Difference between revisions of "Team:TJU China/Notebook/Group1"

 
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     </div>
 
     </div>
 
     <div style=" margin-top:28px;z-index:10; border-top: solid #4e72b8 2px;width: 100%; position: fixed;"></div>
 
     <div style=" margin-top:28px;z-index:10; border-top: solid #4e72b8 2px;width: 100%; position: fixed;"></div>
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     <div class="notebook_month">  
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     <div class="notebook_month">
 
         <div class="notebook_month_p">Mar.</div>
 
         <div class="notebook_month_p">Mar.</div>
 
         <div class="notebook_month_p">Apr.</div>
 
         <div class="notebook_month_p">Apr.</div>
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         <div id="notebook_March">
 
         <div id="notebook_March">
 
             <div class="notebook_Month_head">March</div>
 
             <div class="notebook_Month_head">March</div>
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             <div class="notebook_week">
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             <div class="notebook_week_p">Group1 get started at July.</div>
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                <div>
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−
                    <button id="3week1" class="notebook_information_button1">week1</button>
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                </div>
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                <div id="3notebook_week1" class="notebook_information_word1" style="display:none;">
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                    <div class="notebook_week_p">After a lot of document viewing and brainstorms, we finally came up with a clear idea about how we were
+
−
                        going to carry out our part of project. We decided to construct the RNPs (complex of sgRNA and Cas9
+
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                        protein) and to wrap them with delivery vector in order to improve the gene-editing efficiency of
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                        the system into the cells.</div>
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                </div>
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                <div>
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                    <button id="3week2" class="notebook_information_button2">week2</button>
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                </div>
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                <div id="3notebook_week2" class="notebook_information_word2" style="display:none;">
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                    <div class="notebook_week_p">After daily meetings and discussions, we exchanged knowledge and ideas from the literatures we read.
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                        Comparing different transportation methods, including physical electroporation, chemical nanoparticles,
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                        and even virus methods, we decided to improve the transport efficiency of the system by using two-dimensional
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                        nanomaterials to encapsulate the cas9/sgRNA complex assembled in vitro to transport into the cells.</div>
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                </div>
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                <div>
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                    <button id="3week3" class="notebook_information_button3">week3</button>
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                </div>
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                <div id="3notebook_week3" class="notebook_information_word3" style="display:none;">
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                    <div class="notebook_week_p">We began to delve into more detailed and specific knowledge, such as the structure of cas9 protein and
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                        how it works, the structure and composition of sgRNA and how it is assembled with cas9 protein, the
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                        method of primer design, learning how to design sgRNA, and so on.</div>
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                </div>
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                <div>
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                    <button id="3week4" class="notebook_information_button4">week4</button>
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                </div>
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                <div id="3notebook_week4" class="notebook_information_word4" style="display:none;">
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                    <div class="notebook_week_p">After confirming the type of cas9 protein, we started buying and asking other labs if they had the same
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                        cas9 protein. While waiting for an answer, we are continuing to refine the questions left over from
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                        last week's knowledge and begin to learn the experimental procedures and necessary knowledge related
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                        to protein expression purification.</div>
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                </div>
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             </div>
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         </div>
 
         </div>
 
         <div id="notebook_April">
 
         <div id="notebook_April">
 
             <div class="notebook_Month_head">April</div>
 
             <div class="notebook_Month_head">April</div>
−
             <div class="notebook_week">
+
             <div class="notebook_week_p">Group1 get started at July.</div>
−
                <div>
+
              
−
                    <button id="4week1" class="notebook_information_button1">week1</button>
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                </div>
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                <div id="4notebook_week1" class="notebook_information_word1" style="display:none;">
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                    <div class="notebook_week_p"> We continued to search for related documents in order to choose the appropriate target sequences of gene
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                        for our project. After the final decision of making eGFP as our interested sequence of gene, we immediately
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                        sent out the primers of sgRNA we designed to GENEWIZ, and we also sent lots of emails to different
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                        labs to see if they can offer us a stable cell strain expressing eGFP protein.</div>
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                </div>
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                <div>
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                    <button id="4week2" class="notebook_information_button2">week2</button>
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                </div>
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                <div id="4notebook_week2" class="notebook_information_word2" style="display:none;">
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                    <div class="notebook_week_p">Meanwhile, we successfully transformed the plasmid pET-NLS-Cas9-6xHis (purchased from Addgene) into E.coli
+
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                        (BL21), then we tried to express and purify the cas9 protein according to the protocol[1]. But the
+
−
                        results were not so ideal.
+
−
                        <br> 【1】Ha J S, Lee J S, Jeong J, et al. Poly-sgRNA/siRNA ribonucleoprotein nanoparticles for targeted
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                        gene disruption[J]. Journal of Controlled Release, 2017, 250:27-35.</div>
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                </div>
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             </div>
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         </div>
 
