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| | <b>Ⅰ.Constraction of expression plasmid in CFPS(cell-free protein synthesis)</b><br/> | | <b>Ⅰ.Constraction of expression plasmid in CFPS(cell-free protein synthesis)</b><br/> |
| − | The mutations of PETase gene had been contracted by over-lap PCR and Restriction Enzyme cutting sites(SacⅠ&EcoRⅠ) had been added to it’s ends (5’&3’) separately. | + | The mutants of PETase gene had been contracted by over-lap PCR and Restriction Enzyme cutting sites(SacⅠ&EcoRⅠ) had been added to it’s ends (5’&3’) separately. |
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| | <div class="note-content2"> | | <div class="note-content2"> |
| − | <li>We linked the remained cut <b><i>CpxR-RFP</i></b> fragment into the skeleton and then transformed the recombinant <b><i>pUC57</i></b> and the <b><i>pET21a</i></b> into<b><i> E.coli</i></b> at the same time.</li> | + | <p><li> XbaⅠ&SacⅠdouble restriction endonuclease digestion in <i>CFP</i> gene.</li><br /> |
| | + | <li>XbaⅠ&EcoRⅠdouble restriction endonuclease digestion in pRset_CFP-1<br/> |
| | + | Bands as expected(760bp&3000bp) in agarose gel , gel purification of the digested products(3000bp). |
| | + | </li><br /> |
| | + | <li>Ligaion of digested pRset_CFP-1, digested <i>CFP</i> gene and digested <i>PETase(M154L)</i> gene in accordance with the 1:5:5 molecular ratio. The newly constructed plasmid is called pRset_CFP-1-M154L.<br/> |
| | + | Transformation of E.coli (Trans-T1 Phage Resistant Chemically Competent Cell) with pRset_CFP-1-M154L(with AmpR) and plating on solid LB culture medium with Amp.<br/> |
| | + | Verification : Colony PCR with fast taq Polymerase, primers: PETase-S/PETase -A, template: Colony of E.coli with plasmid pRset_CFP-1-M154L. Bands as expected(about 760bp) in agarose gel. |
| | + | </li><br /> |
| | | | |
| − | <li>The transformation last night turned to be a failure. We tried it again.</li> | + | <img class="aligncenter size-full wp-image-4256" src="https://static.igem.org/mediawiki/2016/5/55/T--Tianjin--cell-free_note-1.png" alt="T--Tianjin--cell-free_note-1" width="800" height="533"> |
| − | <li>The transformation last day seemed to be successful for the colonies were visible in LB+Amp plate. However, we use PCR to verify and it turned out that the fragment had not been linked into the plasmid.</li>
| + | |
| − | <li>We finally gave up the former design and decided to link the<b><i> PETase</i></b> gene into the plasmid<b><i> pUC19</i></b>. However, we did not have the key enzyme <b><i>Sal1</i></b> so we started to construct the TPA positive feedback system.</li>
| + | <li>Cultivation the E.coli cells transformed yesterday in several 5mL liquid LB culture mediums with Amp at 37℃ for 12 hours.</li><br/> |
| − | <li>We first prepared the TPA standard solution (5g/L) for further use. Then we use PCR to amplify the <b><i>TPA-sensing leader sequence</i></b>,<b><i> PGK1 promoter</i></b>, <b><i>CYC1 terminator</i></b>, <b><i>RFP gene</i></b>, TPA regulation protein gene (<b><i>tpaR</i></b>), TPA transporting protein gene (<b><i>tpaK</i></b>). Then we cut the fragments above and plasmid <b><i>pRS413</i></b>, <b><i>pRS415</i></b>, and <b><i>pYES2</i></b> with corresponding enzymes and recycled the fragments from agarose gel.</li>
| + | <li>Plasmid isolation of pRset_CFP-1-M154L.</li><br/> |
| − | <li>We linked the fragments together by this way:<br/>
| + | |
| − | <b><i>1. pYES2-leader-PGK1-RFP.<br/>
| + | </p> |
| − | 2. pRS413-PGK1-tpaK-CYC1.<br/>
| + | |
| − | 3. pRS415-PGK1-tpaR-CYC1</i></b>
| + | |
| − | </li>
| + | |
| − | <li>Then we used PCR to verify the success and all of the plasmids were correctly constructed. Then we transformed the there plasmids into <b><i>Saccharomyces cerevisiae</i></b>. | + | |
