. 2</span></strong>), |
which means the protein of dCas9-SpyCatcher could be successfully expressed, and the related | which means the protein of dCas9-SpyCatcher could be successfully expressed, and the related | ||
gene worked well. | gene worked well. | ||
</span></p> | </span></p> | ||
− | + | <img src="https://static.igem.org/mediawiki/2021/5/5e/T--CPU_CHINA--Engineering_success_fig_2.png"></p> | |
− | + | ||
<p class="imgdescribe"><strong><span>Fig. 2 SDS-PAGE of purified products of dCas9-SpyCatcher. | <p class="imgdescribe"><strong><span>Fig. 2 SDS-PAGE of purified products of dCas9-SpyCatcher. | ||
− | </span></strong><em><span>50 mM | + | </span></strong><em class="reference"><span>50 mM |
imidazole and 500 mM imidazole represent corresponding eluates with different imidazole | imidazole and 500 mM imidazole represent corresponding eluates with different imidazole | ||
concentrations, and | concentrations, and | ||
− | the binding buffer is generated by equilibrating the Ni column after elution with 500 mM | + | the binding buffer is generated by equilibrating the Ni-NTA affinity column after elution with 500 mM |
imidazole.</span></em></p> | imidazole.</span></em></p> | ||
− | < | + | <h4><span> </span><strong><span>1-3. Western Blot</span></strong></h4> |
− | <p><strong><span>Method:</span></strong><span> Since we had introduced | + | <p><strong><span>Method:</span></strong><span> Since we had introduced 6×his-tag at the N-terminus of |
dCas9-SpyCatcher, we used his-antibody as the primary antibody to perform Western Blot analysis | dCas9-SpyCatcher, we used his-antibody as the primary antibody to perform Western Blot analysis | ||
to detect the | to detect the | ||
expression status of the target protein.</span></p> | expression status of the target protein.</span></p> | ||
<p><strong><span>Result:</span></strong><span> The protein band of dCas9-SpyCatcher could be observed | <p><strong><span>Result:</span></strong><span> The protein band of dCas9-SpyCatcher could be observed | ||
− | clearly, | + | clearly (<strong><span>Fig. 3</span></strong>), |
meaning that the related gene functioned well. </span></p> | meaning that the related gene functioned well. </span></p> | ||
− | + | <img src="https://static.igem.org/mediawiki/2021/c/c6/T--CPU_CHINA--Engineering_success_fig_3.jpg"> | |
− | + | ||
<p class="imgdescribe"><strong><span>Fig. 3 Western Blot of dCas9-SpyCatcher.</span></strong><span> | <p class="imgdescribe"><strong><span>Fig. 3 Western Blot of dCas9-SpyCatcher.</span></strong><span> | ||
− | </span><em><span>The concentration | + | </span><em class="reference"><span>The concentration |
of dCas9-SpyCatcher: lane 1: 0.25 mg/ml; lane 2: 0.5 mg/ml; lane 3: 1 mg/ml.</span></em></p> | of dCas9-SpyCatcher: lane 1: 0.25 mg/ml; lane 2: 0.5 mg/ml; lane 3: 1 mg/ml.</span></em></p> | ||
− | < | + | <h4><span> </span><strong><span>1-4. SpyCatcher/SpyTag system combination</span></strong></h4> |
<p><strong><span>Method:</span></strong><span> We incubated SpyTag-MnP and dCas9-SpyCatcher together to | <p><strong><span>Method:</span></strong><span> We incubated SpyTag-MnP and dCas9-SpyCatcher together to | ||
verify the | verify the | ||
− | combination of SpyCatcher and SpyTag </span><sup><span>[1]</span></sup><span>. SpyTag-MnP was | + | combination of SpyCatcher and SpyTag</span><sup><span>[1]</span></sup><span>. SpyTag-MnP was |
mixed with | mixed with | ||
dCas9-SpyCatcher in a ratio of 1 : 1, and they were allowed to conjugate for 1 h at 37℃. | dCas9-SpyCatcher in a ratio of 1 : 1, and they were allowed to conjugate for 1 h at 37℃. | ||
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<p><strong><span>Result:</span></strong><span> As shown in </span><strong><span>Fig. | <p><strong><span>Result:</span></strong><span> As shown in </span><strong><span>Fig. | ||
4</span></strong><span>. The | 4</span></strong><span>. The | ||
− | band of the conjugated dCas9-MnP complex appeared at lane 3, with a slightly higher | + | band of the conjugated dCas9-MnP complex appeared at lane 3, with a slightly higher position |
than | than | ||
dCas9-SpyCatcher; Also, the original SpyTag-MnP band at lane 3 disappeared , meaning the two | dCas9-SpyCatcher; Also, the original SpyTag-MnP band at lane 3 disappeared , meaning the two | ||
proteins had | proteins had | ||
successfully conjugated.</span></p> | successfully conjugated.</span></p> | ||
− | + | <img src="https://static.igem.org/mediawiki/2021/7/75/T--CPU_CHINA--BBa_K3853008_fig_12.png"> | |
