Team:SEU A/Notebook

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<header>
<header>
-
<h1>Safety</h1>
+
<h1>Notebook</h1>
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<span>Aiming to construct a safe and creative lab environment.</span>
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<span>This is our experiment diary.</span>
</header>
</header>
             <table id="toc" class="toc" style="margin:0 0 auto auto; background:none; padding:10px;">
             <table id="toc" class="toc" style="margin:0 0 auto auto; background:none; padding:10px;">
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                   <div id="toctitle"><h2>Index</h2> </div>
                   <div id="toctitle"><h2>Index</h2> </div>
<ul>
<ul>
-
<li class="toclevel-1 tocsection-1"><a href="#.A0"><span class="tocnumber">1</span> <span class="toctext">Biosafety</span></a>
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<li class="toclevel-1 tocsection-1"><a href="#.A0"><span class="tocnumber">1</span> <span class="toctext">Learning Period</span></a>
<ul>
<ul>
<li class="toclevel-2 tocsection-1"><a href="#.A1"><span class="tocnumber">&nbsp;&nbsp;&nbsp;1.1</span> <span class="toctext">Key words</span></a></li>
<li class="toclevel-2 tocsection-1"><a href="#.A1"><span class="tocnumber">&nbsp;&nbsp;&nbsp;1.1</span> <span class="toctext">Key words</span></a></li>
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</ul>
</ul>
</li>
</li>
-
<li class="toclevel-1 tocsection-2"><a href="#.B0"><span class="tocnumber">2</span> <span class="toctext">Laboratory Biosafety</span></a></li>
+
<li class="toclevel-2 tocsection-0"><a href="#.B0"><span class="tocnumber">2</span><span class="toctext">Period 2</span></a></li>
-
<li class="toclevel-1 tocsection-3"><a href="#.C0"><span class="tocnumber">3</span> <span class="toctext">BioBrick Safety</span></a></li>
+
<li class="toclevel-3 tocsection-0"><a href="#.C0"><span class="tocnumber">3</span><span class="toctext">Period 3</span></a></li>
-
<li class="toclevel-1 tocsection-4"><a href="#.D0"><span class="tocnumber">4</span> <span class="toctext">Local biosafety Group\Committee</span></a></li>
+
<li class="toclevel-4 tocsection-0"><a href="#.D0"><span class="tocnumber">4</span><span class="toctext">Period 4</span></a></li>
-
<li class="toclevel-1 tocsection-5"><a href="#.E0"><span class="tocnumber">5</span> <span class="toctext">Biosafety @ SEU</span></a></li>
+
<li class="toclevel-5 tocsection-0"><a href="#.E0"><span class="tocnumber">5</span><span class="toctext">Period 5</span></a></li>
 +
<li class="toclevel-6 tocsection-0"><a href="#.F0"><span class="tocnumber">6</span><span class="toctext">Period 6</span></a></li>
</ul>
</ul>
                 </td>
                 </td>
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             </table>
             </table>
-
       </br><h2> <span class="mw-headline" id=".A0">1  Biosafety</span></h2></br>
+
       </br><h2> <span class="mw-headline" id=".A0">Period 1  Biosafety</span></h2></br>
-
      &nbsp;&nbsp;&nbsp;<span class="mw-headline" id=".A1">1.1 Key words</br></br>
+
-
      Biosafety is used to describe efforts to reduce and eliminate the potential risks resulting from biotechnology and its products. It has similarly been defined as “the avoidance of risk to human health and safety, and to the conservation of the environment, as a result of the use for research and commerce of infectious or genetically modified organisms” (Zaid, 2001).</br></br>
+
-
      &nbsp;&nbsp;&nbsp;<span class="mw-headline" id=".A2">1.2 Researcher Safety</span></br></br>
+
-
      It is impossible to work in a laboratory environment without its risks, so we took a variety of steps to make sure the safety of our team during our lab work. All our team members are students in the school of life sciences and are well trained through teaching and lab rotations. Protective measures involve gloves, masks and lab coats. The labs we use are equipped with safety facilities, so researcher safety is guaranteed.</br></br>
+
-
      &nbsp;&nbsp;&nbsp;<span class="mw-headline" id=".A3">1.3 Public Safety</span></br></br>
+
-
      We just use the common competent cells: BL21 ,DH5αwhich we gain them from invetrogen and HB101 strain from the military medicine research institution in china, all of them are harmless to the public. National standards link:</br>
+
-
<a href="http://www.cnca.gov.cn/cnca/zwxx/xwdt/zxtz/images/20080310/3873.pdf">http://www.cnca.gov.cn/cnca/zwxx/xwdt/zxtz/images/20080310/3873.pdf</a></br>
+
-
      Also, the plasmids and DNA fragments that we use are mostly provided by iGEM. Furthermore, all the stuff will be autoclaved sterilization before we discard them. None of these parts involves direct interaction with the public, so there is little risk to public safety from our project.</br></br>
+
-
      &nbsp;&nbsp;&nbsp;<span class="mw-headline" id=".A4">1.4 Environment Safety</span></br></br>
+
-
      There is no way that our experiment bacteria can spread outside our laboratory. Thus, the external environment will not be contaminated by our experiment bacteria. This is actually a common feature in many other researches. Plus, all plates will be boiled thoroughly to kill the bacteria before disposal. So, our project does not pose a risk to environmental safety.</br></br>
+
-
      </br><h2> <span class="mw-headline" id=".B0">2  Laboratory Biosafety</span></h2></br>
+
2012/04/05-2012/06/23
-
      This part is mainly focused on Health Risks and Personal Safety, Working in the lab always has implicit risks, but we have taken action to minimize them. Everyone engages in the experiment must wear biohazard gloves and mouth-muffles, on one hand to protect themselves, on other hand to prevent the unnecessary enzymes. Before leaving the laboratory, the instructors will guide us to check all the experimental facilities including sterile the various containers, take off the water bath and other risky facilities. We also be required to washing our hands with the disinfectant to ensure the personal safety.</br></br>
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学习阶段:实验技能学习、实验理论学习,掌握从已有工程菌得到希望构建的工程菌的过程</br>
 +
设计从污水中分离培养蛭弧菌方案</br>
 +
Learning stage : Learn about the skill and theory of Molecular Biology Experiment.
 +
Set up the method of isolation and culture BD from sewage.
-
      </br><h2> <span class="mw-headline" id=".C0">3  BioBrick Safety</span></h2></br>
+
             
