Oscarliu117 (Talk | contribs) |
Oscarliu117 (Talk | contribs) |
||
Line 15: | Line 15: | ||
<div class="col-2 side"> | <div class="col-2 side"> | ||
<div id="sidelist" class="list-Fgroup"> | <div id="sidelist" class="list-Fgroup"> | ||
− | <a class="list-group-item list-group-item-action" href="#list-item-1"> | + | <a class="list-group-item list-group-item-action" href="#list-item-1">Construction of Pasr and |
− | <a class="list-group-item list-group-item-action" href="#list-item-2"> | + | Pgada</a> |
− | <a class="list-group-item list-group-item-action" href="#list-item-3"> | + | <a class="list-group-item list-group-item-action" href="#list-item-2">The pH sensor test</a> |
− | <a class="list-group-item list-group-item-action" href="#list-item-4"> | + | <a class="list-group-item list-group-item-action" href="#list-item-3">Construction of CA</a> |
− | <a class="list-group-item list-group-item-action" href="#list-item-5"> | + | <a class="list-group-item list-group-item-action" href="#list-item-4">Construction of RbcL</a> |
− | <a class="list-group-item list-group-item-action" href="#list-item-6"> | + | <a class="list-group-item list-group-item-action" href="#list-item-5">Construction of RbcXS</a> |
− | <a class="list-group-item list-group-item-action" href="#list-item-7"> | + | <a class="list-group-item list-group-item-action" href="#list-item-6">Construction of PRK</a> |
− | <a class="list-group-item list-group-item-action" href="#list-item-8"> | + | <a class="list-group-item list-group-item-action" href="#list-item-7">Whole construction</a> |
+ | <a class="list-group-item list-group-item-action" href="#list-item-8">Total solution</a> | ||
<a class="list-group-item list-group-item-action" href="#"><i class="fa fa-arrow-up fa-1x" | <a class="list-group-item list-group-item-action" href="#"><i class="fa fa-arrow-up fa-1x" | ||
aria-hidden="true"></i> | aria-hidden="true"></i> | ||
Line 32: | Line 33: | ||
<h1 class="head2">Notebook</h1></br></br> | <h1 class="head2">Notebook</h1></br></br> | ||
− | + | ||
− | + | ||
<div id="list-item-1"> | <div id="list-item-1"> | ||
− | + | </br></br></br> | |
+ | <h3>Construction of Pasr and Pgada</h3></br> | ||
− | + | <p class="pcontent">4/26~4/27</br> </p> | |
− | + | <ul> | |
− | + | <li>PCR Pasr and Pgada from E. coli MG1655 chromosome. Confirmed and extracted | |
− | + | products by DNA gel. </li> | |
− | + | </ul></br> | |
− | + | <p class="pcontent">4/28</br> </p> | |
− | + | <ul> | |
− | + | <li>Digested the pSB1C3-sfGFP plasmid. Ligase the plasmid with Pasr / Pgada and | |
− | + | RiboJ by PCR. Confirmed by DNA gel electrophoresis. Transformed the gene in DH5 | |
− | + | alpha and spread the bacteria on LB plates.</li> | |
− | + | </ul></br> | |
− | + | <p class="pcontent">4/30~5/1</br> </p> | |
− | + | <ul> | |
− | + | <li>Confirmed the transformation by colony PCR ,enzyme digestion and DNA gel | |