− | + | ||
<p class="imgdescribe"><strong><span>Fig. 4 SDS-PAGE of the combination of SpyTag-MnP and | <p class="imgdescribe"><strong><span>Fig. 4 SDS-PAGE of the combination of SpyTag-MnP and | ||
dCas9-SpyCatcher.</span></strong><span> | dCas9-SpyCatcher.</span></strong><span> | ||
− | </span><em><span>Lane 1: SpyTag-MnP; Lane 2: dCas9-SpyCatcher; Lane 3: SpyTag-MnP mixed with | + | </span><em class="reference"><span>Lane 1: SpyTag-MnP; Lane 2: dCas9-SpyCatcher; Lane 3: SpyTag-MnP mixed with |
dCas9-SpyCatcher.</span></em></p> | dCas9-SpyCatcher.</span></em></p> | ||
<p> </p> | <p> </p> | ||
− | < | + | <h3 id='2-his-tag-spytag-hfb1-bbak3853010'><span>2. His-tag-SpyTag-HFB1 (<a |
+ | href="http://parts.igem.org/Part:BBa_K3853010">BBa_K3853010</a>) </span></h3> | ||
<p><span>Hydrophobin-1 (HFB1) is a kind of class Ⅱ HFBs derived from </span><em><span>Trichoderma | <p><span>Hydrophobin-1 (HFB1) is a kind of class Ⅱ HFBs derived from </span><em><span>Trichoderma | ||
reesei</span></em><span>, which can exert surface activity at the hydrophilic-hydrophobic | reesei</span></em><span>, which can exert surface activity at the hydrophilic-hydrophobic | ||
interface. We | interface. We | ||
introduced SpyTag at its N-terminus for the assembly of multi-enzyme complexes.</span></p> | introduced SpyTag at its N-terminus for the assembly of multi-enzyme complexes.</span></p> | ||
− | < | + | <h4><span> </span><strong><span>2-1. Agarose Gel Electrophoresis</span></strong></h4> |
<p><strong><span>Method: </span></strong><span>The plasmid carrying the gene of SpyTag-HFB1 was | <p><strong><span>Method: </span></strong><span>The plasmid carrying the gene of SpyTag-HFB1 was | ||
transformed into | transformed into | ||
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<p><strong><span>Result:</span></strong><span> The bands of target gene appeared at the normal position, | <p><strong><span>Result:</span></strong><span> The bands of target gene appeared at the normal position, | ||
the | the | ||
− | result was shown in </span><strong><span>Fig. | + | result was shown in </span><strong><span>Fig. 5</span></strong><span>. Sequencing verification |
results | results | ||
− | (</span><strong><a href="https://static.igem.org/mediawiki/2021/8/8f/T--CPU_CHINA--SpyTag-HFB1_sequencing_results.zip">File 2</a></strong><span>) showed a successful transformation.</span> | + | (</span><strong><a |
+ | href="https://static.igem.org/mediawiki/2021/8/8f/T--CPU_CHINA--SpyTag-HFB1_sequencing_results.zip">File | ||
+ | 2</a></strong><span>) showed a successful transformation.</span> | ||
</p> | </p> | ||
− | + | <img src="https://static.igem.org/mediawiki/2021/f/ff/T--CPU_CHINA--Engineering_success_fig_5.jpg" | |
− | referrerpolicy="no-referrer" | + | referrerpolicy="no-referrer"> |
− | <p class="imgdescribe"><strong><span>Fig. 5 Agarose gel electrophoresis of PCR products.</span></strong> | + | <p class="imgdescribe"><strong><span>Fig. 5 Agarose gel electrophoresis of PCR products of monoclonal colonies of SpyTag-HFB1.</span></strong> |
</p> | </p> | ||
− | < | + | <h4><span> </span><strong><span>2-2. SDS-PAGE</span></strong></h4> |
<p><strong><span>Method: </span></strong><span>We used Ni-NTA affinity column to obtain purified | <p><strong><span>Method: </span></strong><span>We used Ni-NTA affinity column to obtain purified | ||
SpyTag-HFB1. | SpyTag-HFB1. | ||
</span></p> | </span></p> | ||
<p><strong><span>Result:</span></strong><span> Target bands could be observed at the position of about | <p><strong><span>Result:</span></strong><span> Target bands could be observed at the position of about | ||
− | 15 kDa, | + | 15 kDa (<strong><span>Fig. 6</span></strong>), |
which means the protein of SpyTag-HFB1 was successfully expressed, and the related gene worked | which means the protein of SpyTag-HFB1 was successfully expressed, and the related gene worked | ||
well. </span> | well. </span> | ||
</p> | </p> | ||
− | + | <img src="https://static.igem.org/mediawiki/2021/f/f2/T--CPU_CHINA--Engineering_success_fig_6.png" | |
− | + | ></p> | |
<p class="imgdescribe"><strong><span>Fig. 6 SDS-PAGE of purification products of dCas9-SpyCatcher. | <p class="imgdescribe"><strong><span>Fig. 6 SDS-PAGE of purification products of dCas9-SpyCatcher. | ||
− | </span></strong><em><span>flow-through is the liquid flowing out of the column when loading the | + | </span></strong><em class="reference"><span>flow-through is the liquid flowing out of the column when loading the |