-
      Our project consider about exploiting new methods to deal with the antibiotic issues, the biobrick safety matters that we may involved in is that we construct a plasmid with the conjugation function in order to spread our target DNA fragment. For the sake of preventing the plasmid broadcast some unknown function genes, we ensure that every used liquid medium and gel solid medium are sterile before disposal. Well, all the other DNA fragments no matter we used or modified are achieved from the kit plates or commonly employed in laboratory.</br></br>
+
-
       </br><h2> <span class="mw-headline" id=".D0">4 Local Biosafety Group\Committee</span></h2></br>
+
2012/04/05
-
      In China, the main concerns for biosafety are pathogenic microbes and the genetically modified crops. Our experiment is designed and carried out following the standards of national biosafety office, therefore no laws or regulations regarding the biosafety issue are violated.</br>
+
LB培养基的制备、高压蒸汽灭菌锅的使用</br>
-
      &nbsp;&nbsp;&nbsp;<a href="http://www.cnca.gov.cn/cnca/zwxx/xwdt/zxtz/images/20080310/3873.pdf">http://www.cnca.gov.cn/cnca/zwxx/xwdt/zxtz/images/20080310/3873.pdf</a></br>
+
LB  medium preparation . Learn the usage of autoclaves.
-
      &nbsp;&nbsp;&nbsp;The laboratory that we worked in is the State Key Laboratory of Bioelectronics which has a serious biosafety constitution. And all new experiments would be weighed on safety by experienced teachers before execution.</br>
+
细菌的培养
-
      &nbsp;&nbsp;&nbsp;In addition, we have meticulously refer to the rules made by Experiment Center of Biomaterial and Biotechnology. For further information of this set of rules, please visit the website:</br>
+
2012/04/06
-
      &nbsp;&nbsp;&nbsp;<a href="http://www.lmbe.seu.edu.cn/cailiao/guizhangzhidu.html">http://www.lmbe.seu.edu.cn/cailiao/guizhangzhidu.html</a>
+
利用BIOMIGA质粒小量提取试剂盒进行大肠杆菌质粒提取</br>
-
</br></br>
+
2012/04/12
 +
    购买农业用蛭弧菌混合液,从中分离培养蛭弧菌</br>
 +
2012/04/14
 +
    蛭弧菌长出噬菌斑,分离纯化培养</br>
 +
2012/04/21
 +
    关于酶切、酶接相关实验技术学习</br>
 +
2012/05/18
 +
2012年kit寄到,取kit进行转化,第一个转化I13521,命名“小红菌”</br>
 +
线性化质粒骨架酶切</br>
 +
2012/05/19
 +
转化涂板技能练习</br>
 +
2012/05/20
 +
挑单克隆,液体培养基振荡过夜培养</br>
 +
2012/05/22
 +
PCR体系配置和技能练习</br>
 +
2012/05/23
 +
优化酶接是骨架和片段的比例</br>
 +
2012/05/25
 +
蛭弧菌菌液PCR,条带很弱,显示其生长不好或已变种</br>
 +
2012/06/13
 +
所构建的工程菌保种</br>
 +
 
 +
       </br><h2> <span class="mw-headline" id=".B0">Period 2 Biosafety</span></h2></br>
 +
 
 +
Jun 26-Jul 12
 +
整体方案敲定,如下所示:
 +
 +
 
 +
Death gene验证通路确定,如下:
 +
 +
蛭弧菌培养如火如荼</br>
 +
Sweet gene的钓取</br>
 +
2012/06/26
 +
用小红菌作为营养物质培养异养型蛭弧菌</br>
 +
2012/06/28
 +
K117010、I14032kit转化,构建工程菌,并设置对照实验验证其功能</br>
 +
2012/06/29
 +
设置对照组,用活的&死的大肠杆菌培养蛭弧菌</br>
 +
2012/06/30
 +
K117010功能验证实验</br>
 +
I14032质粒小提,凝胶回收</br>
 +
2012/07/01
 +
0630阳性、阴性均未长菌,0628所转菌为假阳性</br>
 +
0630摇R0010质粒小提</br>
 +
2012/07/02
 +
基因组分别钓取groES、groEL基因</br>通过PCR</br>
 +
2012/07/03
 +
转化K117010、B0034、K117000涂板技术及使用菌液量改进</br>
 +
复苏小红菌</br>
 +
2012/07/04
 +
K117010、B0034、K117000、R0010、B0015摇菌</br>
 +
2012/07/05
 +
蛭弧菌双层琼脂培养基分开涂板,计算E.coli菌落扩散速率及蛭弧菌游动并捕食E.coli的速率</br>
 +
2012/07/06
 +
蛭弧菌半定量PCR引物重新设计购买</br>
 +
2012/07/07
 +
跟进蛭弧菌、小红菌菌斑扩增速率</br>
 +
2012/07/09
 +
K117000、B0015双酶切</br>
 +
复苏K117000、K117010、B0034、R0010、B0015</br>
 +
2012/07/10
 +
质粒小提K117000、K117010、B0034、R0010、B0015</br>
 +
K117000使用通用引物PCR,20ul体系</br>
 +
2012/07/11
 +
质粒小提K117000、B0015</br>
 +
小红菌摇菌,用作蛭弧菌的食物</br>
 +
转化K117000,B0015</br>
 +
摇菌K117010、R0010、B0034、B0015.
 +
2012/07/12
 +
蛭弧菌菌液PCR验证</br>
 +
K117010、R0010、B0015菌液PCR</br>
 +
保种B0034、R0010、K117010、B0015,传代培养R0010、B0034、B0015</br>
 +
K117000挑单菌落,BL21摇菌</br>
 +
B0034、B0015酶切</br>
 +
 
 +
      </br><h2> <span class="mw-headline" id=".C0">Period 3  Biosafety</span></h2></br>
 +
 
 +
Jul 13--Jul 21
 +
Conjugation支路的构建:
 +
 +
趋化性支路的构建:
 +
 +
成功钓取groE并含标准组件所需的酶切位点
 +
2012/07/13
 +
0712酶切体系跑胶回收</br>
 +
质粒小提B0034、B0015</br>
 +
R0010、B0034通用引物PCR</br>
 +
酶接体系配制R0010+B0034</br>
 +
转化酶接产物R0010+B0034,转化J01003、K117000</br>
 +
groES、groEL基因钓取</br>
 +
2012/07/14
 +
J01003、R0010+B0034、K117000挑单菌落</br>
 +
2012/07/15
 +
质粒小提K117000、J01003、R0010+B0034
 +
K117000、J01003、R0010+B0034使用通用引物PCR</br>
 +
2012/07/16
 +
R0010、K117010复苏</br>
 +
R0010+B0034菌液PCR</br>
 +
转化J01003</br>
 +
2012/07/17
 +
R0010、K117010质粒小提
 +
K117010、J01003、R0010+B0034质粒、R0010+B0034菌液、K117000 PCR</br>
 +
2012/07/18
 +
J01003、K537001转化</br>
 +
自制感受态DH5α</br>
 +
2012/07/19
 +
K117010、B0015、groEL、K117010(自主设计引物)</br>
 +
2012/07/20
 +
groE基因钓取PCR</br>
 +
J01003质粒小提</br>R0010、K117010转化</br>
 +
 +
图3.从基因组钓取的groE基因电泳图
 +
(中间为D15000 Marker)
 +
2012/07/21
 +
R0010、K117010挑单菌落</br>
 +
R0010、K117010质粒小提</br>
 +
K537001转化</br>
 +
接合转移实验验证</br>
 +
 
 +
 
 +
      </br><h2> <span class="mw-headline" id=".D0">Period 4  Biosafety</span></h2></br>
 +
 