− | + | electrophoresis. Selected the correct colonies for sequencing and cultured them | |
− | + | on LB plates.</li> | |
− | + | </ul></br> | |
− | + | <p class="pcontent">5/9</br> </p> | |
− | + | <ul> | |
− | + | <li>Store the correct colonies in glycerol in -80O C.</li> | |
− | + | </ul></br> | |
− | + | <p class="pcontent">6/1</br> </p> | |
− | + | <ul> | |
− | + | <li>Transformed each plasmid into BL21(DE3)</li> | |
− | + | </ul></br> | |
− | + | </div> | |
− | + | <div id="list-item-2"> | |
− | + | </br></br></br> | |
+ | <h3>The pH sensor test</h3></br> | ||
− | + | <p class="pcontent">5/9</br> </p> | |
− | + | <ul> | |
− | + | <li>Prepared the M9 medium in different pH value.</li> | |
− | + | </ul></br> | |
− | + | <p class="pcontent">5/11</br> </p> | |
− | + | <ul> | |
− | + | <li>Pre-cultured the DH5 alpha-pSB1C3 -Pasr-sfGFP strain.</li> | |
− | + | </ul></br> | |
− | + | <p class="pcontent">5/12</br> </p> | |
− | + | <ul> | |
− | + | <li>Cultured the DH5 alpha-pSB1C3 -Pasr-sfGFP strain in M9 medium in different pH | |
− | + | value. | |
− | + | Measured the OD600 value and fluorescence every hour.</li> | |
+ | </ul></br> | ||
− | + | <p class="pcontent">5/18</br> </p> | |
− | + | <ul> | |
− | + | <li>Pre-cultured DH5 alpha-pSB1C3 –Pgada-RiboJ-sfGFP strain.</li> | |
− | + | <li>Prepared the M9 medium in different pH value.</li> | |
− | + | </ul></br> | |
− | + | <p class="pcontent">5/19</br> </p> | |
− | + | <ul> | |
− | + | <li>Cultured the DH5 alpha-pSB1C3 –Pgada-RiboJ-sfGFP strain in M9 medium in different | |
− | + | pH | |
− | + | value. Measured the OD600 value and fluorescence every hour.</li> | |
+ | </ul></br> | ||
− | + | <p class="pcontent">6/2~6/3</br> </p> | |
− | + | <ul> | |
− | + | <li>Repeated the experiment on 5/11~5/12</li> | |
− | + | </ul></br> | |
− | + | <p class="pcontent">7/29</br> </p> | |
− | + | <ul> | |
− | + | <li>Pre-culture the BL21(DE3)-pSB1C3 –Pasrr-sfGFP strain</li> | |
− | + | </ul></br> | |
− | + | <p class="pcontent">7/30</br> </p> | |
− | + | <ul> | |
− | + | <li>Cultured the BL21(DE3)-pSB1C3 –Pasr -sfGFP strain on 96 well plate in different pH | |
− | + | value. Measure the fluorescence every 5 minutes in 30 minutes.</li> | |
− | + | </ul></br> | |
− | + | <p class="pcontent">9/4</br> </p> | |
− | + | <ul> | |
− | + | <li>Pre-culture the BL21(DE3)-pSB1C3 –Pgada-RiboJ-sfGFP strain</li> | |
− | + | </ul></br> | |
− | + | <p class="pcontent">9/5</br> </p> | |
− | + | <ul> | |
− | + | <li>Cultured the BL21(DE3)-pSB1C3 –Pgada-RiboJ-sfGFP strain strain on 96 well plate in | |
− | + | different pH value. Measure the fluorescence every 5 minutes in 30 minutes.</li> | |
− | + | </ul></br> | |
− | + | </div> | |
Line 133: | Line 138: | ||
− | + | <div id="list-item-3"> | |
− | + | </br></br></br><h3>Construction of CA</h3></br> | |
− | + | <p class="pcontent">7/2</br> </p> | |
− | + | <ul> | |