| − | </li> | + | |
| − | <li>The key enzyme <b><i>Sal1</i></b> arrived and we isolate the plasmid <b><i>pET21a</i></b>. Then we use <b><i>BamH1</i></b> and <b><i>Sal1</i></b> to cut both plasmid and <b><i>PETase</i></b> gene, then linked them together and transformed the recombinant plasmid into <b><i>E.coli</i></b>.</li>
| + | |
| | </div> | | </div> |
| | <a class="expand-btn2">Show More</a> | | <a class="expand-btn2">Show More</a> |
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| − | <li>We cultured the transformed<b><i> E.coli</i></b> and isolated the plasmid. Then we use PCR to amplify the whole fragment in <b><i>pET21a</i></b> from <b><i>T7 promoter</i></b> to <b><i>T7 terminator</i></b>. Then we recycled this fragment from agarose gel.</li> | + | <p><li>Our strategy is to exchange the site-directed mutant PETase-M154L for the other 21 mutants and 1 wild type, separately.<br/> |
| − | <li>The transformed <b><i>Saccharomyces cerevisiae</i></b> had grown to visible colony in Sc-Ura-Leu-His plate. Then we use colony PCR to verify the plasmids had been transformed into the cells. The result is successful so that we streaked more plates.</li>
| + | XbaⅠ&EcoRⅠdouble restriction endonuclease digestion in pRset_CFP-1-M154L<br/> |
| − | <li>We cut the <b><i>T7 promoter-PETase gene-T7 terminator</i></b> fragment with enzymes <b><i>EcoR1</i></b> and <b><i>Sac1</i></b>. Then we linked it to the already cut plasmid <b><i>pUC19</i></b> (cut in August 28th). Then we transformed the recombinant plasmid into<b><i> E.coli</i></b>.</li>
| + | No expected bands(3600bp&750bp) in agarose gel. We don’t find the reason why the plasmid constuced can’t be cut into the two expected bands, so we decided to change the initial strategy. |
| − | <li>We cultured the transformed <b><i>Saccharomyces cerevisiae</i></b> into Sc-Ura-Leu-His culture medium in 30℃. We added TPA standard solution in this way:<br/> | + | </li><br/> |
| − | 1. Group 1: did not add TPA.<br/>
| + | <li>Ligaion of digested pRset_CFP-1, digested <i>CFP</i> gene and the rest 22 digested gene interested (including 21 PETase site-directed mutants geneand 1 wild type ) separately in accordance with the 1:5:5 molecular ratio. The newly constructed plasmid is called pRset_CFP-1-R90I (R90T, R90A, S92A, Q119A, M154A, W159H, W159M, N205L, N205V, S207T, S207A, I208V, S238F, S238V,A240P, S242I.)</li><br/> |
| − | 2. Group 2: added 1000μL TPA standard solution.<br/>
| + | <li>Transformation</li> |
| − | 3. Group 3: added 100μL TPA standard solution.<br/>
| + | <li> Plasmid isolation</li> |
| − | 4. Group 4: added 10μL TPA standard solution.<br/>
| + | <li> Verification</li> |
| − | 5.Group 5: added 1μL TPA standard solution. | + | </p> |
| − | </li>
| + | |
| − | <li>We cultured the transformed <b><i>E.coli</i></b> into LB+Amp culture medium. Then add 1.5μL IPTG to induce the expression of <b><i>PETase</i></b> gene.</li>
| + | |
| − | <li>We first detected the red fluorescence of <b><i>E.coli</i></b>, however, the experiment group had almost no increase of red fluorescence relative to control group. We changed the induction wavelength and scan the whole wavelength of emission, but we did not receive any result we expected.</li>
| + | |
| − | <li>The TPA positive feedback system seemed to have minor effection for there were a little increment of red fluorescence of the 5th group relative to the 1st one.</li>
| + | |
| − | <li>We doubted that it might be the<b><i> RFP</i></b> in the kit was useless. We isolated the <b><i>pET21a</i></b> and used PCR to amplify the <b><i>RFP </i></b>gene.</li> | + | |
| − | <li>We cut the <b><i>RFP</i></b> gene and <b><i>pET21a</i></b> with enzymes <b><i>Xba1</i></b> and <b><i>Sac1</i></b>, then we linked them and transformed it into <b><i>E.coli</i></b>.</li> | + | |