sample, 50 mM | sample, 50 mM | ||
imidazole and 500 mM imidazation are eluates of different imidazole | imidazole and 500 mM imidazation are eluates of different imidazole | ||
concentrations.</span></em></p> | concentrations.</span></em></p> | ||
− | < | + | <h4><span> </span><strong><span>2-3. Functional Verification</span></strong></h4> |
<p><strong><span>Method:</span></strong><span> We dropped the liquid containing SpyTag-HFB1 and the | <p><strong><span>Method:</span></strong><span> We dropped the liquid containing SpyTag-HFB1 and the | ||
liquid without | liquid without | ||
− | SpyTag-HFB1 on a plastic dish, and observed the | + | SpyTag-HFB1 on a plastic dish, and observed the contact angle, shape and motion state of |
different | different | ||
liquids to evaluate the effect of HFB1.</span></p> | liquids to evaluate the effect of HFB1.</span></p> | ||
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hydrophilicity of the | hydrophilicity of the | ||
plastic surface. </span></p> | plastic surface. </span></p> | ||
− | <p class=" | + | <p class="tabledescribe"><strong><span>Table 1. Contact angle measurement among different |
− | < | + | samples.</span></strong></p> |
− | + | <div id='write' class=''> | |
− | + | <figure> | |
− | + | <table> | |
− | + | <thead> | |
− | + | <tr> | |
− | + | <th style='text-align:center;'><span>Sample</span></th> | |
− | + | <th style='text-align:center;'><span>Unmodified (°)</span></th> | |
− | + | <th style='text-align:center;'><span>HFB1-modified (°)</span></th> | |
− | + | </tr> | |
− | + | </thead> | |
− | + | <tbody> | |
− | + | <tr> | |
− | + | <td style='text-align:center;'><strong><span>Polypropylene (PP)</span></strong></td> | |
− | + | <td style='text-align:center;'><span>106.823 ± 1.888</span></td> | |
− | + | <td style='text-align:center;'><span>74.033 ± 1.195<sup>****</sup></span></td> | |
− | + | </tr> | |
− | + | <tr> | |
− | + | <td style='text-align:center;'><strong><span>Polyethylene (PE)</span></strong></td> | |
− | + | <td style='text-align:center;'><span>72.436 ± 1.600</span></td> | |
− | + | <td style='text-align:center;'><span>51.642 ± 3.417<sup>***</sup></span></td> | |
− | + | </tr> | |
− | + | </tbody> | |
− | + | </table> | |
− | + | <p> Significant difference analysis: ***P < 0.001, ****P < 0.0001.</p> | |
− | + | </figure> | |
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</div> | </div> | ||
+ | |||
+ | <img src="https://static.igem.org/mediawiki/2021/4/41/T--CPU_CHINA--Engineering_success_fig_7.jpg" | ||
+ | referrerpolicy="no-referrer"></p> | ||
+ | <p class="imgdescribe"><strong><span>Fig. 7 Contact angle of two types of | ||
+ | liquid.</span></strong><em class="reference"><span>A: Comparison between | ||
+ | buffer and HFB1-buffer mixture after shaking on a PP surface. B: Contact angle on a PP surface | ||
+ | . C: a | ||
+ | HFB1-modified PP surface. D: Contact angle on a PE surface. E: a HFB1-modified PE surface | ||
+ | .</span></em></p> | ||
+ | <video controls style="width: 100%;"> | ||
+ | <source src="https://static.igem.org/mediawiki/2021/c/c1/T--CPU_CHINA--Engineering_success_video_1.mp4" | ||
+ | type="video/mp4"> | ||
+ | </video> | ||
+ | <p class="imgdescribe"><strong><span>Video 1. Comparison of fluid movement status</span></strong></p> | ||
+ | |||
+ | <h3 class="mume-header" id="3-his-tag-spytag-mnp-bba_k3853008"><span>3. his-tag-SpyTag-MnP(<a | ||
+ | href="http://parts.igem.org/Part:BBa_K3853008">BBa_K3853008</a>)</span></h3> | ||
+ | |||
+ | <p>Manganese peroxidase (MnP) is a highly glycosylated, heme-containing<sup>[3]</sup> lignin peroxidase produced by | ||
+ | the white-rot fungus <em>Phanerochaete chrysosporium</em>. Introduce SpyTag at its N-terminus for the assembly of | ||
+ | multi-enzyme complexes.</p> | ||
+ | <h4><strong>3-1. Agarose Gel Electrophoresis</strong></h4> | ||
+ | <p><strong>Method: </strong> The synthetic pPIC9K plasmid was linearized and electrotransformed into <em>Pichia | ||
+ | pastoris</em> strain GS115, and the monoclonal colonies were screened for colony PCR and sequencing | ||
+ | verification.</p> | ||
+ | <p><strong>Result:</strong> The target gene appears in the normal position, and the result is shown in <strong>Fig. | ||
+ | 8</strong>. Sequencing results (</span><strong><a | ||
+ | href="https://static.igem.org/mediawiki/2021/8/89/T--CPU_CHINA--MnP.zip">File | ||
