Difference between revisions of "Team:FAFU-CHINA/Notebook"

 
(7 intermediate revisions by one other user not shown)
Line 1: Line 1:
−
{{BIT-China}}
+
<html lang="zh">
−
<html>
+
<head>
−
<style>
+
<meta charset="UTF-8">
 +
<meta http-equiv="X-UA-Compatible" content="IE=edge,chrome=1">  
 +
<meta name="viewport" content="width=device-width, initial-scale=1.0">
 +
<title>侧边栏</title>
 +
<link rel="stylesheet" href="https://2015.igem.org/Template:FAFU_CHINA/css/homecss?action=raw&amp;ctype=text/css" type="text/css" />
 +
<link rel="stylesheet" type="text/css" href="https://2015.igem.org/Template:FAFU_CHINA/guge/css/styles?action=raw&amp;ctype=text/css" />
 +
</head>
 +
<body>
 +
<div class="com">
 +
<div class="com__content">
 +
<section class="com__section com__section--text">
 +
        <div style="background-color:rgba(255,255,255,0.2);">
 +
  <br>
 +
             
  
−
#container_Notebook{
+
<div id="Notebook"></div>
−
    position: absolute;
+
−
    margin: 0 auto;
+
−
    padding: 0px;
+
−
    left: 140px;
+
−
    right: 20px;
+
−
  font-size: 110%;
+
  
−
    line-height: 1.5em;
+
<br>
−
}
+
<center><div>Notebook</div></center>
−
.Notebook h2{
+
<br>
−
    color:#016cb4;
+
<hr style="height:1px;border:none;border-top:1px solid #555555;" /> 
−
    line-height: 37px;
+
−
}
+
  
−
sup{
 
−
    line-height: 10px;
 
−
}
 
  
−
.Notebook{
+
                <center><div>2015.6.8</div></center>
−
margin: 0 auto;
+
<hr style="height:1px;border:none;border-top:1px solid #555555;" />
−
    width: 90%;
+
                <div style="text-align:left">Today was the first day that we began our project in the lab after we made a scientific plan. Firstly, we extracted the whole RNA of CSBV by Trizol. As we all know, RNA is easy to be digested in the environment. Therefore although we did this quickly, there were still some samples without RNA fragment. But we did again and the result was satisfied at that time. It was encouraging!</div>
−
}
+
<br>
  
−
#progress_bar_container{
 
−
width: 120px;
 
−
overflow-y:scroll;
 
−
background-color: #ffffff;
 
−
padding: 20px 0px;
 
−
}
 
−
/* 设置滚动条的样式 */
 
−
#progress_bar_container::-webkit-scrollbar {width: 10px;}
 
−
/* 滚动槽 */
 
−
::-webkit-scrollbar-track {-webkit-box-shadow: inset 0 0 6px rgba(0,0,0,0.3);border-radius: 10px;}
 
−
/* 滚动条滑块 */
 
−
::-webkit-scrollbar-thumb {border-radius: 10px;background: rgba(34,105,160,0.3);-webkit-box-shadow:inset 0 0 6px rgba(34,105,160,0.7);}
 
  
−
/* 设置滚动条的样式 */
+
                <center><div>2015.6.9</div></center>
−
#progress_bar_container::scrollbar {width: 10px;}
+
<hr style="height:1px;border:none;border-top:1px solid #555555;" />
−
/* 滚动槽 */
+
                <div style="text-align:left">Today we made RT-PCR with four sets of reverse specific primers and the RNA of CSBV to synthesize homologous cDNA(CSBV Helicase、Protease、VP1 and RdRp respectively). Then dsDNAs was amplified with four sets of specific primers by PCR. It went through smoothly. So far, we got the target fragments successfully. Also,we used GFP F/R to amplify GFP gene. </div>
−
::scrollbar-track {box-shadow: inset 0 0 6px rgba(0,0,0,0.3);border-radius: 10px;}
+
<br>
−
/* 滚动条滑块 */
+
 
−
::scrollbar-thumb {border-radius: 10px;background: rgba(34,105,160,0.3);box-shadow:inset 0 0 6px rgba(34,105,160,0.7);}
+
               