| − | <li>We cultured the transformed <b><i>E.coli</i></b> and added IPTG to induce the expression of<b><i> RFP</i></b>, and this time the red fluorescence was clear enough that could be seen directly.</li> | + | |
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| | </div> | | </div> |
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| | <div class="note-content4"> | | <div class="note-content4"> |
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| − | <li>We started to construct another regulation way, the <b><i>E.coli</i></b> lysis regulation pathway. We first used colony PCR to obtain the <b><i>ddpX</i></b> gene from the <b><i>E.coli</i></b> genome and recycled the <b><i>ddpX</i></b> from the agarose gel.</li>
| + | <b>Ⅱ. Expression of the plasmids constracted before</b> |
| | | | |
| − | <li>We found that there was no enzyme cleavage site between the<b><i> CpxR</i></b> promoter and <b><i>RFP</i></b> gene in the part we use. We had to design the primers and amplified the <b><i>CpxR</i></b> promoter by PCR.</li> | + | <li>We choosed 4 tubes of plasmid to express in the cell-free system firstly.<br/> |
| − | <li>We used PCR to amplify the<b><i> CpxR</i></b> promoter. Then we recycled it from agarose gel.</li> | + | The protocol of the cell-free protein synthesis system(50 µL) we used :<br/> |
| − | <li>We cut the<b><i> CpxR</i></b> promoter with enzymes <b><i>Xba1</i></b> and<b><i> BamH1</i></b>, <b><i>ddpX</i></b> gene with enzymes <b><i>BamH1</i></b> and<b><i> EcoR1</i></b>, first batch of <b><i>pET21a</i></b> with<b><i> Xba1</i></b> and<b><i> EcoR1</i></b>, second batch of <b><i>pET21a</i></b> with<b><i> BamH1</i></b> and <b><i>EcoR1</i></b>.</li> | + | MQ H2O 7.9µL<br/> |
| − | <li>Then we linked these fragment in the following two ways:<br/> | + | Feeding buffer 25µL<br/> |
| − | <b><i>1. pET21a-CpxR-ddpX.<br/> | + | Mg2+ solution 1.1µL<br/> |
| − | 2. pET21a-ddpX.</i></b>
| + | Gene( plasmid as template) 1µL<br/> |
| − | </li> | + | Lysate 15µL<br/> |
| | + | (PS: The details of the system are not available.)<br/> |
| | + | </li> |
| | + | |
| | + | <b>Systems were expressed in 96-well plates and put the 96-well plates into the ELIASA (microplate reader) at 30℃ for 12 hours.</b><br/> |
| | + | <img class="aligncenter size-full wp-image-4256" src="https://static.igem.org/mediawiki/2016/3/3d/T--Tianjin--cell-free_note-2.png" alt="T--Tianjin--cell-free_note-2" width="800" height="533"> |
| | + | |
| | + | <li><b>Parallel experiments for expression in CFPS system</b></li><br/> |
| | + | |
| | + | |
| | + | <img class="col-md-4" src="https://static.igem.org/mediawiki/2016/9/97/T--Tianjin--cf-blank.png" alt="T--Tianjin--cf-blank" height="250px"> |
| | + | <img class="col-md-4" src="https://static.igem.org/mediawiki/2016/d/d0/T--Tianjin--cf-m1.png" alt="T--Tianjin--cf-m1" height="250px"> |
| | + | <img class="col-md-4" src="https://static.igem.org/mediawiki/2016/4/44/T--Tianjin--cf-m2.png" alt="T--Tianjin--cf-m2" height="250px"> |
| | + | <img class="col-md-4" src="https://static.igem.org/mediawiki/2016/3/34/T--Tianjin--cf-m3.png" alt="T--Tianjin--cf-m3" height="250px"> |
| | + | <img class="col-md-4" src="https://static.igem.org/mediawiki/2016/7/75/T--Tianjin--cf-m4.png" alt="T--Tianjin--cf-m4" height="250px"> |
| | + | <img class="col-md-4" src="https://static.igem.org/mediawiki/2016/b/be/T--Tianjin--cf-m5.png" alt="T--Tianjin--cf-m5" height="250px"> |
| | + | <img class="col-md-4" src="https://static.igem.org/mediawiki/2016/4/4e/T--Tianjin--cf-m6.png" alt="T--Tianjin--cf-m6" height="250px"> |
| | + | <img class="col-md-4" src="https://static.igem.org/mediawiki/2016/4/4e/T--Tianjin--cf-m7.png" alt="T--Tianjin--cf-m7" height="250px"> |