 +
Jul 22-Aug 17
 +
Death gene 验证通路构建成功</br>
 +
Conjugation通路构建成功,并将其连接至标准载体</br>
 +
Sweet gene表达载体的构建</br>
 +
 
 +
2012/07/22
 +
PCR退火最适温度实验探究</br>
 +
2012/07/23
 +
转化J01001、R0084
 +
0721接合转移菌株质粒检测</br>
 +
2012/07/24
 +
K537001复苏摇菌</br>
 +
R0084、B0015挑单菌落</br>
 +
2012/07/25
 +
半固体培养基配制</br>
 +
质粒小提J01001、R0084</br>
 +
K537001、R0084、J01001、groE菌液PCR</br>
 +
2012/07/26
 +
K537001、K098011转化
 +
2012/07/27
 +
蛭弧菌涂板
 +
2012/07/28
 +
质粒小提R0010、K117010</br>
 +
菌液PCR R0010
 +
2012/07/29
 +
K537001、K098011挑单菌落</br>
 +
2012/07/30
 +
菌液PCR  groE/cheZ</br>
 +
复苏 R0084+B0034,送测序</br>
 +
2012/07/31
 +
转化I13521、K117000、K098011</br>
 +
菌液PCR K117010、R0084+B0034、groE</br>
 +
质粒PCR K117010、R0084+B0034</br>
 +
质粒小提K098011、K117010、R0010、R0084+B0034</br>
 +
 +
质粒小提电泳图
 +
复苏R0010、 R0084、B0034 、K117010、K117000</br>
 +
双酶切R0010、K117010</br>
 +
2012/08/01
 +
保种groES、B0034</br>
 +
双酶切R0084+B0034 、B0034 、K117000</br>
 +
 +
双酶切电泳图
 +
转化J04500</br>
 +
菌液PCR K117000、R0084、groE、R0010</br>
 +
2012/08/02
 +
酶切回收R0084+B0034、B0034</br>
 +
菌液PCR K117000、R0084、K117010</br>
 +
质粒小提groES、B0034</br>
 +
保种K117000</br>
 +
双酶切R0084、B0034</br>
 +
J04500挑单菌落</br>
 +
酶接(R0084+B0034)+ K117000 、R0084+B0034、R0010+B0034</br>
 +
2012/08/03
 +
质粒小提B0015、R0084、B0034、K117000、J04500</br>
 +
保种J04500</br>
 +
J04500、K117000质粒送测序</br>
 +
酶切回收R0084、B0034</br>
 +
双酶切R0084、B0034、J04500</br>
 +
酶接R0084+B0034</br>
 +
2012/08/04
 +
酶切回收R0084、B0034、J04500</br>
 +
酶接R0084+B0034、J04500+ K117000</br>
 +
摇菌R0084、B0034、K117000</br>
 +
2012/08/05
 +
质粒小提R0084、B0034、K117000</br>
 +
双酶切R0084、B0034、K117000</br>
 +
小红菌I13521 挑单菌落</br>
 +
复苏J01003、groES、PET</br>
 +
转化J01001</br>
 +
2012/08/06
 +
菌液PCR groE</br>
 +
保种PET、I13521</br>
 +
酶接R0084+B0034、J04500+ K117000</br>
 +
质粒小提groES、J01003、PET32a、I13521</br>
 +
双酶切groE、J01003、pET32a、I13521</br>
 +
J01001挑单菌落</br>
 +
2012/08/07
 +
质粒小提J01001、J04500、B0034</br>
 +
菌液PCR K117000、R0084、J01001</br>
 +
质粒PCR K117000、J01003</br>
 +
PCR产物回收K117000
 +
双酶切J04500(S.P)、B0034(E.X)、K117000(X.P)、R0084(E.S)、 J01003(E.S)</br>
 +
转化R0084+B0034</br>
 +
2012/08/08
 +
J01003+I13521、groE+pET32a酶接纯化</br>
 +
转化J01003+I13521、groE+pET32a</br>
 +
PCR  J01003+I13521</br>
 +
复苏J04500、groES、pET32a、I13521、 R0084、B0034</br>
 +
保种R0084+B0034</br>
 +
2012/08/09
 +
J01003+I13521 回收送测序
 +
J01003+I13521、groE+pET32a挑单菌落</br>
 +
J04500、groES、pET32a、B0034质粒小提,凝胶回收</br>
 +
菌液PCR J01003+I13521、R0084、J01001、J01003</br>
 +
PCR产物回收J01003+I13521、R0084、J01001、J01003</br>
 +
双酶切J04500、groES、pET32a、B0034、K117000</br>
 +
 +
双酶切电泳图
 +
2012/08/10
 +
0809的PCR产物点样
 +
J01003+I13521、groE+pET32a菌液PCR验证,保种</br>
 +
pET32a保种</br>
 +
酶接J04500 +K117000、groES + pET32a</br>
 +
转化pSB1C3、J04500 +K117000、groES + pET32a</br>
 +
2012/08/11
 +
pSB1C3、J04500 +K117000、groES + pET32a挑单菌落</br>
 +
J04500 +K117000、groES + pET32a酶接纯化</br>
 +
转化J04500 +K117000、groES + pET32a</br>
 +
菌液PCR J04500 +K117000</br>
 +
PCR产物回收J04500 +K117000</br>
 +
复苏B0015</br>
 +
2012/08/12
 +
B0015质粒小提,凝胶回收</br>
 +
菌液PCR验证pSB1C3、J04500 +K117000、groES + pET32a</br>
 +
保种pSB1C3、J04500 +K117000、groES + pET32a</br>
 +
J04500 +K117000、groES + pET32a挑单菌落</br>
 +
2012/08/13
 +
复苏B0034
 +
重新转化pSB1C3
 +
质粒小提(J04500+K117000)、菌液PCR用于酶切(J04500+K117000)
 +
双酶切B0015、(J04500+K117000)
 +
2012/08/14
 +
挑单菌落pSB1C3
 +
重新转化R0084
 +
复苏(J01003+I13521)
 +
0813双酶切回收
 +
酶接体系(J04500+K117000)+B0015
 +
2012/08/15
 +
    挑单菌落R0084
 +
    保种pSB1C3
 +
质粒小提pSB1C3
 +
菌液PCR用于酶切(J01003+I13521)
 +
酶接回收,转化(J04500+K117000)+B0015
 +
双酶切pSB1C3、(J01003+I13521)
 +
2012/08/16
 +
    菌液PCR验证R0084
 +
挑单菌落(J04500+K117000)+B0015
 +
0815双酶切回收
 +
酶接pSB1C3+(J01003+I13521)
 +
复苏pSB1C3
 +
2012/08/17
 +
    保种(J04500+K117000)+B0015
 +
0816酶接回收,转化pSB1C3+(J01003+I13521)
 +
质粒小提pSB1C3
 +
菌液PCR用于酶切(J04500+K117000)+B0015
 +
双酶切pSB1C3、(J04500+K117000)+B0015
 +
 
 +
 
 +
      </br><h2> <span class="mw-headline" id=".E0">Period 5  Biosafety</span></h2></br>
 +
 