+ | 3</a></strong><span>) showed successful transformation.</p> | ||
+ | <img src="https://static.igem.org/mediawiki/2021/0/0b/T--CPU_CHINA--BBa_K3853008_fig_2.jpg" | ||
+ | alt="T--CPU_CHINA--BBa_K3853008_fig_2" style="zoom:30%;"> | ||
+ | <p class="imgdescribe"><strong>Fig. 8 Gel electrophoresis result of colony PCR to detect the insertion of BBa_K3853008 into <em>Pichia | ||
+ | pastoris</em> strain GS115.</strong> <em class="reference">Control refers to the wild-type Pichia pastoris strain GS115 without | ||
+ | electrotransformation.</em></p> | ||
+ | <h4><strong>3-2. SDS-PAGE</strong></h4> | ||
+ | <p><strong>Method: </strong>We set a series of concentration gradients of ammonium sulfate solution to | ||
+ | determine the best salting-out concentration to roughly remove the impurity and our target bands were observed | ||
+ | through SDS-PAGE.</p> | ||
+ | <p><strong>Result:</strong> Target bands can be seen at SDS-PAGE, which means SpyTag-MnP gene is expressed | ||
+ | successfully.</p> | ||
+ | <img src="https://static.igem.org/mediawiki/2021/7/76/T--CPU_CHINA--BBa_K3853008_fig_3.png" | ||
+ | alt="T--CPU_CHINA--BBa_K3853008_fig_3" style="zoom:30%;"> | ||
+ | <p class="imgdescribe"><strong>Fig. 9 SDS-PAGE analysis of SpyTag-MnP after ammonium sulfide salting out.</strong> <em class="reference">Lane 1: | ||
+ | SpyTag-MnP salting out with 40% (NH<sub>4</sub>)<sub>2</sub>SO<sub>4</sub> ; Lane 2: SpyTag-MnP salting out with 50% (NH<sub>4</sub>)<sub>2</sub>SO<sub>4</sub> ; Lane 3: | ||
+ | SpyTag-MnP salting out with 60% (NH<sub>4</sub>)<sub>2</sub>SO<sub>4</sub> ; Lane 4: SpyTag-MnP salting out with 70% (NH<sub>4</sub>)<sub>2</sub>SO<sub>4</sub> . Control refers | ||
+ | to the supernatant of wild-type Pichia pastoris strain GS115 without plasmid transfer. We finally chose 60% | ||
+ | ammonium sulfate as our salting out concentration.</em></p> | ||
+ | <h4><strong>3-3. qRT-PCR</strong></h4> | ||
+ | <p><strong>Method: </strong>For assaying the mRNA expression of SpyTag-MnP, qRT-PCRs were performed. We did | ||
+ | data analysis using a variation of the Livak method. To determine the relative expression of SpyTag-MnP vs. | ||
+ | reference gene <em>ACT1</em>, total RNA was prepared from an equal volume of yeast solution.</p> | ||
+ | <p><strong>Result:</strong> SpyTag-MnP reached its peak to a fold difference of 0.21 after 2% methanol inducing for | ||
+ | 72 h. The C<sub>T</sub> values for the SpyTag-MnP and the reference gene <em>ACT1</em> | ||
+ | were then used to calculate the fold difference with the following equation:</p> | ||
+ | <img src="https://static.igem.org/mediawiki/2021/9/91/T--CPU_CHINA--BBa_K3853008_fig_A.png" | ||
+ | alt="T--CPU_CHINA--BBa_K3853008_fig_A" style="width:60%;margin-left:20%;"> | ||
+ | <img src="https://static.igem.org/mediawiki/2021/4/44/T--CPU_CHINA--BBa_K3853008_fig_4.png" | ||
+ | alt="T--CPU_CHINA--BBa_K3853008_fig_4"> | ||
+ | <p class="imgdescribe"><strong>Fig. 10 qPCR results of SpyTag-MnP using the relative quantitative method</strong>.</p> | ||
+ | <h4><strong>3-4. Enzyme Activity</strong></h4> | ||
+ | <p><strong>Method: </strong>MnP activity of SpyTag-MnP was measured by monitoring the oxidation of | ||
+ | 2,6-dimethyloxyphenol (2,6-DMP) at 469 nm<sup>[5]</sup>. The reaction mixtures contained 0.4 mM MnSO<sub>4</sub>, | ||
+ | 50 mM sodium malonate (pH 4.5), and 1 mM 2, 6-DMP. For a 96-well plate, 140 μl of the above reaction | ||
+ | mixtures and 20 μl enzyme solution were mixed uniformly in advance and then 40 μl 0.1 mM | ||
+ | H<sub>2</sub>O<sub>2</sub> were added to initiate reaction. The concentration of 2, 6-DMP's oxidation | ||
+ | products, 2, 2', 6, 6'-tetramethoxydibenzo-1, 1'-diquinone, were determined using ε469 = 49.6 | ||
+ | mM-1 cm-1. One unit (U) of MnP activity is defined as the amount of enzyme required to convert 1 μM 2, 6-DMP | ||
+ | to 2, 2', 6, 6'-tetramethoxydibenzo-1, 1'-diquinone in 1 minute.</p> | ||
+ | <p><strong>Result:</strong> As shown in <strong>Fig. 11A</strong>, the absorbance of the reaction system with | ||
+ | SpyTag-MnP continued to rise within 1 min, while the absorbance of the control group (without enzyme) did not | ||
+ | change. Through UV-visible spectrum of the reaction system after 1 min, the characteristic absorption at 469 nm | ||
+ | was observed (<strong>Fig. 11B</strong>). Besides, SpyTag-MnP showed the same characteristic as MnP and the enzyme | ||
+ | activity of the former exhibited a slightly higher profile than the latter, which means that SpyTag-MnP | ||