−
#progress_bar_container ul li{
+
              <center><div>2015.6.10</div></center>
−
text-align: center;
+
<hr style="height:1px;border:none;border-top:1px solid #555555;" />
−
line-height: 35px;
+
                <div style="text-align:left">We utilized T7 RiboMAXTM Express RNAi System kit and dsDNAs as templates to synthesize dsRNAs(dsHelicase、dsProtease、dsVP1、dsRdRp and dsGFP), but there were only dsHelicase and dsGFP we could get. We tried again and still failed. It took our whole day to find the reason. Tomorrow we will ask our advisor for some solutions and do it one more time.</div>
−
color: #999999;
+
<br> 
−
text-decoration: none;
+
                <center><div>2015.6.12</div></center>
−
list-style: none;
+
<hr style="height:1px;border:none;border-top:1px solid #555555;" />
−
cursor:pointer;
+
                <div style="text-align:left">After figuring out the problem, we eventually got the all right dsRNAs. Another part, we began to prepare the larvae of Chinese honeybee which was offered by our institute of bees.
 +
</div>
 +
                <center><div>2015.6.13</div></center>
 +
<hr style="height:1px;border:none;border-top:1px solid #555555;" />
 +
                <div style="text-align:left">We put dsRNAs into fodder of the larvae of the honeybee to feed them. Then after 12h, we fed them another fodder with CSBV extracting solution. We did so twice before they got normal fodder. </div>
 +
<br>               
 +
                  <center><div>2015.6.17</div></center>
 +
<hr style="height:1px;border:none;border-top:1px solid #555555;" />
 +
                <div style="text-align:left">During three days, we observed the change of percentage of pupation and collect and analyse data. Meanwhile,RT-qPCR was done to detect the effect of different dsRNAs to CSBV. According to the result, we found that dsRdRp made a remarkable influence on the replication of CSBV. So we decided to use dsRdRp to inference CSBV.</div>
 +
<br>
 +
                <center><div>2015.6.19</div></center>
 +
<hr style="height:1px;border:none;border-top:1px solid #555555;" />
 +
                <div style="text-align:left">We used cDNA of RdRp and specific primers(Not1-F/Pst1-R、EcoR1-F/Pst1-R) to synthesize RdRp gene. However, after agarose gel electrophoresis(AGE), we could not find our target fragment.</div>
 +
<br>
 +
                <center><div>2015.6.20</div></center>
 +
<hr style="height:1px;border:none;border-top:1px solid #555555;" />
 +
                <div style="text-align:left">We changed a better DNA polymerase and set the annealing temperature to 60℃. Finally we got the RdRp gene.</div>
 +
<br>
 +
                <center><div>2015.6.22</div></center>
 +
<hr style="height:1px;border:none;border-top:1px solid #555555;" />
 +
                <div style="text-align:left">We picked the colonies to do PCR and sent the positive sequencing(M13).</div>
 +
<br>
 +
                <center><div>2015.6.24</div></center>
 +
<hr style="height:1px;border:none;border-top:1px solid #555555;" />
 +
                <div style="text-align:left">Today we got the result of sequencing. After comparing with data in NCBI, we only had two 100 percent samples.
 +
We started to do double enzyme digestion(Not1 and Pst1、EcoR1 and Pst1) to cut vector L4440、pSB1C3 and RdRp-T recombinant plasmid.
 +
</div>
 +
<br>               
 +
                <center><div>2015.6.25</div></center>
 +
<hr style="height:1px;border:none;border-top:1px solid #555555;" />
 +
                <div style="text-align:left">We made RdRp gene link with T vector in 25℃, 15min and then transformed it into DH5α,a kind of competent cell, which was cultured in 37℃,overnight.</div>
 +
<br>
 +
                <center><div>2015.6.26</div></center>
 +
<hr style="height:1px;border:none;border-top:1px solid #555555;" />
 +
                <div style="text-align:left">We made it! And we immediately did gel recovery. Then we began to link RdRp with L4440 and pSB1C3 using T4 ligase, 4℃, overnight.
 +
</div>
 +
<br>
 +
                <center><div>2015.6.27</div></center>
 +
<hr style="height:1px;border:none;border-top:1px solid #555555;" />
 +
                <div style="text-align:left">We did transformation. However,we forgot to make LB medium with CmRR. So the transformation of RdRp-pSB1C3 could only be done tomorrow.
 +
</div>
 +
<br>
 +
                <center><div>2015.6.29</div></center>
 +
<hr style="height:1px;border:none;border-top:1px solid #555555;" />
 +
                <div style="text-align:left">Today there was two news. The good one was we successfully got recombinant plasmid RdRp-L4440 while the bad one was nothing grew on the plate with CmRR. We thought maybe it was because RdRp did not link with pSB1C3 successfully. Anyway we would do it again tomorrow.
 +
</div>
 +
<br>
 +
                <center><div>2015.7.2</div></center>
 +
<hr style="height:1px;border:none;border-top:1px solid #555555;" />
 +
                <div style="text-align:left">On the one hand, we still could not link RdRp with pSB1C3 so that there still nothing on the plate. We wonder if it was because of the efficiency of ligase or anything.
 +
On the other hand,we prepared another competent cell,HT115,which included T7 RNAP gene in its genome. It was used as engineering bacteria for expressing dsRdRp.</div>
 +
<div style="text-align:left">Also, considering the biosecurity of E.coli, we decided use yeast as the final transformation target. So we want to recombine another vector,T7 RNAP-pYES2.</div>
 +
<br>
 +
                <center><div>2015.7.3</div></center>
 +
<hr style="height:1px;border:none;border-top:1px solid #555555;" />
 +
                <div style="text-align:left">We transformed RdRp-L4440 into HT115,culturing in 37℃. Overnight. And T7 RNAP gene was amplified by PCR(EcoR1-F/Xho1-R).</div>
 +
<br>
 +
                <center><div>2015.7.4</div></center>
 +
<hr style="height:1px;border:none;border-top:1px solid #555555;" />
 +
                <div style="text-align:left">Today we used IPTG, which was divided into two groups with different concentrations(0.4mmol/ml、0.8mmol/ml), to induce the expression of dsRdRp in HT115 for 5h. Then we collected the bacteria to extract dsRdRp by CTAB. According to the result of AGE, dsRdRp had been expressed in HT115 successfully, even though its concentration was a little bit lower. It was really a good news for us! </div>
 +
<br>
 +
                <center><div>2015.7.5</div></center>
 +
<hr style="height:1px;border:none;border-top:1px solid #555555;" />
 +
                <div style="text-align:left">We added engineering bacteria with expressed dsRdRp, which was divided into three groups with different concentrations(low、mediate and high), into the fodder of the infected swarms. And another infected swarm was fed normal fodder. Since now, we will obverse the change of population of them, the mortality, and the number of sealed brood and collect data every weeks until we get the obvious result.
 +
</div>
 +
<br>
 +
                <center><div>2015.7.7</div></center>
 +
<hr style="height:1px;border:none;border-top:1px solid #555555;" />
 +
                <div style="text-align:left">Considering we have not gotten RdRp-pSB1C3 yet,we decided to link T7 RNAP gene to pSB1C3 first. So we used specific primers(EcoR1-F/Pst1-R) to synthesize T7 RNAP gene by PCR.
 +
</div>
 +
<br>
 +
                <center><div>2015.7.8</div></center>
 +
<hr style="height:1px;border:none;border-top:1px solid #555555;" />
 +
                <div style="text-align:left">Transformation again!</div>
 +
<br>
 +
                <center><div>2015.7.9</div></center>
 +
<hr style="height:1px;border:none;border-top:1px solid #555555;" />
 +
                <div style="text-align:left">As usual, pinking the positive colonies, PCR and sequencing. We hope everything goes smoothly.
 +
</div>
 +
<br>
 +
                <center><div>2015.7.11</div></center>
 +
<hr style="height:1px;border:none;border-top:1px solid #555555;" />
 +
                <div style="text-align:left">According to the result of sequencing, there was no sample 100 percent matching. We did transformation again.
 +
</div>
 +
<br>
 +
                <center><div>2015.7.13</div></center>
 +
<hr style="height:1px;border:none;border-top:1px solid #555555;" />
 +
                <div style="text-align:left">The second sequencing was completely failed because the sequencing company could not get any plasmid. We thought maybe the replication of plasmid in competent cell(DH5α) was inefficient, so we changed another one,T1,bought from Transgene company.
 +
</div>
 +
<br>
 +
                <center><div>2015.7.14</div></center>
 +
<hr style="height:1px;border:none;border-top:1px solid #555555;" />
 +
                <div style="text-align:left">This time we sent plasmid rather than bacteria. Although the concentration of the plasmid was a little bit lower, it was enough to do sequencing.</div>
 +
<br>
 +
                <center><div>2015.7.15</div></center>
 +
<hr style="height:1px;border:none;border-top:1px solid #555555;" />
 +
                <div style="text-align:left">Considering the lower fidelity of the enzyme used for T7 amplification before, we changed into a better one called KD Plus and restarted to PCR, linking and transformation.</div>
 +
<br>
 +
                <center><div>2015.7.16</div></center>
 +
<hr style="height:1px;border:none;border-top:1px solid #555555;" />
 +
                <div style="text-align:left">The third sequencing still showed nothing right. We were a little disappointed, but we exactly knew that failure was a normal phenomenon in the lab. </div>
 +
<br>
 +
                <center><div>2015.7.18</div></center>
 +
<hr style="height:1px;border:none;border-top:1px solid #555555;" />
 +
                <div style="text-align:left">Eventually,we got the matching sample after using KD Plus. Immediately, we began to do double enzyme digestion(EcoR1/Xho1、EcoR1/Pst1).</div>
 +
<br>
 +
                <center><div>2015.7.19</div></center>
 +
<hr style="height:1px;border:none;border-top:1px solid #555555;" />
 +
                <div style="text-align:left">After gel recovery, we linked our target fragments to relative vectors with T4 ligase, 4℃, overnight.</div>
 +
                <div style="text-align:left">Meanwhile, we collected data about the index of the condition of swarms again, which was used to analyze the effect of dsRdRp by Excel later.</div>
 +
<br>
 +
                <center><div>2015.7.20</div></center>
 +
<hr style="height:1px;border:none;border-top:1px solid #555555;" />
 +
                <div style="text-align:left">Transformation, plate coating,37℃, overnight.</div>
 +
<br>
 +
                <center><div>2015.7.21</div></center>
 +
<hr style="height:1px;border:none;border-top:1px solid #555555;" />
 +
                <div style="text-align:left">Pinking the positive colonies, PCR detection, and then plasmid extraction. It was smooth! So, we got T7 RNAP-pYES2 and T7 RNAP-pSB1C3 successfully. We stored them in -20℃.</div>
 +
<img src="https://static.igem.org/mediawiki/2015/8/81/FAFU_CHINA_RESULT.jpg" alt="">
 +
<br>
 +
                <center><div>2015.7.23</div></center>
 +
<hr style="height:1px;border:none;border-top:1px solid #555555;" />
 +
                <div style="text-align:left">Today we went to observe the condition of swarms, and it was happy to see that the experimental groups turned to a healthier development comparing with control group. It meant that using dsRdRp expressed in E.coli to prevent and cure CSBV was working. Next, we would continue to collect more data until we draw the scientific conclusion.</div>
 +
<br>
 +
                <center><div>2015.7.26</div></center>
 +
<hr style="height:1px;border:none;border-top:1px solid #555555;" />
 +
                <div style="text-align:left">With the development of our project, we gradually have realized that even if using yeast can solve the problem of biosecurity, there are still other troubles, such as less expression of dsRdRp in yeast, easier to loss plasmids in yeast and harder to digest the cytoderm of yeast for Chinese honeybees.</div>
 +
<div style="text-align:left">So we need a better plan to deal with these to make a more practical production. That was what we would consider deeply in future work.</div>
 +
<br>
 +
                <center><div>2015.7.27</div></center>
 +
<hr style="height:1px;border:none;border-top:1px solid #555555;" />
 +
                <div style="text-align:left">Today we decided to constract RdRp-pSB1C3 again, because we thought T7 RNAP-pSB1C3 may be not a new part any more. So, we did linking. We decided if it is failed just like before this time. We had to change T4 into a faster ligase..</div>
 +
<br>
 +
                <center><div>2015.7.29</div></center>
 +
<hr style="height:1px;border:none;border-top:1px solid #555555;" />
 +
                <div style="text-align:left">As predicted, there was still nothing on the plate.(please tell us why you cannot grow out,bacteria!)</div>
 +
<br>
 +
                <center><div>2015.8.1</div></center>
 +
<hr style="height:1px;border:none;border-top:1px solid #555555;" />
 +
                <div style="text-align:left">We got the preliminary result of index of swarms. Next few days we would detect the expression quantity of CSBV in offspring.</div>
 +
<br>
 +
                <center><div>2015.8.2</div></center>
 +
<hr style="height:1px;border:none;border-top:1px solid #555555;" />
 +
                <div style="text-align:left">We made the figure of the effect of dsRdRp on the number of sac-like larvae. It showed that the number of infected larvae which were fed with HT115-dsRdRp in mediate and high concentration was remarkably less than the control group.</div>
 +
<br>
 +
                <center><div>2015.8.5</div></center>
 +
<hr style="height:1px;border:none;border-top:1px solid #555555;" />
 +
                <div style="text-align:left">Other two figures were made out, which showed the effect of dsRdRp on the number of sealed brood and the population of a colony respectively. They demonstrated the similar conclusion. </div>
 +
 