         </div>
 
         <div id="notebook_May">
 
         <div id="notebook_May">
 
             <div class="notebook_Month_head">May</div>
 
             <div class="notebook_Month_head">May</div>
 +
            <div class="notebook_week_p">Group1 get started at July.</div>
 +
           
 +
        </div>
 +
        <div id="notebook_June">
 +
            <div class="notebook_Month_head">June</div>
 +
            <div class="notebook_week_p">Group1 get started at July.</div>
 +
           
 +
        </div>
 +
        <div id="notebook_July">
 +
            <div class="notebook_Month_head">July</div>
 
             <div class="notebook_week">
 
             <div class="notebook_week">
 
                 <div>
 
                 <div>
−
                     <button id="5week1" class="notebook_information_button1">week1</button>
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                     <button id="7week2" class="notebook_information_button2">week2</button>
 
                 </div>
 
                 </div>
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                 <div id="5notebook_week1" class="notebook_information_word1" style="display:none;">
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                 <div id="7notebook_week2" class="notebook_information_word2" style="display:none;">
−
                     <div class="notebook_week_p">PCR and Gel Purification (TIANgel Midi Purification Kit) of the template of sgRNA, we tried different
+
                     <div class="notebook_week_p">Activate smURFP glycerol.</div>
−
                        Tm temperatures.</div>
+
                     <img src="https://static.igem.org/mediawiki/2018/8/88/T--TJU_China--g1.1.png">
−
                     <img src="./img/group3May1pic1.PNG">
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                    <div class="notebook_week_p">
−
                     <img src="./img/group3May1pic2.PNG">
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                        <b>Figure 1.</b> Incubate to activate the smURFP plasmid PCR amplification of smURFP fragments.</div>
 +
                     <img src="https://static.igem.org/mediawiki/2018/6/67/T--TJU_China--g1.2.png">
 +
                    <div class="notebook_week_p">
 +
                        <b>Figure 2.</b> The result of nucleic acid gel electrophoresis of smURFP after PCR. Lane M, Marker.
 +
                        Lane1-8，smURFP.
 +
                        <br>Gel Extraction purification of the PCR product.
 +
                        <br>Find the arsenic ion plasmid inside the box and transform the plasmid into E.coli DH5α.</div>
 
                 </div>
 
                 </div>
 +
 +
 
                 <div>
 
                 <div>
−
                     <button id="5week2" class="notebook_information_button2">week2</button>
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                     <button id="7week3" class="notebook_information_button3">week3</button>
 
                 </div>
 
                 </div>
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                 <div id="5notebook_week2" class="notebook_information_word2" style="display:none;">
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                 <div id="7notebook_week3" class="notebook_information_word3" style="display:none;">
−
                     <div class="notebook_week_p">After two weeks of hard working, we got our first batch of Cas9 protein.
+
                     <div class="notebook_week_p">Transform the plasmid（Bba-J33201） inside the box.
−
                         <br> At the same time, we tried our first transcription and preparation of sgRNA using T7 High Efficiency
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                        <br>Amplification of PCR fragments.
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                         Transcription Kit and EasyPure RNA Purification Kit (TransGene Biotech) but failed. We supposed that
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                         <br>PCR amplification of arsenic ion loop.</div>
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                         the template might be polluted by RNase during the process.</div>
+
                    <img src="https://static.igem.org/mediawiki/2018/2/2b/T--TJU_China--g1.3.png">
 +
                    <div class="notebook_week_p">
 +
                         <b>Figure 3.</b> The result of nucleic acid gel electrophoresis of Bba-J33201 after PCR. Lane M, Marker.
 +
                        Lane 1-6，BBa-J33201.</div>
 +
                    <div class="notebook_week_p">PCR amplification of ArsR promoter and ArsR protein fragment.
 +
                         <br>Transform plasmid pKM586 into E.coli BL21.
 +
                        <br>Activate and extract pKM586 plasmid.</div>
 
                 </div>
 
                 </div>
 +
 
                 <div>
 
                 <div>
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                     <button id="5week3" class="notebook_information_button3">week3</button>
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                     <button id="7week4" class="notebook_information_button4">week4</button>
 