+ | <strong>retained the functions of the original MnP and had the potential to surpass the latter (Fig. 11)</strong>. | ||
+ | </p> | ||
+ | <img src="https://static.igem.org/mediawiki/2021/5/53/T--CPU_CHINA--BBa_K3853008_fig_5.png" | ||
+ | alt="T--CPU_CHINA--BBa_K3853008_fig_5" style="zoom:24%;"> | ||
+ | <p class="imgdescribe"><strong>Fig. 11 The detection of 2, 2', 6, 6'-tetramethoxydibenzo-1, 1'-diquinone. Control group | ||
+ | refers to the reaction system without enzyme</strong>. A: The absorbance change at 469 nm in the reaction system | ||
+ | within 1 min. B: UV-visible spectrum of the reaction system after 1 min.</p> | ||
+ | <h4><strong>3-5. the conjugation of SpyTag-MnP to dCas9-SpyCatcher</strong></h4> | ||
+ | <p><strong>Method: </strong>For assembling the dCas9-SpyCather/SpyTag-MnP complex, SpyTag-MnP was mixed with | ||
+ | dCas9-SpyCather in a ratio of 1 : 1 and allowed to conjugate for 1 h at 37℃<sup>[6]</sup>.</p> | ||
+ | <p><strong>Result:</strong> As shown in <strong>Fig. 12</strong>, <strong>the band of the complex appeared</strong>, | ||
+ | which was higher than that of dCas9-SpyCather and the original SpyTag-MnP band had disappeared. Then we compared | ||
+ | the difference in MnP activity between SpyTag-MnP and the complex, and, as shown in <strong>Fig. 13</strong>, | ||
+ | there was no significant change. This result suggested that the assembly of SpyTag-MnP and dCas9-SpyCather will | ||
+ | <strong>not affect the enzyme activity.</strong></p> | ||
+ | <img src="https://static.igem.org/mediawiki/2021/7/75/T--CPU_CHINA--BBa_K3853008_fig_12.png" | ||
+ | alt="T--CPU_CHINA--BBa_K3853008_fig_12"> | ||
+ | <p class="imgdescribe"><strong>Fig. 12 SDS-PAGE showing the conjugation of SpyTag-MnP to dCas9-SpyCatcher</strong>. <em class="reference">Lane 1: | ||
+ | SpyTag-MnP (0.3 μM); Lane 2: dCas9-SpyCatcher (0.3 μM); Lane 3: SpyTag-MnP (0.3 μM) mixed with | ||
+ | dCas9-SpyCatcher (0.3 μM).</em></p> | ||
+ | <p>Upon mixing the two components, the upward shift in the band corresponding to dCas9-SpyCatcher as well as the | ||
+ | disappearance of the band corresponding to SpyTag-MnP were observed, indicating successful conjugation. Note that | ||
+ | the conjugation is unaffected by the SDS-PAGE conditions due to covalent isopeptide bond formation.</p> | ||
+ | <img src="https://static.igem.org/mediawiki/2021/2/24/T--CPU_CHINA--BBa_K3853008_fig_13.png" | ||
+ | alt="T--CPU_CHINA--BBa_K3853008_fig_13" style="zoom:10%;width:60%;margin-left:15%"> | ||
+ | <p class="imgdescribe"><strong>Fig. 13 Comparison of MnP activity between SpyTag-MnP and dCas9-SpyCather/SpyTag-MnP complex</strong>. | ||
+ | <em class="reference">Complex refers to the dCas9-SpyCather/SpyTag-MnP complex. p > 0.05.</em></p> | ||
+ | <h4 class="mume-header" id="references">References</h4> | ||
+ | |||
+ | <p class="reference">[1] Reddington, S. C. & Howarth, M. Secrets of a covalent interaction for biomaterials and biotechnology: | ||
+ | SpyTag and SpyCatcher. <em>Current opinion in chemical biology</em> <strong>29</strong>, 94-99, | ||
+ | doi:10.1016/j.cbpa.2015.10.002 (2015).</p> | ||
+ | <p class="reference">[2] Jankowski, N., Koschorreck, K. & Urlacher, V. B. High-level expression of aryl-alcohol oxidase 2 from | ||
+ | Pleurotus eryngii in Pichia pastoris for production of fragrances and bioactive precursors. <em>Applied | ||
+ | microbiology and biotechnology</em> <strong>104</strong>, 9205-9218, doi:10.1007/s00253-020-10878-4 (2020)</p> | ||
+ | <p class="reference">[3] Martínez, A. T. et al. Oxidoreductases on their way to industrial biotransformations. <em>Biotechnol | ||
+ | Adv</em> <strong>35</strong>, 815-831, doi:10.1016/j.biotechadv.2017.06.003 (2017).</p> | ||
+ | <p class="reference">[4] Sáez-Jiménez, V. et al. Demonstration of Lignin-to-Peroxidase Direct Electron Transfer: A | ||
+ | TRANSIENT-STATE KINETICS, DIRECTED MUTAGENESIS, EPR, AND NMR STUDY. <em>J Biol Chem</em> <strong>290</strong>, | ||
+ | 23201-23213, doi:10.1074/jbc.M115.665919 (2015).</p> | ||
+ | <p class="reference">[5] Wariishi, H., Valli, K. & Gold, M. H. Manganese(II) oxidation by manganese peroxidase from the | ||
+ | basidiomycete Phanerochaete chrysosporium. Kinetic mechanism and role of chelators. <em>The Journal of biological | ||
+ | chemistry</em> <strong>267</strong>, 23688-23695 (1992).</p> | ||
+ | <p class="reference">[6] Lim, S., Kim, J., Kim, Y., Xu, D. & Clark, D. S. CRISPR/Cas-directed programmable assembly of | ||