| | + | <img class="col-md-4" src="https://static.igem.org/mediawiki/2016/9/97/T--Tianjin--cf-m8.png" alt="T--Tianjin--cf-m8" height="250px"> |
| | + | <img class="col-md-4" src="https://static.igem.org/mediawiki/2016/d/d1/T--Tianjin--cf-m9.png" alt="T--Tianjin--cf-m9" height="250px"> |
| | + | <img class="col-md-4" src="https://static.igem.org/mediawiki/2016/6/61/T--Tianjin--cf-m10.png" alt="T--Tianjin--cf-m10" height="250px"> |
| | + | <img class="col-md-4" src="https://static.igem.org/mediawiki/2016/c/c9/T--Tianjin--cf-m11.png" alt="T--Tianjin--cf-m11" height="250px"> |
| | + | <img class="col-md-4" src="https://static.igem.org/mediawiki/2016/3/36/T--Tianjin--cf-m12.png" alt="T--Tianjin--cf-m12"height="250px"> |
| | + | <img class="col-md-4" src="https://static.igem.org/mediawiki/2016/d/d2/T--Tianjin--cf-m13.png" alt="T--Tianjin--cf-m13" height="250px"> |
| | + | <img class="col-md-4" src="https://static.igem.org/mediawiki/2016/3/35/T--Tianjin--cf-m14.png" alt="T--Tianjin--cf-m14" height="250px"> |
| | + | <img class="col-md-4" src="https://static.igem.org/mediawiki/2016/b/b3/T--Tianjin--cf-m15.png" alt="T--Tianjin--cf-m15" height="250px"> |
| | + | <img class="col-md-4" src="https://static.igem.org/mediawiki/2016/2/2c/T--Tianjin--cf-m16.png" alt="T--Tianjin--cf-m16" height="250px"> |
| | + | <img class="col-md-4" src="https://static.igem.org/mediawiki/2016/9/91/T--Tianjin--cf-m17.png" alt="T--Tianjin--cf-m17" height="250px"> |
| | + | <img class="col-md-4" src="https://static.igem.org/mediawiki/2016/3/3f/T--Tianjin--cf-m18.png" alt="T--Tianjin--cf-m18" height="250px"> |
| | + | <img class="col-md-4" src="https://static.igem.org/mediawiki/2016/a/ad/T--Tianjin--cf-m19.png" alt="T--Tianjin--cf-m19" height="250px"> |
| | + | <img class="col-md-4" src="https://static.igem.org/mediawiki/2016/0/03/T--Tianjin--cf-m20.png" alt="T--Tianjin--cf-m20" height="250px"> |
| | + | <img class="col-md-4" src="https://static.igem.org/mediawiki/2016/b/b9/T--Tianjin--cf-m21.png" alt="T--Tianjin--cf-m21" height="250px"> |
| | + | <img class="col-md-4" src="https://static.igem.org/mediawiki/2016/3/3d/T--Tianjin--cf-m22.png" alt="T--Tianjin--cf-m22" height="250px"> |
| | + | <img class="col-md-4" src="https://static.igem.org/mediawiki/2016/3/3e/T--Tianjin--cf-m-no.png" alt="T--Tianjin--cf-m-no"height="250px"> |
| | + | |
| | + | <br/> |
| | + | |
| | + | <p> |
| | + | <b>The figures above show that the plasmids can express in the CFPS system, although the results of the parallel experimennts indicate that the CFPS system is unstable.</b> |
| | + | </p> |
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| | <div class="note-content5"> | | <div class="note-content5"> |
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| − | <li>We used PCR to amplify the whole fragments in<b><i> pET21a</i></b> (from <b><i>CpxR </i></b>to <b><i>T7 terminator</i></b>). However, the band in the agarose gel was disperse so that we were unable to recycle it.</li>
| + | <b>Ⅲ.Detection of Enzyme Activity</b> |
| | + | <p> |
| | + | |
| | + | <li>Substrate: 1mM pNPA Acetonitrile solution.<br/> |
| | + | 10 times diluted Enzyme solution(unpurified) mixed with the equal volume substrate.<br/> |
| | + | This attempt of detection failed because our enzyme precipitated in acetonitrile. |
| | + | </li> |
| | | | |
| − | <li>We used colony PCR to verify if the <b><i>pET21a</i></b> had been correctly constructed, the result is yes.</li> | + | <li>Subsrtrate: 1mM pNPA Tris-HCl buffer.<br/> |
| − | <li>We changed the DNA polymerase and annealing temperature several times and redid the PCR, however, the disperse band were always existed.</li>
| + | 10 times diluted Enzyme solution(unpurified) mixed with the equal volume substrate.<br/> |