                 </div>
 
                 </div>
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                 <div id="5notebook_week3" class="notebook_information_word3" style="display:none;">
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                 <div id="7notebook_week4" class="notebook_information_word4" style="display:none;">
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                     <div class="notebook_week_p">We searched for information and knowledge of experiments dealing with RNA and then retried the PCR and
+
                     <div class="notebook_week_p">Activate smURFP glycerol.</div>
−
                        Gel Purification of the template according strictly to the RNA principles. However, we failed again.
+
                     <img src="https://static.igem.org/mediawiki/2018/7/74/T--TJU_China--g1.4.png">
−
                        <br> On the other hand, we managed to get plasmid encoding AcGFP by using TIANprep Mini Plasmid Kit (TIANGEN).</div>
+
                    <div class="notebook_week_p">
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                     <img src="./img/group3May3pic1.png">
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                        <b>Figure 4.</b> The result of nucleic acid gel electrophoresis after overlapping of ArsR Promoter and
−
                </div>
+
                         smURFP. Lane M, Marker. Lane 1-10, ArsR Promoter＋smURFP</div>
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                <div>
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                    <button id="5week4" class="notebook_information_button4">week4</button>
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                </div>
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                <div id="5notebook_week4" class="notebook_information_word4" style="display:none;">
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                    <div class="notebook_week_p">We confirmed the best Tm temperature of PCR was 67.2℃ after several preliminary experiments. And this
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                         time, there were lines shown on the RNA gel, but they were not clear. And the RNA marker (TAKARA)
+
−
                        didn’t appear exactly as the instruction. Then we moved forward to the In vitro digestion of DNA
+
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                        but failed. Since that the result of the SDS-PAGE of Cas9 protein showed no problem, we considered
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                        that the sgRNA might degraded.</div>
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                    <img src="./img/group3May4pic1.png">
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                    <img src="./img/group3May4pic2.png">
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                    <div class="notebook_week_p">Line1，plasmid；2，cleavage result</div>
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                 </div>
 
                 </div>
  
 
             </div>
 
             </div>
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        </div>
 
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        <div id="notebook_June">
 
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            <div class="notebook_Month_head">June</div>
 
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            <div class="notebook_week">
 
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                <div style="text-align: left;font-size: 20px;text-indent: 2em;">Preparation for the final-term exams. </div>
 
  
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            </div>
 
 
         </div>
 
         </div>
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        <div id="notebook_July">
 
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            <div class="notebook_Month_head">July</div>
 
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            <div class="notebook_week">
 
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                <div style="text-align: left;font-size: 20px;text-indent: 0;">week1-3
 
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                    <br> Most members joined a summer camp as a part of school lessons.
 
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                    <br> week4
 
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                    <br> In order to reduce the influence of RNase on the subsequent experiment, the phenol-chloroform method
 
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                    was used to remove RNase. But it turned out that quantity of DNA was not sufficient enough to be extracted.
 
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                    We tried for several times but the results of the nucleic acid gel electrophoresis showed that we got
 
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                    nothing after the purification.</div>
 
  
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            </div>
 
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        </div>
 
 
         <div id="notebook_August">
 
         <div id="notebook_August">
 
             <div class="notebook_Month_head">August</div>
 
             <div class="notebook_Month_head">August</div>
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                 <div id="8notebook_week1" class="notebook_information_word1" style="display:none;">
 
                 <div id="8notebook_week1" class="notebook_information_word1" style="display:none;">
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                     <div class="notebook_week_p">Double digestion of pKM586 plasmid.</div>
 +
                    <img src="https://static.igem.org/mediawiki/2018/7/74/T--TJU_China--g1.5.png">
 +
                    <div class="notebook_week_p">
 +
                        <b>Figure 5.</b> Double digestion of pKM586 with AatII and BamHI. Lane M, Marker. Lane 1，pKM586; Lane
 +
                        2-3, plasmid pKM586 after double enzyme digestion
 +
 
 +
                        <br>Overlap ArsR + Arsr Promoter,but failed.
 +
 
 +
                        <br>Overlap ArsR Promoter + smURFP, failed.
 +
                        <br>Enzyme digestion of pwj66.</div>
 
                 </div>
 
                 </div>
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                 <div>
 
                 <div>
 
                     <button id="8week2" class="notebook_information_button2">week2</button>
 
                     <button id="8week2" class="notebook_information_button2">week2</button>
 
                 </div>
 
                 </div>
 
                 <div id="8notebook_week2" class="notebook_information_word2" style="display:none;">
 