+ | multi-enzyme complexes. <em>Chem Commun (Camb)</em> <strong>56</strong>, 4950-4953, doi:10.1039/d0cc01174f (2020). | ||
+ | </p> | ||
</div> | </div> | ||
+ | </div> | ||
</div> | </div> | ||
<footer> | <footer> | ||
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<div id="connect"> | <div id="connect"> | ||
− | < | + | <h3>Contact us</h3> |
<ul id="contact"> | <ul id="contact"> | ||
<li> | <li> |
Latest revision as of 12:31, 23 November 2021
![](https://static.igem.org/mediawiki/2021/c/c4/T--CPU_CHINA--loading.gif)
![](https://static.igem.org/mediawiki/2021/c/cf/T--CPU_CHINA--Parts--background.png)
ENGINEERING SUCCESS
![](https://static.igem.org/mediawiki/2021/3/38/T--CPU_CHINA--flying.png)
OVERVIEW
In the project of CPU_CHINA 2021, We designed a multi-enzyme complex to degrade plastics and used synthetic biology tools to produce the expected results. 30 new BioBricks were designed for our project (See the Parts and Design details in Parts and Design.)
We have done a lot of amazing work to make sure they can realize the goals we set. Moreover, it is worth mentioning that BBa_K3853011, BBa_K3853010 and BBa_K3853008 are the representatives among them. These three BioBricks work well to perform their functions and occupations as expected. All the related data are recorded below. We hope they will make some contributions to the iGEM community.
ENGINEERING SUCCESS
1. dCas9-SpyCatcher (BBa_K3853011)
In our 2021 project, we fused deactivated CRISPR-associated protein 9 (dCas9) with SpyCatcher, turning it into a biological module that could fuse with various "standardized elements" link to SpyTag. T7 promoter was used to construct the express circuit, and the dCas9-SpyCatcher protein was expressed and purified to test its function.
1-1. Agarose Gel Electrophoresis
Method: We used PCR to obtain three homologous recombination fragments (dCas9; vector of pET-28a; G3S*4-SpyCatcher) for the construction of expression plasmid. The successfully recombined plasmid was transformed into E.coli BL21(DE3), and the monoclonal colonies with positive transformation results were selected for subsequent sequencing verification.
Result: The three fragments used for homologous recombination were successfully obtained by PCR, and the result was shown in Fig. 1. The sequencing results of the E.coli BL21(DE3) monoclonal colonies (File 1) showed a successful transformation.
![](https://static.igem.org/mediawiki/2021/d/dd/T--CPU_CHINA--Engineering_success_fig_1.jpg)
Fig. 1 Agarose gel electrophoresis of PCR products. Vector refers to plasmid pET-28a. G3S*4 refers to quadruple Ser/Gly link.
1-2. SDS-PAGE
Method: We used Ni-NTA affinity column to purify dCas9-SpyCatcher.
Result: Target bands could be observed at the position of about 176.2 kDa (Fig. 2), which means the protein of dCas9-SpyCatcher could be successfully expressed, and the related gene worked well.
![](https://static.igem.org/mediawiki/2021/5/5e/T--CPU_CHINA--Engineering_success_fig_2.png)
Fig. 2 SDS-PAGE of purified products of dCas9-SpyCatcher. 50 mM imidazole and 500 mM imidazole represent corresponding eluates with different imidazole concentrations, and the binding buffer is generated by equilibrating the Ni-NTA affinity column after elution with 500 mM imidazole.
1-3. Western Blot
Method: Since we had introduced 6×his-tag at the N-terminus of dCas9-SpyCatcher, we used his-antibody as the primary antibody to perform Western Blot analysis to detect the expression status of the target protein.
Result: The protein band of dCas9-SpyCatcher could be observed clearly (Fig. 3), meaning that the related gene functioned well.
![](https://static.igem.org/mediawiki/2021/c/c6/T--CPU_CHINA--Engineering_success_fig_3.jpg)
Fig. 3 Western Blot of dCas9-SpyCatcher. The concentration of dCas9-SpyCatcher: lane 1: 0.25 mg/ml; lane 2: 0.5 mg/ml; lane 3: 1 mg/ml.
1-4. SpyCatcher/SpyTag system combination
Method: We incubated SpyTag-MnP and dCas9-SpyCatcher together to verify the combination of SpyCatcher and SpyTag[1]. SpyTag-MnP was mixed with dCas9-SpyCatcher in a ratio of 1 : 1, and they were allowed to conjugate for 1 h at 37℃. Followed by SDS-PAGE verification.
Result: As shown in Fig. 4. The band of the conjugated dCas9-MnP complex appeared at lane 3, with a slightly higher position than dCas9-SpyCatcher; Also, the original SpyTag-MnP band at lane 3 disappeared , meaning the two proteins had successfully conjugated.