| − | <li>We cultured the <b><i>E.coli</i></b> transformed into the <b><i>pET21a-ddpX</i></b> fragment and detect the OD600 in order to verify the lysis effection of <b><i>ddpX</i></b>. </li>
| + | This attempt of detection failed, too. Because we found that Tris can react with pNPA , and pNPA would be transfered to pNP, too. |
| − | <li>Considering the <b><i>pYES2</i></b> is multicopy plasmid so that the copy number would affect the <b><i>RFP</i></b> expression level, we decided to change the <b><i>pYES2</i></b> to single-copy plasmid <b><i>pRS416</i></b>. Since the <b><i>pRS416</i></b> does not have terminator in its backbone, we used PCR to amplify the <b><i>CYC1</i></b> terminator from plasmid <b><i>pYES2</i></b>.</li>
| + | </li></p> |
| − | <li>We cut the <b><i>pYES2</i></b> with enzyme <b><i>Hind3</i></b> and <b><i>EcoR1</i></b>, <b><i>CYC1</i></b> with<b><i> EcoR1</i></b> and <b><i>Sal1</i></b>,<b><i> pRS416</i></b> with <b><i>Hind3</i></b> and<b><i> Sal1</i></b>. Then we linked the three part together.</li>
| + | |
| − | <li>We transformed the three plasmids into <b><i>Saccharomyces cerevisiae</i></b> together. </li>
| + | |
| − | <li>The new primers using to amplify the<b><i> CpxR-ddpX-T7</i></b> terminator fragment arrived and we redid the PCR. However, the disperse band was still existed. </li>
| + | |
| − | <li>The transformation of Saccharomyces cerevisiae turned out to be a failure because no colony was found on the Sc-Ura-Leu-His plate. </li>
| + | |
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| | <div class="note-content6"> | | <div class="note-content6"> |
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| − | <li>We redid the inclusion body reporting experiment, and this time we directly observed the color of bacterial after centrifugation (12000rpm, 1min). The group with <b><i>PETase</i></b> gene and <b><i>CpxR-RFP</i></b> fragment showed the deepest red.</li> | + | <p> |
| | + | <li>Substrate:0.2mM pNPA water solution.<br/> |
| | + | 10 times diluted Enzyme solution(unpurified) mixed with the equal volume substrate.<br/> |
| | + | After static reaction at 39℃ for 8 hours, we detected the characteristic adsorption peak of the product ,pNP, which has no other characteristic adsorption peak except in 400nm. |
| | + | </li> |
| | + | |
| | + | <img class="aligncenter size-full wp-image-4256" src="https://static.igem.org/mediawiki/2016/f/f4/T--Tianjin--cell-free_note-3.png" alt="T--Tianjin--cf-m-no" width="800" height="533"> |
| | + | |
| | + | <b>The control group in the image above indicate that something else in the CFPS system can cause characteristic adsorption peak except in 400nm with the sunstrate.</b><br/><br/><br/> |
| | + | |
| | + | <li>Substrate:PET.<br/> |
| | + | PET was been put into 20 times diluted Enzyme solution(unpurified).<br/> |
| | + | After static reaction at 39℃ for 5 days, we detected the characteristic adsorption peak of the product ,MHET, which has no other characteristic adsorption peak except in 260nm. Besides, we also deteected the product by Multispectral Scanning. |
| | + | </li></p> |
| | + | |
| | + | Please visit our Result part for more detailed result. |
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| − | </div> | + | </div><!-- |
| | <div class="side-nav"> | | <div class="side-nav"> |
| | <ul> | | <ul> |
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| | <li><a class="topLink" href="#Week7" style="color:#5555FF">Week7</a></li> | | <li><a class="topLink" href="#Week7" style="color:#5555FF">Week7</a></li> |
| | </ul> | | </ul> |
| − | </div> | + | </div> --> |
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The control group in the image above indicate that something else in the CFPS system can cause characteristic adsorption peak except in 400nm with the sunstrate.