− | + | <li>PCR IDT products (RbcL, RbcXS, PRK, CA). Confirmed by DNA gel electrophoresis. | |
− | + | Extracted the gene from DNA gel. | |
− | + | </li> | |
− | + | </ul></br> | |
− | + | <p class="pcontent">7/5</br> </p> | |
− | + | <ul> | |
− | + | <li>PCR the CA. Confirmed by DNA gel electrophoresis and extracted the gene from DNA | |
+ | gel. | ||
− | + | </li> | |
− | + | </ul></br> | |
− | + | <p class="pcontent"> | |
− | + | 7/6 | |
− | + | </br> </p> | |
− | + | <ul> | |
− | + | <li>Digested and ligase the pSB1C3 plasmid and CA gene. | |
− | + | </li> | |
− | + | </ul></br> | |
− | + | <p class="pcontent">7/10~7/11</br> </p> | |
− | + | <ul> | |
− | + | <li>Repeated the PCR of CA, digestion, ligation.</li> | |
− | + | </ul></br> | |
− | + | <p class="pcontent">7/12</br> </p> | |
− | + | <ul> | |
− | + | <li>Transformed the pSB1C3-PT7-CA into DH5 alpha. </li> | |
− | + | </ul></br> | |
− | + | <p class="pcontent">7/19</br> </p> | |
− | + | <ul> | |
− | + | <li>Send the plasmid to sequencing</li> | |
− | + | </ul></br> | |
− | + | </div> | |
− | + | <div id="list-item-4"> | |
− | + | </br></br></br> | |
+ | <h3>Construction of RbcL</h3></br> | ||
− | + | <p class="pcontent">7/2</br> </p> | |
− | + | <ul> | |
− | + | <li>PCR IDT products (RbcL, RbcXS, PRK, CA). Confirmed by DNA gel electrophoresis.</li> | |
− | + | <li>Extracted the gene from DNA gel.</li> | |
− | + | </ul></br> | |
− | + | <p class="pcontent">7/11</br> </p> | |
− | + | <ul> | |
− | + | <li>PCR the RbcL with new primer.</li> | |
− | + | </ul></br> | |
− | + | <p class="pcontent">7/20</br> </p> | |
− | + | <ul> | |
− | + | <li>PCR the RbcL with old and new primer.</li> | |
− | + | </ul></br> | |
− | + | <p class="pcontent">7/21~24</br> </p> | |
− | + | <ul> | |
− | + | <li>Repeated the PCR of RbcL</li> | |
− | + | </ul></br> | |
− | + | <p class="pcontent">8/1</br> </p> | |
− | + | <ul> | |
− | + | <li>Ligate the RbcL with PlacI.</li> | |
− | + | <li>Transformed the pSB1C3-PT7-RbcL and pSB1C3-PlacI-RbcL into DH5 alpha.</li> | |
− | + | </ul></br> | |
− | + | <p class="pcontent">8/3 </br> </p> | |
− | + | <ul> | |
− | + | <li>Confirmed the pSB1C3-PT7-RbcL construct by colony PCR.</li> | |
− | + | <li>Sent the gene to sequencing</li> | |
− | + | </ul></br> | |
− | + | </div> | |
− | + | <div id="list-item-5"> | |
− | + | </br></br></br> | |
+ | <h3>Construction of RbcXS</h3></br> | ||
− | + | <p class="pcontent">7/2</br> </p> | |
− | + | <ul> | |
− | + | <li>Prepared the M9 medium in different pH value.</li> | |
− | + | </ul></br> | |
− | + | <p class="pcontent">7/3~7/21</br> </p> | |
− | + | <ul> | |
− | + | <li>PCR IDT products (RbcL, RbcXS, PRK, CA). Confirmed by DNA gel electrophoresis.</li> | |
− | + | <li> Extracted the gene from DNA gel.</li> | |
− | + | </ul></br> | |
− | + | <p class="pcontent">7/23</br> </p> | |
− | + | <ul> | |
− | + | <li>PCR normal/mutant RbcXS with old and new primer.</li> | |