 +
Aug 18-Sep 02
 +
趋化性通路构建OK
 +
Sweet gene 验证开幕
 +
Death gene验证开幕
 +
Conjugation验证开幕
 +
HSP promoter验证开幕
 +
2012/08/18
 +
复苏groE+pET32a、pET32a各一瓶
 +
接着重新转化R0084
 +
复苏pSB1C3、(J01003+I13521)
 +
2012/08/19
 +
挑单菌落R0084
 +
Sweet gene验证实验Version 1.0(第一天)
 +
质粒小提pSB1C3
 +
菌液PCR用于酶切 (J01003+I13521)
 +
双酶切pSB1C3、(J01003+I13521)
 +
2012/08/20
 +
菌液PCR验证R0084,送测序
 +
Sweet gene验证实验Version 1.0(第二天)
 +
含RP4质粒的HB101菌株寄到,复苏</br>
 +
0820酶切回收
 +
酶接pSB1C3+(J01003+I13521)
 +
复苏(J04500+K117000+B0015)
 +
2012/08/21
 +
保种HB101、R0084
 +
Death gene验证实验version 1.0(第一天)
 +
转化pSB1C3+(J01003+I13521)
 +
复苏B0034、R0084
 +
2012/08/22
 +
    菌液PCR用于酶切R0084
 +
质粒小提B0034
 +
挑单菌落pSB1C3+(J01003+I13521)
 +
Death gene验证实验version 1.0(第二天)
 +
 +
双酶切R0084、B0034
 +
转化E0240
 +
2012/08/23
 +
0822酶切回收
 +
菌液PCR验证pSB1C3+(J01003+I13521)
 +
保种pSB1C3+(J01003+I13521)
 +
质粒小提pSB1C3+(J01003+I13521)
 +
酶接R0084+B0034
 +
挑单菌落E0240
 +
Promoter合成完成,复苏菌种
 +
2012/08/24
 +
酶接纯化,转化R0084+B0034
 +
菌液PCR验证E0240
 +
保种E0240
 +
质粒小提E0240、HSP Promoter
 +
双酶切E0240、HSP Promoter
 +
复苏pSB1C3+(J01003+I13521)(BL21)、HB101、DH5α菌株
 +
2012/08/25
 +
0824酶切回收
 +
挑单菌落R0084+B0034
 +
Conjugation验证实验version 1.0(第一天)
 +
酶接HSP promoter+E0240
 +
复苏pSB1C3
 +
2012/08/26
 +
菌液PCR验证R0084+B0034
 +
 +
菌液PCR验证构建质粒是否成功
 +
(所挑五个单克隆中,只有中间一个加上了目的片段)
 +
 
 +
保种R0084+B0034
 +
菌液PCR用于酶切R0084+B0034(E& P)、cheZ(E&P)、cheZ(X&P)
 +
 +
从基因组钓取的cheZ基因
 +
(第一泳道为D15000 Marker,第三至第八泳道为cheZ基因)
 +
质粒小提pSB1C3(E&P)、R0084+B0034(S&P)
 +
Conjugation验证实验version 1.0(第二天)
 +
酶接回收
 +
转化HSP promoter+E0240
 +
双酶切R0084+B0034(E& P)、cheZ(E&P)、cheZ(X&P)、pSB1C3(E&P)、R0084+B0034(S&P)
 +
2012/08/27
 +
0826酶切回收
 +
酶接pSB1C3+(R0084+B0034)、pSB1C3+cheZ、(R0084+B0034)+ cheZ
 +
挑单菌落HSP promoter+E0240
 +
复苏pSB1C3、HSP promoter
 +
2012/08/28
 +
0827酶接回收
 +
酶接转化pSB1C3+(R0084+B0034)、pSB1C3+cheZ、(R0084+B0034)+ cheZ
 +
 +
转化cheZ
 +
菌液PCR验证HSP promoter+E0240
 +
 +
菌液PCR验证
 +
(从Marker左数第二个为构建好的目的载体,左数第一个可能没有加上目的片段HSP promoter)
 +
保种HSP promoter+E0240
 +
质粒小提pSB1C3、HSP promoter
 +
双酶切pSB1C3(E&P)、HSP promoter(E&P)
 +
复苏pSB1C3+(J01003+I13521)(BL21)、HB101、DH5α菌株
 +
2012/08/29
 +
0828酶切回收pSB1C3(E&P)、HSP promoter(E&P)
 +
挑单菌落pSB1C3+(R0084+B0034)、pSB1C3+cheZ、(R0084+B0034)+ cheZ
 +
酶接pSB1C3+HSP promoter
 +
Conjugation验证实验version 2.0(第一天)
 +
复苏B0015
 +
2012/08/29
 +
0828酶接回收
 +
转化pSB1C3+HSP promoter
 +
菌液PCR验证pSB1C3+(R0084+B0034)、pSB1C3+cheZ、(R0084+B0034)+ cheZ
 +
保种pSB1C3+(R0084+B0034)、pSB1C3+cheZ、(R0084+B0034)+ cheZ
 +
菌液PCR用于酶切(R0084+B0034)+ cheZ
 +
质粒小提B0015、pSB1C3+(R0084+B0034)、pSB1C3+cheZ
 +
双酶切(R0084+B0034)+ cheZ(E&S)、B0015(E&X)
 +
Conjugation验证实验version 2.0(第二天)
 +
2012/08/30
 +
挑单菌落pSB1C3+HSP promoter
 +
酶切回收(R0084+B0034)+ cheZ(E&S)、B0015(E&X)
 +
酶接(R0084+B0034+ cheZ)+B0015
 +
2012/08/31
 +
菌液PCR验证pSB1C3+HSP promoter
 +
保种pSB1C3+HSP promoter
 +
质粒小提pSB1C3+HSP promoter
 +
酶接纯化(R0084+B0034+ cheZ)+B0015
 +
转化(R0084+B0034+ cheZ)+B0015
 +
复苏HSP promoter+E0240
 +
2012/09/01
 +
HSP promoter验证实验version 1.0(第一天)
 +
挑单菌落(R0084+B0034+ cheZ)+B0015
 +
复苏(J04500+K117000+B0015)
 +
2012/09/02
 +
菌液PCR验证(R0084+B0034+ cheZ)+B0015
 +
保种(R0084+B0034+ cheZ+B0015)
 +
HSP promoter验证实验version 1.0(第二天)
 +
复苏pSB1C3+(J01003+I13521)、K117010、(HSP promoter+E0240)、pSB1C3
 +
转化R0040
 +
 
 +
      </br><h2> <span class="mw-headline" id=".F0">Period 6  Biosafety</span></h2></br>
 +
 