 +
<br>
 +
                <center><div>2015.8.8</div></center>
 +
<hr style="height:1px;border:none;border-top:1px solid #555555;" />
 +
                <div style="text-align:left">We detected the CSBV-carried rate of offspring by RT-PCR. After AGE, it showed that the CSBV-carried rate of first and second filial generation was 30% and 20% respectively, which was obviously declined.</div>
 +
<br>
 +
                <center><div>2015.8.12</div></center>
 +
<hr style="height:1px;border:none;border-top:1px solid #555555;" />
 +
                <div style="text-align:left">We detected the expression quantity of CSBV in offspring by RT-qPCR. We found with the decreasing of CSBV. The expression quantity of RdRp gene declined either, which meant that dsRdRp really could inhibit the replication of CSBV.</div>
 +
<br>
 +
                <center><div>Last final month</div></center>
 +
<hr style="height:1px;border:none;border-top:1px solid #555555;" />
 +
                <div style="text-align:left">Even though we were succeed in using dsRdRp expressed in proeukaryotic system to prevent and cure CSBV, we still had to take care of some problem we met. So next stage we will try to constract another plasmid with suicide gene to control the concentration of engineering bacteria. Further more, we want to use CRISPR-Cas9 system to make RdRp gene inserted into the genome of E.coli. So that we can solve the problem of biosecurity.</div>
 +
<br>
 +
<br>
 +
 
 +
             
 +
 
 +
        </div>
 +
</section>
 +
 +
</div>
 +
 
 +
</div>
 +
<style>
 +
.com__section--text
 +
{
 +
  height:auto;
 +
  overflow:auto;
 
}
 
}
−
#progress_bar_container ul li:hover{
+
.com
−
color: #000000;
+
{
 +
  background-image:url(https://static.igem.org/mediawiki/2015/f/f9/Fafu_bg22.jpg);
 +
  background-size:100%;
 +
  overflow-y:auto;
 
}
 
}
−
#maintext h2{
+
.com__content
−
float: left;
+
{
−
margin-left: -10px;
+
  background-image:none;
−
margin-top: 20px;
+
 
}
 
}
−
#maintext hr{
+
table
−
clear: both;
+
{
−
margin-left: -10px;
+
  background-color:rgba(0,0,0,0);
 