− | + | </ul></br> | |
− | + | <p class="pcontent">7/24 </br> </p> | |
− | + | <ul> | |
− | + | <li>Confirmed by DNA gel.</li> | |
− | + | </ul></br> | |
− | + | </div> | |
− | + | <div id="list-item-6"> | |
− | + | </br></br></br> | |
+ | <h3>Construction of PRK</h3></br> | ||
− | + | <p class="pcontent">7/2</br> </p> | |
− | + | <ul> | |
− | + | <li>PCR IDT products (RbcL, RbcXS, PRK, CA). Confirmed by DNA gel electrophoresis.</li> | |
− | + | <li> Extracted the gene from DNA gel.</li> | |
− | + | </ul></br> | |
− | + | <p class="pcontent">7/3~8/2</br> </p> | |
− | + | <ul> | |
− | + | <li>PCR PRK.</li> | |
− | + | </ul></br> | |
− | + | <p class="pcontent">8/1</br> </p> | |
− | + | <ul> | |
− | + | <li>Ligated PRK with PlacI and PT7.</li> | |
− | + | <li>Transformed into DH5 alpha. | |
− | + | </li> | |
− | + | </ul></br> | |
− | + | <p class="pcontent">8/2</br> </p> | |
− | + | <ul> | |
− | + | <li>Confirmed by colony PCR. Selected the colony with correct length and sent to | |
− | + | sequencing.</li> | |
− | + | </ul></br> | |
− | + | </div> | |
− | + | <div id="list-item-7"> | |
− | + | </br></br></br> | |
+ | <h3>Whole construction</h3></br> | ||
− | + | <p class="pcontent">8/8</br> </p> | |
− | + | <ul> | |
− | + | <li>Ligated PT7-CA with PLacI-PRK on pSB1C3</li> | |
− | + | </ul></br> | |
− | + | <p class="pcontent">8/9</br> </p> | |
− | + | <ul> | |
− | + | <li>Ligated PT7-RbcL with PT7-RbcXS on pSB1C3</li> | |
− | + | </ul></br> | |
− | + | <p class="pcontent">8/28</br> </p> | |
− | + | <ul> | |
− | + | <li>Inserted PT7-CA with PLacI-PRK into pSB3K3</li> | |
− | + | </ul></br> | |
− | + | </div> | |
− | + | <div id="list-item-8"> | |
− | + | </br></br></br> | |
+ | <h3>SDS PAGE, Functional test and Total solution</h3></br> | ||
− | + | <p class="pcontent">7/25~7/30, 8/4~8/5, 8/11~8/12</br> </p> | |
− | + | <ul> | |
− | + | <li>Ran SDS-PAGE of CA, normal-RbcXS </li> | |
− | + | </ul></br> | |
− | + | <p class="pcontent">8/7, 8/9, 8/10</br> </p> | |
− | + | <ul> | |
− | + | <li> Ran SDS-PAGE of RbcL and PRK</li> | |
− | + | </ul></br> | |
− | + | <p class="pcontent">8/16 </br> </p> | |
− | + | <ul> | |
− | + | <li>Ran the SDS-PAGE of RbcL, RbcXS and PRK</li> | |
− | + | </ul></br> | |
− | + | <p class="pcontent">8/18~8/19</br> </p> | |
− | + | <ul> | |
− | + | <li>Activity test of CA</li> | |
− | + | </ul></br> | |
− | + | <p class="pcontent">9/2</br> </p> | |
− | + | <ul> | |
− | + | <li> Ran SDS-PAGE of pSB1C3-PT7-RbcL-PT7-RbcXS and pSB3K3-Plac-PRK-PT7-CA</li> | |
− | + | </ul></br> | |
− | + | <p class="pcontent">9/3</br> </p> | |
− | + | <ul> | |
− | + | <li> Transformed pSB1C3-PT7-RbcL-PT7-RbcXS and pSB3K3-Plac-PRK-PT7-CA into W3110</li> | |
− | + | </ul></br> | |
− | + | <p class="pcontent">9/5~9/17</br> </p> | |
− | + | <ul> | |
− | + | <li> Total solution.</li> | |
− | + | </ul></br> | |
− | + | </div> | |
Revision as of 05:47, 22 September 2018