 +
Sep 03-Sep 22
 +
验证实验升级中~~
 +
尝试构建终极通路:
 +
 +
准备标准组件
 +
    2012/09/03
 +
Death gene验证实验version 2.0(第一天)
 +
质粒小提pSB1C3+(J01003+I13521)、pSB1C3
 +
菌液PCR用于酶切K117010、(HSP promoter+E0240)
 +
双酶切pSB1C3+(J01003+I13521)(S&P)、K117010(X&P)、pSB1C3(E&P)、(HSP promoter+E0240)(E&P)
 +
挑单菌落R0040
 +
2012/09/04
 +
0903酶切回收
 +
酶接pSB1C3+(J01003+I13521)+K117010、pSB1C3+(HSP promoter+E0240)
 +
Death gene验证实验version 2.0(第二天)
 +
复苏B0015
 +
菌液PCR验证R0040
 +
保种R0040
 +
2012/09/05
 +
酶接纯化
 +
转化pSB1C3+(J01003+I13521)+K117010、pSB1C3+(HSP promoter+E0240)
 +
菌液PCR用于酶切groE
 +
质粒小提B0015
 +
双酶切groE(E&S)、B0015(E&X)
 +
2012/09/06
 +
挑单菌落pSB1C3+(J01003+I13521)+K117010、pSB1C3+(HSP promoter+E0240)
 +
酶切回收groE(E&S)、B0015(E&X)
 +
酶接groE+B0015
 +
复苏B0034、R0040
 +
2012/09/07
 +
酶接纯化groE+B0015
 +
转化groE+B0015
 +
菌液PCR验证pSB1C3+(J01003+I13521)+K117010、pSB1C3+(HSP promoter+E0240)
 +
保种pSB1C3+(J01003+I13521)+K117010、pSB1C3+(HSP promoter+E0240)
 +
质粒小提B0034、pSB1C3+(J01003+I13521)+K117010、pSB1C3+(HSP promoter+E0240)
 +
菌液PCR用于酶切R0040
 +
双酶切R0040(E&S)、B0034(E&X)
 +
复苏pSB1C3+(R0084+B0034)
 +
2012/09/08
 +
挑单菌落groE+B0015
 +
酶切回收R0040(E&S)、B0034(E&X)
 +
酶接R0040+B0034
 +
质粒小提pSB1C3+(HSP promoter+E0240)
 +
菌液PCR用于酶切pSB1C3+(R0084+B0034)
 +
双酶切pSB1C3+(HSP promoter+E0240)(S&P)、pSB1C3+(R0084+B0034)(X&P)
 +
2012/09/09
 +
酶接纯化
 +
转化R0040+B0034
 +
酶切回收pSB1C3+(HSP promoter+E0240)(S&P)、pSB1C3+(R0084+B0034)(X&P)
 +
酶接pSB1C3+(HSP promoter+E0240)+(R0084+B0034)
 +
菌液PCR验证groE+B0015
 +
保种(groE+B0015)
 +
复苏J04500
 +
2012/09/10
 +
挑单菌落R0040+B0034
 +
酶接纯化pSB1C3+(HSP promoter+E0240)+(R0084+B0034)
 +
转化pSB1C3+(HSP promoter+E0240)+(R0084+B0034)
 +
质粒小提(groE+B0015)
 +
双酶切(groE+B0015)(E&X)
 +
复苏B0015、pSB1C3
 +
2012/09/11
 +
菌液PCR验证R0040+B0034
 +
保种(R0040+B0034)
 +
酶切回收(groE+B0015)(E&X)
 +
菌液PCR用于酶切(R0040+B0034)、groES
 +
质粒小提B0015、pSB1C3
 +
双酶切(R0040+B0034)(E&P)、groES(E&P)、pSB1C3(E&P)、groES(E&S)、B0015(E&X)
 +
挑单菌落pSB1C3+(HSP promoter+E0240)+(R0084+B0034)
 +
2012/09/12
 +
菌液PCR验证pSB1C3+(HSP promoter+E0240)+(R0084+B0034)
 +
保种pSB1C3+(HSP promoter+E0240)+(R0084+B0034)
 +
质粒小提pSB1C3+(HSP promoter+E0240)+(R0084+B0034)
 +
酶切回收(R0040+B0034)(E&S)、(R0040+B0034)(E&S)、groES(E&P)、pSB1C3(E&P)、groES(E&S)、B0015(E&X)
 +
酶接pSB1C3+(R0040+B0034)、groES+pSB1C3、groES+B0015、(R0040+B0034)+(groE+B0015)
 +
复苏pSB1C3
 +
2012/09/13
 +
酶接纯化pSB1C3+(R0040+B0034)、groES+pSB1C3、groES+B0015、(R0040+B0034)+(groE+B0015)
 +
转化pSB1C3+(R0040+B0034)、groES+pSB1C3、groES+B0015、(R0040+B0034)+(groE+B0015)
 +
质粒小提pSB1C3
 +
质粒PCR用于酶切groES+B0015
 +
双酶切pSB1C3(E&P)、groES+B0015(E&P)
 +
2012/09/14
 +
挑单菌落pSB1C3+(R0040+B0034)、groES+pSB1C3、groES+B0015、(R0040+B0034)+(groE+B0015)
 +
酶切回收pSB1C3(E&P)、groES+B0015(E&P)
 +
酶接pSB1C3+(groES+B0015)
 +
2012/09/15
 +
    酶接纯化pSB1C3+(groES+B0015)
 +
    转化pSB1C3+(groES+B0015)
 +
菌液PCR验证pSB1C3+(R0040+B0034)、groES+pSB1C3、groES+B0015、(R0040+B0034)+(groE+B0015)
 +
保种(pSB1C3+R0040+B0034)、(groES+pSB1C3)、(groES+B0015)、(R0040+B0034+groE+B0015)
 +
质粒小提(pSB1C3+R0040+B0034)、(groES+pSB1C3)
 +
2012/09/16
 +
挑单菌落pSB1C3+(groES+B0015)
 +
菌液PCR验证pSB1C3+(groES+B0015)
 +
保种pSB1C3+(groES+B0015)
 +
质粒小提pSB1C3+(groES+B0015)
 +
寄出所构建的标准组件</br>
 +
2012/09/17
 +
休息
 +
2012/09/18
 +
休息
 +
2012/09/19
 +
复苏验证实验所需菌种</br>
 +
2012/09/20
 +
Death gene 验证实验 version 3.0 (第一天)
 +
HSP promoter验证实验 version 2.0 (第一天)
 +
Conjugation 验证实验 version 3.0 (第一天)
 +
Sweet gene 验证实验 version 1.0 (第一天)
 +
2012/09/21
 +
Death gene 验证实验 version 3.0 (第二天)
 +
HSP promoter验证实验 version 2.0 (第二天)
 +
Conjugation 验证实验 version 3.0 (第二天)
 +
Sweet gene 验证实验 version 1.0 (第二天)
 +
2012/09/22
 +
Death gene 验证实验 version 3.0 (第三天)
 +
Conjugation 验证实验 version 3.0 (第三天)
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Latest revision as of 11:17, 25 September 2012

iGEM 2012 SEU_A Safety


Notebook

This is our experiment diary.