}
 
}
−
#maintext div{
+
</style>
−
float: left;
+
<script src="js/jquery-2.1.1.min.js" type="https://2015.igem.org/Template:FAFU_CHINA/guge/js/jquery-2-1-1-min?action=raw&amp;ctype=text/javascript"></script>
−
clear: right;
+
<script>
−
margin-top: 20px;
+
var link = $('.com__nav-link');
−
width: 100%;
+
var linkParent = link.parent('li');
 +
var section = $('.com__section');
 +
var sectionContent = section.find('*');
 +
var switchTab = function () {
 +
var p = $(this).parent('li');
 +
var i = p.index();
 +
var s = section.eq(i);
 +
var c = s.find('*');
 +
section.removeClass('active');
 +
sectionContent.removeAttr('style');
 +
s.addClass('active');
 +
c.css({
 +
transform: 'none',
 +
opacity: 1
 +
});
 +
linkParent.removeClass('active');
 +
p.addClass('active');
 +
return false;
 +
};
 +
link.on('click', switchTab);
 +
function activeFirst() {
 +
section.first().addClass('active');
 +
section.first().find('*').css({
 +
transform: 'none',
 +
opacity: 1
 +
});
 +
linkParent.first().addClass('active');
 
}
 
}
−
#maintext div img{
+
activeFirst();
−
float: left;
+
</script>
−
width: 7%;
+
<style>
−
margin-top:6px;
+
.firstHeading
−
margin-right: 1%;
+
{
−
}
+
  display:none;
−
#maintext div p{
+
−
float: left;
+
−
width: 92%;
+
 
}
 
}
 
</style>
 
</style>
−
<script type="text/javascript">
+
</body>
−
$(document).ready(function(){
+
−
var win=$(window); //得到窗口对象
+
−
+
−
$("#progress_bar_container ul li").click(function(){
+
−
var scrollTo = $('#h2_'+this.id);
+
−
 
+
−
$('html,body').animate({ scrollTop:scrollTo.offset().top -100},  500);
+
−
})
+
−
 
+
−
});
+
−
</script>
+
−
</p>
+
−
<div id="progress_bar_container" style="position:fixed;left:0px;top:100px;bottom:0px;background-image:url('https://static.igem.org/mediawiki/2015/c/c8/BIT_China_bg-content-blue.png');background-repeat:repeat;">
+
−
<ul>
+
−
<li id="anchor_week1"> Week 1</li>
+
−
<li id="anchor_week2">Week 2</li>
+
−
<li id="anchor_week3">Week 3</li>
+
−
<li id="anchor_week4">Week 4</li>
+
−
<li id="anchor_week5">Week 5</li>
+
−
<li id="anchor_week6">Week 6</li>
+
−
<li id="anchor_week7">Week 7</li>
+
−
<li id="anchor_week8">Week 8</li>
+
−
<li id="anchor_week9">Week 9</li>
+
−
<li id="anchor_week10">Week 10</li>
+
−
<li id="anchor_week11">Week 11</li>
+
−
<li id="anchor_week12">Week 12</li>
+
−
<li id="anchor_week13">Week 13</li>
+
−
<li id="anchor_week14">Week 14</li>
+
−
<li id="anchor_week15">Week 15</li>
+
−
<li id="anchor_week16">Week 16</li>
+
−
<li id="anchor_week17">Week 17</li>
+
−
<li id="anchor_week18">Week 18</li>
+
−
<li id="anchor_week19">Week 19</li>
+
−
<li id="anchor_week20">Week 20</li>
+
−
<li id="anchor_week21">Week 21</li>
+
−
<li id="anchor_week22">Week 22</li>
+
−
<li id="anchor_week23">Week 23</li>
+
−
</ul>
+
−
</div>
+
−
<div class="container" id="container_Notebook" >
+
−
+
−
<img src="https://static.igem.org/mediawiki/2015/6/64/BIT_China_title_Notebook.png" style="display:block;margin:0 auto;padding-top:120px;width:60%"/>
+
−
 
+
−
<div class="Notebook" >
+
−
+
−
<div id="maintext" style="float:left;">
+
−
<p>
+
−
We started our project in March, and we have three parts of experiment, basic circuits part, regulation part and Error-prone PCR part. Basic circuits part was finished by Zongzhi Lv's group (including Hui Luo) and Wenxin Bai's group (including Changxin Zhang and Jiaqi Xu), Regulation part was finished by Jinlin Li's group (including Jiadong Zhong and Pengcheng Zhang). Error-prone PCR was finished by Jing Yang's group(including Chengyi Li, Mingming Cao, Xiao Peng and Zeyan Li). The experiments of Regulation part consist of the construction and verification of two pH-induce promoters (P-atp2 and P-asr) and recombinase system. The main work of error-prone PCR (EP-PCR) gruop is to select different promoters by error-prone PCR. The duty of Basic circuits group is to construct our basic resistance sub-system and basic regulation system.
+
−
</p>
+
−
<h2 id="h2_anchor_week1">Week1 (Mar 16 -Mar 22)</h2>
+
−
<hr style="border:1px solid #cccccc;" />
+
−
<div>
+
−
<img src="https://static.igem.org/mediawiki/2014/2/28/Cornell-nb-dry.png" />
+
−
<p>
+
−
We extracted the genome of <i>Escherichia coli K12</i> strain and got the gene LdhA through PCR.
+
−
</p>
+
−
</div>
+
−
<div>
+
−
<img src="https://static.igem.org/mediawiki/2014/6/6f/Cornell-nb-wet.png" />
+
−
<p>
+
−
We have successfully got the genome of <i>E.coli K-12 DH5α</i>, preparing for the later use of cloning functional genes.<br/>
+
−
We have successfully got the functional gene glsA from the genome of E.coli K-12 DH5α by PCR, which encodes an acid-activated glutaminase.<br/>
+
−
Through digestion, ligation, transformation and cPCR, we have successfully selected the expected strain with glsA. The plasmid is pSB1A3.<br/>
+
−
</p>
+
−
</div>
+
−
 
+
−
<h2 id="h2_anchor_week2">Week2 (Mar 23 -Mar 29)</h2>
+
−
<hr style="border:1px solid #cccccc;" />
+
−
<div >
+
−
<img src="https://static.igem.org/mediawiki/2014/2/28/Cornell-nb-dry.png" />
+
−
<p>
+
−
We constructed the gene LdhA to the vector pET28a(we use restriction enzyme cutting sites EcoRI and XhoI), and transformed it into the BMTOP10.
+
−
</p>
+
−
</div>
+
−
<div>
+
−
<img src="https://static.igem.org/mediawiki/2014/6/6f/Cornell-nb-wet.png" />
+
−
<p>
+
−
We have successfully got the acid-induced promoter J100071 from the genome of E.coli K-12 DH5α.<br/>
+
−
Through digestion, ligation, transformation and cPCR, we have successfully selected the expected strain with J100071. The plasmid is pSB1A3.
+
−
</p>
+
−
</div>
+
−
 