![](https://static.igem.org/mediawiki/2021/7/75/T--CPU_CHINA--BBa_K3853008_fig_12.png)
Fig. 4 SDS-PAGE of the combination of SpyTag-MnP and dCas9-SpyCatcher. Lane 1: SpyTag-MnP; Lane 2: dCas9-SpyCatcher; Lane 3: SpyTag-MnP mixed with dCas9-SpyCatcher.
2. His-tag-SpyTag-HFB1 (BBa_K3853010)
Hydrophobin-1 (HFB1) is a kind of class Ⅱ HFBs derived from Trichoderma reesei, which can exert surface activity at the hydrophilic-hydrophobic interface. We introduced SpyTag at its N-terminus for the assembly of multi-enzyme complexes.
2-1. Agarose Gel Electrophoresis
Method: The plasmid carrying the gene of SpyTag-HFB1 was transformed into E.coli Rosetta(DE3) for heterogenous expression. Colony PCR was applied to screen monoclonal colonies that had positive transformation results for subsequent gene sequencing verification.
Result: The bands of target gene appeared at the normal position, the result was shown in Fig. 5. Sequencing verification results (File 2) showed a successful transformation.
![](https://static.igem.org/mediawiki/2021/f/ff/T--CPU_CHINA--Engineering_success_fig_5.jpg)
Fig. 5 Agarose gel electrophoresis of PCR products of monoclonal colonies of SpyTag-HFB1.
2-2. SDS-PAGE
Method: We used Ni-NTA affinity column to obtain purified SpyTag-HFB1.
Result: Target bands could be observed at the position of about 15 kDa (Fig. 6), which means the protein of SpyTag-HFB1 was successfully expressed, and the related gene worked well.
![](https://static.igem.org/mediawiki/2021/f/f2/T--CPU_CHINA--Engineering_success_fig_6.png)
Fig. 6 SDS-PAGE of purification products of dCas9-SpyCatcher. flow-through is the liquid flowing out of the column when loading the sample, 50 mM imidazole and 500 mM imidazation are eluates of different imidazole concentrations.
2-3. Functional Verification
Method: We dropped the liquid containing SpyTag-HFB1 and the liquid without SpyTag-HFB1 on a plastic dish, and observed the contact angle, shape and motion state of different liquids to evaluate the effect of HFB1.
Result: The results were shown in Table 1, Fig. 7, Video 1. The contact angle of the liquid containing SpyTag-HFB1 was smaller, and the droplets were dispersed. Compared to SpyTag-HFB1-free liquid, the droplets containing SpyTag-HFB1 were not easy to move on the plastic surface, which showed that HFB1 effectively improved the hydrophilicity of the plastic surface.
Table 1. Contact angle measurement among different samples.
Sample | Unmodified (°) | HFB1-modified (°) |
---|---|---|
Polypropylene (PP) | 106.823 ± 1.888 | 74.033 ± 1.195**** |
Polyethylene (PE) | 72.436 ± 1.600 | 51.642 ± 3.417*** |
Significant difference analysis: ***P < 0.001, ****P < 0.0001.
![](https://static.igem.org/mediawiki/2021/4/41/T--CPU_CHINA--Engineering_success_fig_7.jpg)
Fig. 7 Contact angle of two types of liquid.A: Comparison between buffer and HFB1-buffer mixture after shaking on a PP surface. B: Contact angle on a PP surface . C: a HFB1-modified PP surface. D: Contact angle on a PE surface. E: a HFB1-modified PE surface .
Video 1. Comparison of fluid movement status
3. his-tag-SpyTag-MnP(BBa_K3853008)
Manganese peroxidase (MnP) is a highly glycosylated, heme-containing[3] lignin peroxidase produced by the white-rot fungus Phanerochaete chrysosporium. Introduce SpyTag at its N-terminus for the assembly of multi-enzyme complexes.
3-1. Agarose Gel Electrophoresis
Method: The synthetic pPIC9K plasmid was linearized and electrotransformed into Pichia pastoris strain GS115, and the monoclonal colonies were screened for colony PCR and sequencing verification.
Result: The target gene appears in the normal position, and the result is shown in Fig. 8. Sequencing results (File 3) showed successful transformation.
![T--CPU_CHINA--BBa_K3853008_fig_2](https://static.igem.org/mediawiki/2021/0/0b/T--CPU_CHINA--BBa_K3853008_fig_2.jpg)
Fig. 8 Gel electrophoresis result of colony PCR to detect the insertion of BBa_K3853008 into Pichia pastoris strain GS115. Control refers to the wild-type Pichia pastoris strain GS115 without electrotransformation.
3-2. SDS-PAGE
Method: We set a series of concentration gradients of ammonium sulfate solution to determine the best salting-out concentration to roughly remove the impurity and our target bands were observed through SDS-PAGE.
Result: Target bands can be seen at SDS-PAGE, which means SpyTag-MnP gene is expressed successfully.
![T--CPU_CHINA--BBa_K3853008_fig_3](https://static.igem.org/mediawiki/2021/7/76/T--CPU_CHINA--BBa_K3853008_fig_3.png)
Fig. 9 SDS-PAGE analysis of SpyTag-MnP after ammonium sulfide salting out. Lane 1: SpyTag-MnP salting out with 40% (NH4)2SO4 ; Lane 2: SpyTag-MnP salting out with 50% (NH4)2SO4 ; Lane 3: SpyTag-MnP salting out with 60% (NH4)2SO4 ; Lane 4: SpyTag-MnP salting out with 70% (NH4)2SO4 . Control refers to the supernatant of wild-type Pichia pastoris strain GS115 without plasmid transfer. We finally chose 60% ammonium sulfate as our salting out concentration.