Index


Period 1 Biosafety


2012/04/05-2012/06/23 学习阶段:实验技能学习、实验理论学习,掌握从已有工程菌得到希望构建的工程菌的过程
设计从污水中分离培养蛭弧菌方案
Learning stage : Learn about the skill and theory of Molecular Biology Experiment. Set up the method of isolation and culture BD from sewage. 2012/04/05 LB培养基的制备、高压蒸汽灭菌锅的使用
LB medium preparation . Learn the usage of autoclaves. 细菌的培养 2012/04/06 利用BIOMIGA质粒小量提取试剂盒进行大肠杆菌质粒提取
2012/04/12 购买农业用蛭弧菌混合液,从中分离培养蛭弧菌
2012/04/14 蛭弧菌长出噬菌斑,分离纯化培养
2012/04/21 关于酶切、酶接相关实验技术学习
2012/05/18 2012年kit寄到,取kit进行转化,第一个转化I13521,命名“小红菌”
线性化质粒骨架酶切
2012/05/19 转化涂板技能练习
2012/05/20 挑单克隆,液体培养基振荡过夜培养
2012/05/22 PCR体系配置和技能练习
2012/05/23 优化酶接是骨架和片段的比例
2012/05/25 蛭弧菌菌液PCR,条带很弱,显示其生长不好或已变种
2012/06/13 所构建的工程菌保种

Period 2 Biosafety


Jun 26-Jul 12 整体方案敲定,如下所示: Death gene验证通路确定,如下: 蛭弧菌培养如火如荼
Sweet gene的钓取
2012/06/26 用小红菌作为营养物质培养异养型蛭弧菌
2012/06/28 K117010、I14032kit转化,构建工程菌,并设置对照实验验证其功能
2012/06/29 设置对照组,用活的&死的大肠杆菌培养蛭弧菌
2012/06/30 K117010功能验证实验
I14032质粒小提,凝胶回收
2012/07/01 0630阳性、阴性均未长菌,0628所转菌为假阳性
0630摇R0010质粒小提
2012/07/02 基因组分别钓取groES、groEL基因
通过PCR
2012/07/03 转化K117010、B0034、K117000涂板技术及使用菌液量改进
复苏小红菌
2012/07/04 K117010、B0034、K117000、R0010、B0015摇菌
2012/07/05 蛭弧菌双层琼脂培养基分开涂板,计算E.coli菌落扩散速率及蛭弧菌游动并捕食E.coli的速率
2012/07/06 蛭弧菌半定量PCR引物重新设计购买
2012/07/07 跟进蛭弧菌、小红菌菌斑扩增速率
2012/07/09 K117000、B0015双酶切
复苏K117000、K117010、B0034、R0010、B0015
2012/07/10 质粒小提K117000、K117010、B0034、R0010、B0015
K117000使用通用引物PCR,20ul体系
2012/07/11 质粒小提K117000、B0015
小红菌摇菌,用作蛭弧菌的食物
转化K117000,B0015
摇菌K117010、R0010、B0034、B0015. 2012/07/12 蛭弧菌菌液PCR验证
K117010、R0010、B0015菌液PCR
保种B0034、R0010、K117010、B0015,传代培养R0010、B0034、B0015
K117000挑单菌落,BL21摇菌
B0034、B0015酶切

Period 3 Biosafety


Jul 13--Jul 21 Conjugation支路的构建: 趋化性支路的构建: 成功钓取groE并含标准组件所需的酶切位点 2012/07/13 0712酶切体系跑胶回收
质粒小提B0034、B0015
R0010、B0034通用引物PCR
酶接体系配制R0010+B0034
转化酶接产物R0010+B0034,转化J01003、K117000
groES、groEL基因钓取
2012/07/14 J01003、R0010+B0034、K117000挑单菌落
2012/07/15 质粒小提K117000、J01003、R0010+B0034 K117000、J01003、R0010+B0034使用通用引物PCR
2012/07/16 R0010、K117010复苏
R0010+B0034菌液PCR
转化J01003
2012/07/17 R0010、K117010质粒小提 K117010、J01003、R0010+B0034质粒、R0010+B0034菌液、K117000 PCR
2012/07/18 J01003、K537001转化
自制感受态DH5α
2012/07/19 K117010、B0015、groEL、K117010(自主设计引物)
2012/07/20 groE基因钓取PCR
J01003质粒小提
R0010、K117010转化
图3.从基因组钓取的groE基因电泳图 (中间为D15000 Marker) 2012/07/21 R0010、K117010挑单菌落
R0010、K117010质粒小提
K537001转化
接合转移实验验证

Period 4 Biosafety


Jul 22-Aug 17 Death gene 验证通路构建成功
Conjugation通路构建成功,并将其连接至标准载体
Sweet gene表达载体的构建
2012/07/22 PCR退火最适温度实验探究
2012/07/23 转化J01001、R0084 0721接合转移菌株质粒检测
2012/07/24 K537001复苏摇菌
R0084、B0015挑单菌落
2012/07/25 半固体培养基配制
质粒小提J01001、R0084
K537001、R0084、J01001、groE菌液PCR
2012/07/26 K537001、K098011转化 2012/07/27 蛭弧菌涂板 2012/07/28 质粒小提R0010、K117010
菌液PCR R0010 2012/07/29 K537001、K098011挑单菌落
2012/07/30 菌液PCR groE/cheZ
复苏 R0084+B0034,送测序
2012/07/31 转化I13521、K117000、K098011
菌液PCR K117010、R0084+B0034、groE
质粒PCR K117010、R0084+B0034
质粒小提K098011、K117010、R0010、R0084+B0034
质粒小提电泳图 复苏R0010、 R0084、B0034 、K117010、K117000
双酶切R0010、K117010
2012/08/01 保种groES、B0034
双酶切R0084+B0034 、B0034 、K117000
双酶切电泳图 转化J04500
菌液PCR K117000、R0084、groE、R0010
2012/08/02 酶切回收R0084+B0034、B0034
菌液PCR K117000、R0084、K117010
质粒小提groES、B0034
保种K117000
双酶切R0084、B0034
J04500挑单菌落
酶接(R0084+B0034)+ K117000 、R0084+B0034、R0010+B0034
2012/08/03 质粒小提B0015、R0084、B0034、K117000、J04500
保种J04500
J04500、K117000质粒送测序
酶切回收R0084、B0034
双酶切R0084、B0034、J04500
酶接R0084+B0034
2012/08/04 酶切回收R0084、B0034、J04500
酶接R0084+B0034、J04500+ K117000
摇菌R0084、B0034、K117000
2012/08/05 质粒小提R0084、B0034、K117000
双酶切R0084、B0034、K117000
小红菌I13521 挑单菌落
复苏J01003、groES、PET
转化J01001
2012/08/06 菌液PCR groE
保种PET、I13521
酶接R0084+B0034、J04500+ K117000
质粒小提groES、J01003、PET32a、I13521
双酶切groE、J01003、pET32a、I13521
J01001挑单菌落
2012/08/07 质粒小提J01001、J04500、B0034
菌液PCR K117000、R0084、J01001
质粒PCR K117000、J01003
PCR产物回收K117000 双酶切J04500(S.P)、B0034(E.X)、K117000(X.P)、R0084(E.S)、 J01003(E.S)
转化R0084+B0034
2012/08/08 J01003+I13521、groE+pET32a酶接纯化
转化J01003+I13521、groE+pET32a
PCR J01003+I13521
复苏J04500、groES、pET32a、I13521、 R0084、B0034
保种R0084+B0034
2012/08/09 J01003+I13521 回收送测序 J01003+I13521、groE+pET32a挑单菌落
J04500、groES、pET32a、B0034质粒小提,凝胶回收
菌液PCR J01003+I13521、R0084、J01001、J01003
PCR产物回收J01003+I13521、R0084、J01001、J01003
双酶切J04500、groES、pET32a、B0034、K117000
双酶切电泳图 2012/08/10 0809的PCR产物点样 J01003+I13521、groE+pET32a菌液PCR验证,保种
pET32a保种
酶接J04500 +K117000、groES + pET32a
转化pSB1C3、J04500 +K117000、groES + pET32a
2012/08/11 pSB1C3、J04500 +K117000、groES + pET32a挑单菌落
J04500 +K117000、groES + pET32a酶接纯化
转化J04500 +K117000、groES + pET32a
菌液PCR J04500 +K117000
PCR产物回收J04500 +K117000
复苏B0015
2012/08/12 B0015质粒小提,凝胶回收
菌液PCR验证pSB1C3、J04500 +K117000、groES + pET32a
保种pSB1C3、J04500 +K117000、groES + pET32a
J04500 +K117000、groES + pET32a挑单菌落
2012/08/13 复苏B0034 重新转化pSB1C3 质粒小提(J04500+K117000)、菌液PCR用于酶切(J04500+K117000) 双酶切B0015、(J04500+K117000) 2012/08/14 挑单菌落pSB1C3 重新转化R0084 复苏(J01003+I13521) 0813双酶切回收 酶接体系(J04500+K117000)+B0015 2012/08/15 挑单菌落R0084 保种pSB1C3 质粒小提pSB1C3 菌液PCR用于酶切(J01003+I13521) 酶接回收,转化(J04500+K117000)+B0015 双酶切pSB1C3、(J01003+I13521) 2012/08/16 菌液PCR验证R0084 挑单菌落(J04500+K117000)+B0015 0815双酶切回收 酶接pSB1C3+(J01003+I13521) 复苏pSB1C3 2012/08/17 保种(J04500+K117000)+B0015 0816酶接回收,转化pSB1C3+(J01003+I13521) 质粒小提pSB1C3 菌液PCR用于酶切(J04500+K117000)+B0015 双酶切pSB1C3、(J04500+K117000)+B0015