+
−
<h2 id="h2_anchor_week3">Week3 (Mar 30 -April 5)</h2>
+
−
<hr style="border:1px solid #cccccc;" />
+
−
<div >
+
−
<img src="https://static.igem.org/mediawiki/2014/2/28/Cornell-nb-dry.png" />
+
−
<p>
+
−
We sequenced the gene LdhA and the result is right. Then we transform the plasmid LdhA-pET28a into the BL21DE3 and verified it firstly. The result of verification is success.
+
−
</p>
+
−
</div>
+
−
<div>
+
−
<img src="https://static.igem.org/mediawiki/2014/6/6f/Cornell-nb-wet.png" />
+
−
<p>
+
−
Through PCR, we constructed the device J23119+B0034+glsA(JBG) for further testing. The plasmid has been replaced by pSB1C3.<br/>
+
−
Through 3A assembly, we constructed the device J100071+B0034+E1010(RFP) for further testing. The plasmid has been replaced by pSB1C3.
+
−
</p>
+
−
</div>
+
−
 
+
−
<h2 id="h2_anchor_week4">Week4 (Apr 6 -April 12)</h2>
+
−
<hr style="border:1px solid #cccccc;" />
+
−
<div >
+
−
<img src="https://static.igem.org/mediawiki/2014/2/28/Cornell-nb-dry.png" />
+
−
<p>
+
−
We used same method to got and constructed other acid production gene, such as PflB(formic acid production), Pta and AckA(acetic acid production) and so on. At the same time, we verified the gene LdhA again.
+
−
</p>
+
−
</div>
+
−
<div>
+
−
<img src="https://static.igem.org/mediawiki/2014/6/6f/Cornell-nb-wet.png" />
+
−
<p>
+
−
Comparing to the strain with the plasmid carrying J23119, we tested the acid resistance ability of the strain with the plasmid carrying JBG.<br/>
+
−
We set a pH gradient pH=3, 4, 5, 6, 7 and measured the OD600.<br/>
+
−
However, the result is not ideal.<br/>
+
−
</p>
+
−
</div>
+
−
 
+
−
<h2 id="h2_anchor_week5">Week5 (Apr 13 -April 19)</h2>
+
−
<hr style="border:1px solid #cccccc;" />
+
−
<div >
+
−
<img src="https://static.igem.org/mediawiki/2014/2/28/Cornell-nb-dry.png" />
+
−
<p>
+
−
We finished the verification of LdhA(lactic acid production), PflB(formic acid production), Pta and AckA(acetic acid production) three times.
+
−
</p>
+
−
</div>
+
−
<div>
+
−
<img src="https://static.igem.org/mediawiki/2014/6/6f/Cornell-nb-wet.png" />
+
−
<p>
+
−
We measured the intensity of red florescence to test the function of J10071 promoter.
+
−
</p>
+
−
</div>
+
−
 
+
−
<h2 id="h2_anchor_week6">Week6 (Apr 20 -April 26)</h2>
+
−
<hr style="border:1px solid #cccccc;" />
+
−
<div >
+
−
<img src="https://static.igem.org/mediawiki/2014/2/28/Cornell-nb-dry.png" />
+
−
<p>
+
−
We finished the verification of DhaB(3-hydroxypropionic acid production), PhaC, PhaA and PhaB(3-hydroxybutyric acid production) three times.
+
−
</p>
+
−
</div>
+
−
<div>
+
−
<img src="https://static.igem.org/mediawiki/2014/6/6f/Cornell-nb-wet.png" />
+
−
<p>
+
−
We constructed the standard part with the functional gene glsA. The plasmid is pSB1C3.<br/>
+
−
We constructed the standard part with the acid-induced promoter J10071. The plasmid is pSB1C3.
+
−
</p>
+
−
</div>
+
−
 
+
−
<h2 id="h2_anchor_week7">Week7 (Apr 27 -May 3)</h2>
+
−
<hr style="border:1px solid #cccccc;" />
+
−
<div >
+
−
<img src="https://static.igem.org/mediawiki/2014/2/28/Cornell-nb-dry.png" />
+
−
<p>
+
−
We constructed all of acid production genes to the pSB1C3, as standard parts.
+
−
</p>
+
−
</div>
+
−
<div>
+
−
<img src="https://static.igem.org/mediawiki/2014/6/6f/Cornell-nb-wet.png" />
+
−
<p>
+
−
We did the second cell culture to test the function of glsA, but again, we didn’t get the expected results.<br/>
+
−
Comparing to the strain with the plasmid carrying J23119, we measured the intensity of proteins expressed in the strain with the plasmid carrying JBG.
+
−
</p>
+
−
</div>
+
−
 
+
−
<h2 id="h2_anchor_week8">Week8 (May 4 -May 10)</h2>
+
−
<hr style="border:1px solid #cccccc;" />
+
−
<div>
+
−
<img src="https://static.igem.org/mediawiki/2014/2/28/Cornell-nb-dry.png" />
+
−
<p>
+
−
We looked around many articles and decided to use P-atp2 and P-asr as pH-induced promoters. At the same time, we extracted the genome of Corynebacterium glutamicum ATCC 13032 strain.
+
−
</p>
+
−
</div>
+
−
<div>
+
−
<img src="https://static.igem.org/mediawiki/2014/6/6f/Cornell-nb-wet.png" />
+
−
<p>
+
−
We did the second cell culture to test the function of glsA, but again, we didn’t get the expected results.<br/>
+
−
Comparing to the strain with the plasmid carrying J23119, we measured the intensity of proteins expressed in the strain with the plasmid carrying JBG.
+
−
</p>
+
−
</div>
+
−
 
+
−
<h2 id="h2_anchor_week9">Week9 (May 11 -May 17)</h2>
+
−
<hr style="border:1px solid #cccccc;" />
+
−
<div>
+
−
<img src="https://static.igem.org/mediawiki/2014/2/28/Cornell-nb-dry.png" />
+
−
<p>
+
−
We obtained the sequence of P-atp2 and P-asr by articles. We tried to constructed P-atp2 to pSB1C3, but there is an EcoRI sites exist in P-atp2. So we chose another vector pXMJ19 to construct it.
+
−
</p>
+
−
</div>
+
−
<div>
+
−
<img src="https://static.igem.org/mediawiki/2014/6/6f/Cornell-nb-wet.png" />
+
−
<p>
+
−
We did the third cell culture to test the function of glsA, but again, we didn’t get the expected results. We have improved our experimental method, but there are still a lot of difficulties. It’s hard to realize the synchronous culture between the two strain because the different activities of preserved bacteria.
+
−
</p>
+
−
</div>
+
−
 