3-3. qRT-PCR
Method: For assaying the mRNA expression of SpyTag-MnP, qRT-PCRs were performed. We did data analysis using a variation of the Livak method. To determine the relative expression of SpyTag-MnP vs. reference gene ACT1, total RNA was prepared from an equal volume of yeast solution.
Result: SpyTag-MnP reached its peak to a fold difference of 0.21 after 2% methanol inducing for 72 h. The CT values for the SpyTag-MnP and the reference gene ACT1 were then used to calculate the fold difference with the following equation:
![T--CPU_CHINA--BBa_K3853008_fig_A](https://static.igem.org/mediawiki/2021/9/91/T--CPU_CHINA--BBa_K3853008_fig_A.png)
![T--CPU_CHINA--BBa_K3853008_fig_4](https://static.igem.org/mediawiki/2021/4/44/T--CPU_CHINA--BBa_K3853008_fig_4.png)
Fig. 10 qPCR results of SpyTag-MnP using the relative quantitative method.
3-4. Enzyme Activity
Method: MnP activity of SpyTag-MnP was measured by monitoring the oxidation of 2,6-dimethyloxyphenol (2,6-DMP) at 469 nm[5]. The reaction mixtures contained 0.4 mM MnSO4, 50 mM sodium malonate (pH 4.5), and 1 mM 2, 6-DMP. For a 96-well plate, 140 μl of the above reaction mixtures and 20 μl enzyme solution were mixed uniformly in advance and then 40 μl 0.1 mM H2O2 were added to initiate reaction. The concentration of 2, 6-DMP's oxidation products, 2, 2', 6, 6'-tetramethoxydibenzo-1, 1'-diquinone, were determined using ε469 = 49.6 mM-1 cm-1. One unit (U) of MnP activity is defined as the amount of enzyme required to convert 1 μM 2, 6-DMP to 2, 2', 6, 6'-tetramethoxydibenzo-1, 1'-diquinone in 1 minute.
Result: As shown in Fig. 11A, the absorbance of the reaction system with SpyTag-MnP continued to rise within 1 min, while the absorbance of the control group (without enzyme) did not change. Through UV-visible spectrum of the reaction system after 1 min, the characteristic absorption at 469 nm was observed (Fig. 11B). Besides, SpyTag-MnP showed the same characteristic as MnP and the enzyme activity of the former exhibited a slightly higher profile than the latter, which means that SpyTag-MnP retained the functions of the original MnP and had the potential to surpass the latter (Fig. 11).
![T--CPU_CHINA--BBa_K3853008_fig_5](https://static.igem.org/mediawiki/2021/5/53/T--CPU_CHINA--BBa_K3853008_fig_5.png)
Fig. 11 The detection of 2, 2', 6, 6'-tetramethoxydibenzo-1, 1'-diquinone. Control group refers to the reaction system without enzyme. A: The absorbance change at 469 nm in the reaction system within 1 min. B: UV-visible spectrum of the reaction system after 1 min.
3-5. the conjugation of SpyTag-MnP to dCas9-SpyCatcher
Method: For assembling the dCas9-SpyCather/SpyTag-MnP complex, SpyTag-MnP was mixed with dCas9-SpyCather in a ratio of 1 : 1 and allowed to conjugate for 1 h at 37℃[6].
Result: As shown in Fig. 12, the band of the complex appeared, which was higher than that of dCas9-SpyCather and the original SpyTag-MnP band had disappeared. Then we compared the difference in MnP activity between SpyTag-MnP and the complex, and, as shown in Fig. 13, there was no significant change. This result suggested that the assembly of SpyTag-MnP and dCas9-SpyCather will not affect the enzyme activity.
![T--CPU_CHINA--BBa_K3853008_fig_12](https://static.igem.org/mediawiki/2021/7/75/T--CPU_CHINA--BBa_K3853008_fig_12.png)
Fig. 12 SDS-PAGE showing the conjugation of SpyTag-MnP to dCas9-SpyCatcher. Lane 1: SpyTag-MnP (0.3 μM); Lane 2: dCas9-SpyCatcher (0.3 μM); Lane 3: SpyTag-MnP (0.3 μM) mixed with dCas9-SpyCatcher (0.3 μM).
Upon mixing the two components, the upward shift in the band corresponding to dCas9-SpyCatcher as well as the disappearance of the band corresponding to SpyTag-MnP were observed, indicating successful conjugation. Note that the conjugation is unaffected by the SDS-PAGE conditions due to covalent isopeptide bond formation.
![T--CPU_CHINA--BBa_K3853008_fig_13](https://static.igem.org/mediawiki/2021/2/24/T--CPU_CHINA--BBa_K3853008_fig_13.png)
Fig. 13 Comparison of MnP activity between SpyTag-MnP and dCas9-SpyCather/SpyTag-MnP complex. Complex refers to the dCas9-SpyCather/SpyTag-MnP complex. p > 0.05.
References
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