                 <div id="8notebook_week2" class="notebook_information_word2" style="display:none;">
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                     <div class="notebook_week_p">Enzyme digestion of pwj66.
 +
                        <br>Connect pwj66+ TARGETDNA.
 +
                        <br>Double digestion of pKM586.
 +
                        <br>Overlap ArsR + Arsr Promoter.
 +
                        <br>Overlap ArsR Promoter + smURFP.
 +
                        <br>PCR of ParsR promoter ArsR protein fragment.</div>
 +
                    <img src="https://static.igem.org/mediawiki/2018/1/13/T--TJU_China--g1.6.png">
 +
                    <div class="notebook_week_p">
 +
                        <b>Figure 6.</b>LB media of pKM586 after transformed</div>
 
                 </div>
 
                 </div>
 +
 +
 
                 <div>
 
                 <div>
 
                     <button id="8week3" class="notebook_information_button3">week3</button>
 
                     <button id="8week3" class="notebook_information_button3">week3</button>
 
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                 </div>
 
                 <div id="8notebook_week3" class="notebook_information_word3" style="display:none;">
 
                 <div id="8notebook_week3" class="notebook_information_word3" style="display:none;">
−
                      
+
                     <div class="notebook_week_p">Enzyme digestion of pwj66.
 +
                        <br>Connect pwj66+ spacer.
 +
                        <br>Double digestion pkm586.
 +
                        <br>Overlap ArsR + Arsr Promoter.
 +
                        <br>Overlap ArsR Promoter + smURFP.</div>
 +
                    <img src="https://static.igem.org/mediawiki/2018/6/64/T--TJU_China--g1.7.png">
 +
                    <div class="notebook_week_p">
 +
                        <b>Figure 7.</b>The result of nucleic acid gel electrophoresis after overlapping of ArsR Promoter and
 +
                        smURFP. Lane M, Marker. Lane 1-7, ArsR Promoter＋smURFP. PCR of ParsR promoter ArsR protein fragment.</div>
 
                 </div>
 
                 </div>
 
                 <div>
 
                 <div>
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                 </div>
 
                 </div>
 
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−
                      
+
                     <div class="notebook_week_p">Anneal two J23104 oligo chains.
 +
                        <br>PCR amplification of Bba-J23104 fragments. .
 +
                        <br>Overlap J23104 + ArsR protein fragment.
 +
                        <br>Overlap smURFP + ArsR promoter.</div>
 +
                    <img src="https://static.igem.org/mediawiki/2018/9/92/T--TJU_China--g1.8.png">
 +
                    <div class="notebook_week_p">
 +
                        <b>Figure 8.</b> The result of nucleic acid gel electrophoresis after overlapping of ArsR Promoter and
 +
                        smURFP. Lane M, Marker. Lane 1, smURFP. Lane 2-4, ArsR Promoter＋smURFP.</div>
 +
                    <div class="notebook_week_p">Overlapping the two overlapped fragments.
 +
                        <br>Gel Extraction purification.
 +
                        <br>Double enzyme digestion of the entire arsenic loop.
 +
                        <br>Double digestion of pKM586.</div>
 +
                    <img src="https://static.igem.org/mediawiki/2018/4/47/T--TJU_China--g1.9.png">
 +
                    <div class="notebook_week_p">
 +
                        <b>Figure 9.</b>Double digestion of pKM586 with AatII and BamHI. Lane M, Marker. Lane 1,Plasmid pKM586.
 +
                        Lane 2, Plasmid pKM586 after double enzyme digestion
 +
                        <br>Connection the two fragments after enzyme digestion
 +
                        <br>Transform the product into E.coli DH5α</div>
 
                 </div>
 
                 </div>
  
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                 </div>
 
                 </div>
 
                 <div id="9notebook_week1" class="notebook_information_word1" style="display:none;">
 
                 <div id="9notebook_week1" class="notebook_information_word1" style="display:none;">
−
                      
+
                     <div class="notebook_week_p">We did a full fragment overlap of the arsenic loop and the result failed. Double digestion of pKM586
 +
                        vector, plasmid extraction results and double enzyme digestion results are not ideal</div>
 +
                    <img src="https://static.igem.org/mediawiki/2018/3/3e/T--TJU_China--g1.10.png">
 +
                    <div class="notebook_week_p">
 +
                        <b>Figure 10.</b>Double digestion of pKM586 with AatII and BamHI. Lane M, Marker. Lane 1, pKM586; Lane
 +
                        2-3, plasmid pKM586 after double enzyme digestion</div>
 