+
−
<h2 id="h2_anchor_week10">Week10 (May 18 -May 24)</h2>
+
−
<hr style="border:1px solid #cccccc;" />
+
−
<div>
+
−
<img src="https://static.igem.org/mediawiki/2014/2/28/Cornell-nb-dry.png" />
+
−
<p>
+
−
We got P-atp2(Corynebacterium glutamicum ATCC 13032) and P-asr(Escherichia coli K12) through PCR. And linked them to pXMJ19 and pSB1C3 respectively. Then transformed them into BMTOP10.
+
−
</p>
+
−
</div>
+
−
 
+
−
<h2 id="h2_anchor_week11">Week11 (May 25 -May 31)</h2>
+
−
<hr style="border:1px solid #cccccc;" />
+
−
<div>
+
−
<img src="https://static.igem.org/mediawiki/2014/2/28/Cornell-nb-dry.png" />
+
−
<p>
+
−
We used LacZ alpha as a report gene and we verified the strength of two promoters under the different pH environment. The result is success.
+
−
</p>
+
−
</div>
+
−
+
−
 
+
−
<h2 id="h2_anchor_week12">Week12 (Jun 1 -Jun 7)</h2>
+
−
<hr style="border:1px solid #cccccc;" />
+
−
<div>
+
−
<img src="https://static.igem.org/mediawiki/2014/2/28/Cornell-nb-dry.png" />
+
−
<p>
+
−
We designed recombinase system to fine-regulation. Meanwhile, we wanted to build a library of pH-induced promoters by error-prone PCR. So we divided to two groups to finish these works. And this week, we searched and looked around the articles to find suitable recombinase and fit condition of EP-PCR.
+
−
</p>
+
−
</div>
+
−
<div>
+
−
<img src="https://static.igem.org/mediawiki/2014/6/6f/Cornell-nb-wet.png" />
+
−
<p>
+
−
Comparing to the strain with the plasmid carrying J23119, we measured the intensity of proteins expressed in the strain with the plasmid carrying JBG again.
+
−
</p>
+
−
</div>
+
−
 
+
−
<h2 id="h2_anchor_week13">Week13 (Jun 8 -Jun 14)</h2>
+
−
<hr style="border:1px solid #cccccc;" />
+
−
<div>
+
−
<img src="https://static.igem.org/mediawiki/2014/2/28/Cornell-nb-dry.png" />
+
−
<p>
+
−
This week, we confirmed the recombinase(Cre, Flp, Bxb1 and FimE) we used in our gene circuits and the condition of EP-PCR.
+
−
</p>
+
−
</div>
+
−
+
−
<h2 id="h2_anchor_week14">Week14 (Jun 15 -Jun 21)</h2>
+
−
<hr style="border:1px solid #cccccc;" />
+
−
<div>
+
−
<img src="https://static.igem.org/mediawiki/2014/2/28/Cornell-nb-dry.png" />
+
−
<p>
+
−
We extracted the genome of Saccharomyces cerevisiae and transformed mRFP, GFP, K137007(FimE), K907000(Bxb1 gp35) and K907001(Bxb1 gp47) from iGEM kit plates into BMTOP10. Simultaneously, we did the mutation of P-atp2 firstly and selected 157 positive bacteria from more than 500 bacteria
+
−
</p>
+
−
</div>
+
−
 
+
−
<h2 id="h2_anchor_week15">Week15 (Jun 22 -Jun 28)</h2>
+
−
<hr style="border:1px solid #cccccc;" />
+
−
<div>
+
−
<img src="https://static.igem.org/mediawiki/2014/2/28/Cornell-nb-dry.png" />
+
−
<p>
+
−
This week, we had final examination. We didn't have enough time to do any experiments. So we searched and looked around large amount of articles to optimize our experiments.
+
−
</p>
+
−
</div>
+
−
 
+
−
<h2 id="h2_anchor_week16">Week16 (Jun 29 -Jul 5)</h2>
+
−
<hr style="border:1px solid #cccccc;" />
+
−
<div>
+
−
<img src="https://static.igem.org/mediawiki/2014/2/28/Cornell-nb-dry.png" />
+
−
<p>
+
−
We constructed the device K137058(ILL+pTac+ILR+GFP) according to iGEM and linked it with mRFP. We found a better approach of EP-PCR, so we did again.
+
−
</p>
+
−
</div>
+
−
<div>
+
−
<img src="https://static.igem.org/mediawiki/2014/6/6f/Cornell-nb-wet.png" />
+
−
<p>
+
−
We measured the intensity of red florescence at different pH to test the function of acid-induced promoter J100071 again.
+
−
</p>
+
−
</div>
+
−
 
+
−
<h2 id="h2_anchor_week17">Week17 (Jul 6 -Jul 12)</h2>
+
−
<hr style="border:1px solid #cccccc;" />
+
−
<div>
+
−
<img src="https://static.igem.org/mediawiki/2014/2/28/Cornell-nb-dry.png" />
+
−
<p>
+
−
We constructed a new part P-atp2+B0034+K137007(FimE) by over-lap extension PCR(OE-PCR) and transformed it into BMTOP10. In this week, we did amounts of EP-PCR, and were ready to select them next week.
+
−
</p>
+
−
</div>
+
−
<div>
+
−
<img src="https://static.igem.org/mediawiki/2014/6/6f/Cornell-nb-wet.png" />
+
−
<p>
+
−
Through PCR, digestion, ligation, transformation and cPCR, we have successfully constructed the standard part glsA on pSB1C3.
+
−
</p>
+
−
</div>
+
−
 
+
−
<h2 id="h2_anchor_week18">Week18 (Jul 13 -Jul 19)</h2>
+
−
<hr style="border:1px solid #cccccc;" />
+
−
<div>
+
−
<img src="https://static.igem.org/mediawiki/2014/2/28/Cornell-nb-dry.png" />
+
−
<p>
+
−
We constructed a new part P-atp2+B0034+K907000(Bxb1 gp35)+K907001(Bxb1 gp47) by OE-PCR and transformed it into BMTOP10. And we started to selected the positive bacteria from our transformant.
+
−
</p>
+
−
</div>
+
−
<div>
+
−
<img src="https://static.igem.org/mediawiki/2014/6/6f/Cornell-nb-wet.png" />
+
−
<p>
+
−
We did the fourth cell culture to increase the experimental data and analyzed the common features among the four experiments. However, we didn’t get the expected results again. But we have learned some lessons.
+
−
</p>
+
−
</div>
+
−
 