Period 5 Biosafety


Aug 18-Sep 02 趋化性通路构建OK Sweet gene 验证开幕 Death gene验证开幕 Conjugation验证开幕 HSP promoter验证开幕 2012/08/18 复苏groE+pET32a、pET32a各一瓶 接着重新转化R0084 复苏pSB1C3、(J01003+I13521) 2012/08/19 挑单菌落R0084 Sweet gene验证实验Version 1.0(第一天) 质粒小提pSB1C3 菌液PCR用于酶切 (J01003+I13521) 双酶切pSB1C3、(J01003+I13521) 2012/08/20 菌液PCR验证R0084,送测序 Sweet gene验证实验Version 1.0(第二天) 含RP4质粒的HB101菌株寄到,复苏
0820酶切回收 酶接pSB1C3+(J01003+I13521) 复苏(J04500+K117000+B0015) 2012/08/21 保种HB101、R0084 Death gene验证实验version 1.0(第一天) 转化pSB1C3+(J01003+I13521) 复苏B0034、R0084 2012/08/22 菌液PCR用于酶切R0084 质粒小提B0034 挑单菌落pSB1C3+(J01003+I13521) Death gene验证实验version 1.0(第二天) 双酶切R0084、B0034 转化E0240 2012/08/23 0822酶切回收 菌液PCR验证pSB1C3+(J01003+I13521) 保种pSB1C3+(J01003+I13521) 质粒小提pSB1C3+(J01003+I13521) 酶接R0084+B0034 挑单菌落E0240 Promoter合成完成,复苏菌种 2012/08/24 酶接纯化,转化R0084+B0034 菌液PCR验证E0240 保种E0240 质粒小提E0240、HSP Promoter 双酶切E0240、HSP Promoter 复苏pSB1C3+(J01003+I13521)(BL21)、HB101、DH5α菌株 2012/08/25 0824酶切回收 挑单菌落R0084+B0034 Conjugation验证实验version 1.0(第一天) 酶接HSP promoter+E0240 复苏pSB1C3 2012/08/26 菌液PCR验证R0084+B0034 菌液PCR验证构建质粒是否成功 (所挑五个单克隆中,只有中间一个加上了目的片段) 保种R0084+B0034 菌液PCR用于酶切R0084+B0034(E& P)、cheZ(E&P)、cheZ(X&P) 从基因组钓取的cheZ基因 (第一泳道为D15000 Marker,第三至第八泳道为cheZ基因) 质粒小提pSB1C3(E&P)、R0084+B0034(S&P) Conjugation验证实验version 1.0(第二天) 酶接回收 转化HSP promoter+E0240 双酶切R0084+B0034(E& P)、cheZ(E&P)、cheZ(X&P)、pSB1C3(E&P)、R0084+B0034(S&P) 2012/08/27 0826酶切回收 酶接pSB1C3+(R0084+B0034)、pSB1C3+cheZ、(R0084+B0034)+ cheZ 挑单菌落HSP promoter+E0240 复苏pSB1C3、HSP promoter 2012/08/28 0827酶接回收 酶接转化pSB1C3+(R0084+B0034)、pSB1C3+cheZ、(R0084+B0034)+ cheZ 转化cheZ 菌液PCR验证HSP promoter+E0240 菌液PCR验证 (从Marker左数第二个为构建好的目的载体,左数第一个可能没有加上目的片段HSP promoter) 保种HSP promoter+E0240 质粒小提pSB1C3、HSP promoter 双酶切pSB1C3(E&P)、HSP promoter(E&P) 复苏pSB1C3+(J01003+I13521)(BL21)、HB101、DH5α菌株 2012/08/29 0828酶切回收pSB1C3(E&P)、HSP promoter(E&P) 挑单菌落pSB1C3+(R0084+B0034)、pSB1C3+cheZ、(R0084+B0034)+ cheZ 酶接pSB1C3+HSP promoter Conjugation验证实验version 2.0(第一天) 复苏B0015 2012/08/29 0828酶接回收 转化pSB1C3+HSP promoter 菌液PCR验证pSB1C3+(R0084+B0034)、pSB1C3+cheZ、(R0084+B0034)+ cheZ 保种pSB1C3+(R0084+B0034)、pSB1C3+cheZ、(R0084+B0034)+ cheZ 菌液PCR用于酶切(R0084+B0034)+ cheZ 质粒小提B0015、pSB1C3+(R0084+B0034)、pSB1C3+cheZ 双酶切(R0084+B0034)+ cheZ(E&S)、B0015(E&X) Conjugation验证实验version 2.0(第二天) 2012/08/30 挑单菌落pSB1C3+HSP promoter 酶切回收(R0084+B0034)+ cheZ(E&S)、B0015(E&X) 酶接(R0084+B0034+ cheZ)+B0015 2012/08/31 菌液PCR验证pSB1C3+HSP promoter 保种pSB1C3+HSP promoter 质粒小提pSB1C3+HSP promoter 酶接纯化(R0084+B0034+ cheZ)+B0015 转化(R0084+B0034+ cheZ)+B0015 复苏HSP promoter+E0240 2012/09/01 HSP promoter验证实验version 1.0(第一天) 挑单菌落(R0084+B0034+ cheZ)+B0015 复苏(J04500+K117000+B0015) 2012/09/02 菌液PCR验证(R0084+B0034+ cheZ)+B0015 保种(R0084+B0034+ cheZ+B0015) HSP promoter验证实验version 1.0(第二天) 复苏pSB1C3+(J01003+I13521)、K117010、(HSP promoter+E0240)、pSB1C3 转化R0040