                 </div>
 
                 </div>
 
                 <div>
 
                 <div>
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                 </div>
 
                 </div>
 
                 <div id="9notebook_week2" class="notebook_information_word2" style="display:none;">
 
                 <div id="9notebook_week2" class="notebook_information_word2" style="display:none;">
−
                 
+
                    <div class="notebook_week_p">We modified the arsenic loop full-segment overlap system and the PCR program, and the results were successful.</div>
 +
                    <img src="https://static.igem.org/mediawiki/2018/7/79/T--TJU_China--g1.11.png">
 +
                    <div class="notebook_week_p">
 +
                        <b>Figure 11.</b>The result of nucleic acid gel electrophoresis after overlapping the whole arsenic
 +
                        loop. Lane M, Marker. Lane1-7 the whole arsenic loop.</div>
 +
                    <div class="notebook_week_p">Reconstruction of pKM586 original plasmid, the quality of plasmid extraction is not high</div>
 
                 </div>
 
                 </div>
 
                 <div>
 
                 <div>
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                 </div>
 
                 </div>
 
                 <div id="9notebook_week3" class="notebook_information_word3" style="display:none;">
 
                 <div id="9notebook_week3" class="notebook_information_word3" style="display:none;">
−
                      
+
                     <div class="notebook_week_p">Try pKM586 step-by-step double enzyme digestion pre-experiment, resulting in significant improvement
 +
                        in cutting efficiency. The arsenic loop complete fragment double digestion and pKM586 double digestion
 +
                        product gel recovery, as well as ligation transformation.</div>
 +
                    <img src="https://static.igem.org/mediawiki/2018/8/83/T--TJU_China--g1.12.png">
 +
                    <div class="notebook_week_p">
 +
                        <b>Figure 12.</b>the arsenic loop after transformed into DH5α</div>
 
                 </div>
 
                 </div>
 
                 <div>
 
                 <div>
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                 </div>
 
                 </div>
 
                 <div id="9notebook_week4" class="notebook_information_word4" style="display:none;">
 
                 <div id="9notebook_week4" class="notebook_information_word4" style="display:none;">
−
                      
+
                     <div class="notebook_week_p">The plasmid was extracted and verified by double enzyme digestion. The result was successful.</div>
 +
                    <img src="https://static.igem.org/mediawiki/2018/f/f0/T--TJU_China--g1.13.png">
 +
                    <div class="notebook_week_p">
 +
                        <b>Figure 13.</b>Double digestion to verify the ligation product. Lane M, Marker. Lane 1, Plasmid pKM586.
 +
                        Lane 2, Plasmid pKM586 single digestion with BamHI. Lane 3, Plasmid pKM586 double digestion with
 +
                        AatII and BamHI. Lane 4, Plasmid ArS. Lane 5, Plasmid ArS single digestion with BamHI. Lane 6, Plasmid
 +
                        ArS double gigestion with AatII and BamHI. Lane 7, Plasmid ArS. Lane 8, Plasmid ArS single digestion
 +
                        with BamHI. Lane 9, Plasmid ArS double digestion with AatII and BamHI.</div>
 
                 </div>
 
                 </div>
  
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                 </div>
 
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                 <div id="10notebook_week1" class="notebook_information_word1" style="display:none;">
−
                      
+
                     <div class="notebook_week_p">Transform the successfully constructed plasmid into E. coli BL21, pick a single colony, incubate the
 +
                        tube for the first arsenic ion delivery experiment, but the result shows no fluorescence.</div>
 +
                    <img src="https://static.igem.org/mediawiki/2018/9/94/T--TJU_China--g1.14.png">
 +
                    <div class="notebook_week_p">
 +
                        <b>Figure 14.</b>the arsenic loop after transformed into BL21</div>
 
                 </div>
 
                 </div>
 
                 <div>
 
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−
                      
+
                     <div class="notebook_week_p">After the successful construction of the plasmid, we began to try to test whether the transformed E.
−
                </div>
+
                        coli can express fluorescence after adding arsenic ions. We chose to culture the tube for 6 hours
−
                <div>
+
                        and then add different concentrations of arsenic ions for detection. For the second time, we failed
−
                    <button id="10week3" class="notebook_information_button3">week3</button>
+
                        again.
−
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+
                        <br>And we continue to try to change the condition to make it express fluorescence.</div>
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Latest revision as of 12:11, 17 October 2018

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