+
−
<h2 id="h2_anchor_week19">Week19 (Jul 20 -Jul 26)</h2>
+
−
<hr style="border:1px solid #cccccc;" />
+
−
<div>
+
−
<img src="https://static.igem.org/mediawiki/2014/2/28/Cornell-nb-dry.png" />
+
−
<p>
+
−
This week, most members of our two groups attended the iGEM conference held by National Chiao Tung University. So the plenty of members selected others bacteria together.
+
−
</p>
+
−
</div>
+
−
<div>
+
−
<img src="https://static.igem.org/mediawiki/2014/6/6f/Cornell-nb-wet.png" />
+
−
<p>
+
−
We added a terminator downstream the gene glsA. Through 3A assembly, we have successfully constructed the complete standard part. Through the result of sequence measuring, it turns out to be completely right.
+
−
</p>
+
−
</div>
+
−
 
+
−
<h2 id="h2_anchor_week20">Week20 (Jul 27 -Aug 2)</h2>
+
−
<hr style="border:1px solid #cccccc;" />
+
−
<div>
+
−
<img src="https://static.igem.org/mediawiki/2014/2/28/Cornell-nb-dry.png" />
+
−
<p>
+
−
We artificially synthesized attP and attB, the recognized sites of Bxb1, by Genwiz, so that we should wait for them. And we totally selected 1082 positive bacteria. We used LacZ alpha as a report gene and started the verification of them.
+
−
</p>
+
−
</div>
+
−
<div>
+
−
<img src="https://static.igem.org/mediawiki/2014/6/6f/Cornell-nb-wet.png" />
+
−
<p>
+
−
We have improved our experiment methods and carried out the fifth cell culture. Considering the pH might change after cell culture, we added buffer to control the pH. Moreover, we tried to realize the the synchronous culture by choosing the single bacteria on the plate. Unluckily, the results didn’t meet our requirements.
+
−
</p>
+
−
</div>
+
−
 
+
−
 
+
−
<h2 id="h2_anchor_week21">Week21 (Aug 3 -Aug 9)</h2>
+
−
<hr style="border:1px solid #cccccc;" />
+
−
<div>
+
−
<img src="https://static.igem.org/mediawiki/2014/2/28/Cornell-nb-dry.png" />
+
−
<p>
+
−
This week, we got a measure that we helped Beijing Normal University(BNU) to finish their gene circuit. So we transformed the gene I15008(ho1) and I15009(PcyA) from iGEM kit plates into BMTOP10. And we still did the verification of our mutants.
+
−
</p>
+
−
</div>
+
−
<div>
+
−
<img src="https://static.igem.org/mediawiki/2014/6/6f/Cornell-nb-wet.png" />
+
−
<p>
+
−
We measured the intensity of proteins again. It showed that the expression of glsA is critically low. So, we planned to change a strong promoter T7.
+
−
</p>
+
−
</div>
+
−
 
+
−
<h2 id="h2_anchor_week22">Week22 (Aug 10 -Aug 16)</h2>
+
−
<hr style="border:1px solid #cccccc;" />
+
−
<div>
+
−
<img src="https://static.igem.org/mediawiki/2014/2/28/Cornell-nb-dry.png" />
+
−
<p>
+
−
We got the BNU's part (J23100+B0034+PcyA+B0034+ho1+B0015), and constructed it to the pSB1C3. We still verified the mutants this week.
+
−
</p>
+
−
</div>
+
−
<div>
+
−
<img src="https://static.igem.org/mediawiki/2014/6/6f/Cornell-nb-wet.png" />
+
−
<p>
+
−
Referring to the paper we read, we did a small experiment to measure the proportion of living cells under extreme pH 3.0, both the testing group and the comparison group didn’t grow well. The difference is not evident.
+
−
</p>
+
−
</div>
+
−
 
+
−
<h2 id="h2_anchor_week23">Week23 (Aug 17 -Aug 23)</h2>
+
−
<hr style="border:1px solid #cccccc;" />
+
−
<div>
+
−
<img src="https://static.igem.org/mediawiki/2014/2/28/Cornell-nb-dry.png" />
+
−
<p>
+
−
We finished the gene circuits of BNU and constructed the new device P-atp2+B0034+K137007(FimE)+B0015+mRFP+ILL+pTac+ILR+B0034+GFP+B0015 to pSB1C3. We sorted part of our verification data and we were ready to verify the optimize mutants.
+
−
</p>
+
−
</div>
+
−
<div>
+
−
<img src="https://static.igem.org/mediawiki/2014/6/6f/Cornell-nb-wet.png" />
+
−
<p>
+
−
Through OE-PCR, digestion, ligation, transformation and cPCR, we planned to ligate p-atp2+B0034+LdhA. However, we cannot select the right one. After analysis, there were some problems with our plasmid, so we replace it and plan to try again.
+
−
</p>
+
−
</div>
+
−
 
+
−
 
+
−
</div>
+
−
</div>
+
−
</div>
+
−
 
+
−
</div></div><!---->
+
 
</html>
 
</html>

Latest revision as of 21:19, 18 September 2015

侧边栏



Notebook

2015.6.8
Today was the first day that we began our project in the lab after we made a scientific plan. Firstly, we extracted the whole RNA of CSBV by Trizol. As we all know, RNA is easy to be digested in the environment. Therefore although we did this quickly, there were still some samples without RNA fragment. But we did again and the result was satisfied at that time. It was encouraging!

2015.6.9
Today we made RT-PCR with four sets of reverse specific primers and the RNA of CSBV to synthesize homologous cDNA(CSBV Helicase、Protease、VP1 and RdRp respectively). Then dsDNAs was amplified with four sets of specific primers by PCR. It went through smoothly. So far, we got the target fragments successfully. Also,we used GFP F/R to amplify GFP gene.

2015.6.10
We utilized T7 RiboMAXTM Express RNAi System kit and dsDNAs as templates to synthesize dsRNAs(dsHelicase、dsProtease、dsVP1、dsRdRp and dsGFP), but there were only dsHelicase and dsGFP we could get. We tried again and still failed. It took our whole day to find the reason. Tomorrow we will ask our advisor for some solutions and do it one more time.

2015.6.12
After figuring out the problem, we eventually got the all right dsRNAs. Another part, we began to prepare the larvae of Chinese honeybee which was offered by our institute of bees.
2015.6.13
We put dsRNAs into fodder of the larvae of the honeybee to feed them. Then after 12h, we fed them another fodder with CSBV extracting solution. We did so twice before they got normal fodder.

2015.6.17
During three days, we observed the change of percentage of pupation and collect and analyse data. Meanwhile,RT-qPCR was done to detect the effect of different dsRNAs to CSBV. According to the result, we found that dsRdRp made a remarkable influence on the replication of CSBV. So we decided to use dsRdRp to inference CSBV.

2015.6.19
We used cDNA of RdRp and specific primers(Not1-F/Pst1-R、EcoR1-F/Pst1-R) to synthesize RdRp gene. However, after agarose gel electrophoresis(AGE), we could not find our target fragment.