Period 6 Biosafety


Sep 03-Sep 22 验证实验升级中~~ 尝试构建终极通路: 准备标准组件 2012/09/03 Death gene验证实验version 2.0(第一天) 质粒小提pSB1C3+(J01003+I13521)、pSB1C3 菌液PCR用于酶切K117010、(HSP promoter+E0240) 双酶切pSB1C3+(J01003+I13521)(S&P)、K117010(X&P)、pSB1C3(E&P)、(HSP promoter+E0240)(E&P) 挑单菌落R0040 2012/09/04 0903酶切回收 酶接pSB1C3+(J01003+I13521)+K117010、pSB1C3+(HSP promoter+E0240) Death gene验证实验version 2.0(第二天) 复苏B0015 菌液PCR验证R0040 保种R0040 2012/09/05 酶接纯化 转化pSB1C3+(J01003+I13521)+K117010、pSB1C3+(HSP promoter+E0240) 菌液PCR用于酶切groE 质粒小提B0015 双酶切groE(E&S)、B0015(E&X) 2012/09/06 挑单菌落pSB1C3+(J01003+I13521)+K117010、pSB1C3+(HSP promoter+E0240) 酶切回收groE(E&S)、B0015(E&X) 酶接groE+B0015 复苏B0034、R0040 2012/09/07 酶接纯化groE+B0015 转化groE+B0015 菌液PCR验证pSB1C3+(J01003+I13521)+K117010、pSB1C3+(HSP promoter+E0240) 保种pSB1C3+(J01003+I13521)+K117010、pSB1C3+(HSP promoter+E0240) 质粒小提B0034、pSB1C3+(J01003+I13521)+K117010、pSB1C3+(HSP promoter+E0240) 菌液PCR用于酶切R0040 双酶切R0040(E&S)、B0034(E&X) 复苏pSB1C3+(R0084+B0034) 2012/09/08 挑单菌落groE+B0015 酶切回收R0040(E&S)、B0034(E&X) 酶接R0040+B0034 质粒小提pSB1C3+(HSP promoter+E0240) 菌液PCR用于酶切pSB1C3+(R0084+B0034) 双酶切pSB1C3+(HSP promoter+E0240)(S&P)、pSB1C3+(R0084+B0034)(X&P) 2012/09/09 酶接纯化 转化R0040+B0034 酶切回收pSB1C3+(HSP promoter+E0240)(S&P)、pSB1C3+(R0084+B0034)(X&P) 酶接pSB1C3+(HSP promoter+E0240)+(R0084+B0034) 菌液PCR验证groE+B0015 保种(groE+B0015) 复苏J04500 2012/09/10 挑单菌落R0040+B0034 酶接纯化pSB1C3+(HSP promoter+E0240)+(R0084+B0034) 转化pSB1C3+(HSP promoter+E0240)+(R0084+B0034) 质粒小提(groE+B0015) 双酶切(groE+B0015)(E&X) 复苏B0015、pSB1C3 2012/09/11 菌液PCR验证R0040+B0034 保种(R0040+B0034) 酶切回收(groE+B0015)(E&X) 菌液PCR用于酶切(R0040+B0034)、groES 质粒小提B0015、pSB1C3 双酶切(R0040+B0034)(E&P)、groES(E&P)、pSB1C3(E&P)、groES(E&S)、B0015(E&X) 挑单菌落pSB1C3+(HSP promoter+E0240)+(R0084+B0034) 2012/09/12 菌液PCR验证pSB1C3+(HSP promoter+E0240)+(R0084+B0034) 保种pSB1C3+(HSP promoter+E0240)+(R0084+B0034) 质粒小提pSB1C3+(HSP promoter+E0240)+(R0084+B0034) 酶切回收(R0040+B0034)(E&S)、(R0040+B0034)(E&S)、groES(E&P)、pSB1C3(E&P)、groES(E&S)、B0015(E&X) 酶接pSB1C3+(R0040+B0034)、groES+pSB1C3、groES+B0015、(R0040+B0034)+(groE+B0015) 复苏pSB1C3 2012/09/13 酶接纯化pSB1C3+(R0040+B0034)、groES+pSB1C3、groES+B0015、(R0040+B0034)+(groE+B0015) 转化pSB1C3+(R0040+B0034)、groES+pSB1C3、groES+B0015、(R0040+B0034)+(groE+B0015) 质粒小提pSB1C3 质粒PCR用于酶切groES+B0015 双酶切pSB1C3(E&P)、groES+B0015(E&P) 2012/09/14 挑单菌落pSB1C3+(R0040+B0034)、groES+pSB1C3、groES+B0015、(R0040+B0034)+(groE+B0015) 酶切回收pSB1C3(E&P)、groES+B0015(E&P) 酶接pSB1C3+(groES+B0015) 2012/09/15 酶接纯化pSB1C3+(groES+B0015) 转化pSB1C3+(groES+B0015) 菌液PCR验证pSB1C3+(R0040+B0034)、groES+pSB1C3、groES+B0015、(R0040+B0034)+(groE+B0015) 保种(pSB1C3+R0040+B0034)、(groES+pSB1C3)、(groES+B0015)、(R0040+B0034+groE+B0015) 质粒小提(pSB1C3+R0040+B0034)、(groES+pSB1C3) 2012/09/16 挑单菌落pSB1C3+(groES+B0015) 菌液PCR验证pSB1C3+(groES+B0015) 保种pSB1C3+(groES+B0015) 质粒小提pSB1C3+(groES+B0015) 寄出所构建的标准组件
2012/09/17 休息 2012/09/18 休息 2012/09/19 复苏验证实验所需菌种
2012/09/20 Death gene 验证实验 version 3.0 (第一天) HSP promoter验证实验 version 2.0 (第一天) Conjugation 验证实验 version 3.0 (第一天) Sweet gene 验证实验 version 1.0 (第一天) 2012/09/21 Death gene 验证实验 version 3.0 (第二天) HSP promoter验证实验 version 2.0 (第二天) Conjugation 验证实验 version 3.0 (第二天) Sweet gene 验证实验 version 1.0 (第二天) 2012/09/22 Death gene 验证实验 version 3.0 (第三天) Conjugation 验证实验 version 3.0 (第三天)



Southeast University

Biomedical Engineer School, SEU | iGEM 2012

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