2015.6.20
We changed a better DNA polymerase and set the annealing temperature to 60℃. Finally we got the RdRp gene.

2015.6.22
We picked the colonies to do PCR and sent the positive sequencing(M13).

2015.6.24
Today we got the result of sequencing. After comparing with data in NCBI, we only had two 100 percent samples. We started to do double enzyme digestion(Not1 and Pst1、EcoR1 and Pst1) to cut vector L4440、pSB1C3 and RdRp-T recombinant plasmid.

2015.6.25
We made RdRp gene link with T vector in 25℃, 15min and then transformed it into DH5α,a kind of competent cell, which was cultured in 37℃,overnight.

2015.6.26
We made it! And we immediately did gel recovery. Then we began to link RdRp with L4440 and pSB1C3 using T4 ligase, 4℃, overnight.

2015.6.27
We did transformation. However,we forgot to make LB medium with CmRR. So the transformation of RdRp-pSB1C3 could only be done tomorrow.

2015.6.29
Today there was two news. The good one was we successfully got recombinant plasmid RdRp-L4440 while the bad one was nothing grew on the plate with CmRR. We thought maybe it was because RdRp did not link with pSB1C3 successfully. Anyway we would do it again tomorrow.

2015.7.2
On the one hand, we still could not link RdRp with pSB1C3 so that there still nothing on the plate. We wonder if it was because of the efficiency of ligase or anything. On the other hand,we prepared another competent cell,HT115,which included T7 RNAP gene in its genome. It was used as engineering bacteria for expressing dsRdRp.
Also, considering the biosecurity of E.coli, we decided use yeast as the final transformation target. So we want to recombine another vector,T7 RNAP-pYES2.

2015.7.3
We transformed RdRp-L4440 into HT115,culturing in 37℃. Overnight. And T7 RNAP gene was amplified by PCR(EcoR1-F/Xho1-R).

2015.7.4
Today we used IPTG, which was divided into two groups with different concentrations(0.4mmol/ml、0.8mmol/ml), to induce the expression of dsRdRp in HT115 for 5h. Then we collected the bacteria to extract dsRdRp by CTAB. According to the result of AGE, dsRdRp had been expressed in HT115 successfully, even though its concentration was a little bit lower. It was really a good news for us!

2015.7.5
We added engineering bacteria with expressed dsRdRp, which was divided into three groups with different concentrations(low、mediate and high), into the fodder of the infected swarms. And another infected swarm was fed normal fodder. Since now, we will obverse the change of population of them, the mortality, and the number of sealed brood and collect data every weeks until we get the obvious result.

2015.7.7
Considering we have not gotten RdRp-pSB1C3 yet,we decided to link T7 RNAP gene to pSB1C3 first. So we used specific primers(EcoR1-F/Pst1-R) to synthesize T7 RNAP gene by PCR.

2015.7.8
Transformation again!

2015.7.9
As usual, pinking the positive colonies, PCR and sequencing. We hope everything goes smoothly.

2015.7.11
According to the result of sequencing, there was no sample 100 percent matching. We did transformation again.

2015.7.13
The second sequencing was completely failed because the sequencing company could not get any plasmid. We thought maybe the replication of plasmid in competent cell(DH5α) was inefficient, so we changed another one,T1,bought from Transgene company.

2015.7.14
This time we sent plasmid rather than bacteria. Although the concentration of the plasmid was a little bit lower, it was enough to do sequencing.

2015.7.15
Considering the lower fidelity of the enzyme used for T7 amplification before, we changed into a better one called KD Plus and restarted to PCR, linking and transformation.

2015.7.16
The third sequencing still showed nothing right. We were a little disappointed, but we exactly knew that failure was a normal phenomenon in the lab.

2015.7.18
Eventually,we got the matching sample after using KD Plus. Immediately, we began to do double enzyme digestion(EcoR1/Xho1、EcoR1/Pst1).

2015.7.19
After gel recovery, we linked our target fragments to relative vectors with T4 ligase, 4℃, overnight.
Meanwhile, we collected data about the index of the condition of swarms again, which was used to analyze the effect of dsRdRp by Excel later.

2015.7.20
Transformation, plate coating,37℃, overnight.

2015.7.21
Pinking the positive colonies, PCR detection, and then plasmid extraction. It was smooth! So, we got T7 RNAP-pYES2 and T7 RNAP-pSB1C3 successfully. We stored them in -20℃.

2015.7.23
Today we went to observe the condition of swarms, and it was happy to see that the experimental groups turned to a healthier development comparing with control group. It meant that using dsRdRp expressed in E.coli to prevent and cure CSBV was working. Next, we would continue to collect more data until we draw the scientific conclusion.

2015.7.26
With the development of our project, we gradually have realized that even if using yeast can solve the problem of biosecurity, there are still other troubles, such as less expression of dsRdRp in yeast, easier to loss plasmids in yeast and harder to digest the cytoderm of yeast for Chinese honeybees.
So we need a better plan to deal with these to make a more practical production. That was what we would consider deeply in future work.

2015.7.27
Today we decided to constract RdRp-pSB1C3 again, because we thought T7 RNAP-pSB1C3 may be not a new part any more. So, we did linking. We decided if it is failed just like before this time. We had to change T4 into a faster ligase..

2015.7.29
As predicted, there was still nothing on the plate.(please tell us why you cannot grow out,bacteria!)

2015.8.1
We got the preliminary result of index of swarms. Next few days we would detect the expression quantity of CSBV in offspring.

2015.8.2
We made the figure of the effect of dsRdRp on the number of sac-like larvae. It showed that the number of infected larvae which were fed with HT115-dsRdRp in mediate and high concentration was remarkably less than the control group.

2015.8.5
Other two figures were made out, which showed the effect of dsRdRp on the number of sealed brood and the population of a colony respectively. They demonstrated the similar conclusion.

2015.8.8
We detected the CSBV-carried rate of offspring by RT-PCR. After AGE, it showed that the CSBV-carried rate of first and second filial generation was 30% and 20% respectively, which was obviously declined.

2015.8.12
We detected the expression quantity of CSBV in offspring by RT-qPCR. We found with the decreasing of CSBV. The expression quantity of RdRp gene declined either, which meant that dsRdRp really could inhibit the replication of CSBV.

Last final month
Even though we were succeed in using dsRdRp expressed in proeukaryotic system to prevent and cure CSBV, we still had to take care of some problem we met. So next stage we will try to constract another plasmid with suicide gene to control the concentration of engineering bacteria. Further more, we want to use CRISPR-Cas9 system to make RdRp gene inserted into the genome of E.coli. So that we can solve the problem of biosecurity.