Difference between revisions of "Part:BBa K1864000"
 
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<partinfo>BBa_K1864000 short</partinfo>
 
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Our part is CSBV's RdRP,RdRP gene cloned into the L4440 vector,and transformed into E.coli strain HT115 to express dsRdRP.
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Our part is the gene of CSBV's RNA dependent RNA polymerase(RdRp). We transferred the gene of RdRp into plasmid L4440 ,and transformed the recombinant plasmid into E.coli strain HT115 to express double stranded RNA of RdRp.
  
 
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<partinfo>BBa_K1864000 parameters</partinfo>
 
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<h3>Application </h3>
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<h3>Application: To produce double stranded RNA of RNA dependent RNA polymerase which is able to initiate the process of RNA-interference </h3>
 
<h3>Group:  FAFU-CHINA </h3>
 
<h3>Group:  FAFU-CHINA </h3>
 
<h3>Author:  Ruicheng Dai & Changlong Lu </h3>
 
<h3>Author:  Ruicheng Dai & Changlong Lu </h3>
 
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<h3>Summary: The control efficiency of dsRdRp in Chinese Scabrood Virus（CSBV）</h3>
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<h3>Summary: We used this part to produce dsRNA of RdRp, which is essential in our project to silence the RdRp gene of Sacbrood virus（CSBV）, preventing it from producing a protein. </h3>
 
<h3>Design</h3>
 
<h3>Design</h3>
 
<p style="margin-right:100px" align="justify">
 
<p style="margin-right:100px" align="justify">
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In our project,we have been aiming to control CSBV through silence of CSBV’s RdRp (RNA-dependent RNA polymerase) gene by using RNA-interference technology. We built recombinant L4440 plasmids which contained dual T7 promoter and we transformed it to HT115 strain.
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In our project,we have been aiming to control Chinese Sacbrood virus through silence of CSBV’s RdRp (RNA-dependent RNA polymerase) gene by using RNA-interference technology. We built recombinant L4440 plasmid containing dual T7 promoter and the gene of RNA Dependent RNA polymerase, and we transformed it to HT115 strain.
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After the enlage cultivation of HT115, we extracted dsRdRp from bacteria. And we added different concentrations into the honeybees' food.
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After the enlarge cultivation of HT115, we added the engineered bacteria to the forage of honeybees. To test the effect of dsRdRp, we then did a infectious experiment by feeding infected hives with dsRdRp, and collected data to study the pupation rate of infected hives under different concentrations.
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Meanwhile,We noticed that the induction of IPTG could improve the expression of dsRdRp. Therefore,we also tested the inductive effect of IPTG.
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<h3>The control efficiency of dsRdRp in Chinese Scabrood Virus</h3>
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<h3>We expected this part to express homologous double-stranded RNA of RdRp.  And the result of infectious experiment indicated that this part works as expected.</h3>
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In apiculture, scientists regard the number of sealed brood as an important index which means normal developing embryo.Therefore,we gathered datas about the effects in different dsRdRp concentrations and administration time.
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[[File:FAFU-CHINA Parts information (1).png |200px|thumb|left|Figure.1 The effect of dsRdRp on the number of sealed brood. After feeding with dsRdRp , the groups of medium concentration and high concentration are performing better.]]
  
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[[File:FAFU-CHINA Parts information (3).png |200px|thumb|left|Figure.2 Detection the virus-infected rate of offsprings of by RT-PCR. The infection rate is sharply decreased after  feeding A.c.creana.]]
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<h3>IPTG inductive effect</h3>
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We tested the IPTG inductive effect in T7 RNA polymerase. In our project,we built the yeast which contained two parts to produce dsRNA. Meanwhile,IPTG could induce the operation of T7 RNA polymerase.Therefore,we could test the concentration of dsRNA to estimate the inductive effect.
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<h3>The inductive effect of different concentration IPTG</h3>
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We tested dsRNA concentration with the induction of 0.1,0.2,0.3,0.4,0.5,0.6 mmol/L IPTG after 4 hours
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[[File:FAFU-CHINA PART (1).png|200px|thumb|left|Figure 1.Concentration of dsRdRp differs under different concentration of IPTG. The concentration of dsRdRp continues to increase till the concentration of IPTG reaches to 0.4 mmol/L.]]
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<h3>The inductive effect of different time length</h3>
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We tested dsRNA concentration with the induction time length from 0 to 9 hours. And we tested three group which involved concentration.
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[[File:FAFU-CHINA PART (2).png|200px|thumb|left|Figure 2.Concentration of dsRdRp changes with three different concentration of IPTG over time. Induced by IPTG about 4 hours, the concentration of dsRdRp appears to decrease or remain unchanged]]
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Latest revision as of 23:04, 26 September 2015

CSBV-RdRP

Our part is the gene of CSBV's RNA dependent RNA polymerase(RdRp). We transferred the gene of RdRp into plasmid L4440 ,and transformed the recombinant plasmid into E.coli strain HT115 to express double stranded RNA of RdRp.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 837
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 110
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Application: To produce double stranded RNA of RNA dependent RNA polymerase which is able to initiate the process of RNA-interference

Group: FAFU-CHINA

Author: Ruicheng Dai & Changlong Lu


Summary: We used this part to produce dsRNA of RdRp, which is essential in our project to silence the RdRp gene of Sacbrood virus（CSBV）, preventing it from producing a protein.

Design

In our project,we have been aiming to control Chinese Sacbrood virus through silence of CSBV’s RdRp (RNA-dependent RNA polymerase) gene by using RNA-interference technology. We built recombinant L4440 plasmid containing dual T7 promoter and the gene of RNA Dependent RNA polymerase, and we transformed it to HT115 strain. After the enlarge cultivation of HT115, we added the engineered bacteria to the forage of honeybees. To test the effect of dsRdRp, we then did a infectious experiment by feeding infected hives with dsRdRp, and collected data to study the pupation rate of infected hives under different concentrations.



We expected this part to express homologous double-stranded RNA of RdRp. And the result of infectious experiment indicated that this part works as expected.


Figure.1 The effect of dsRdRp on the number of sealed brood. After feeding with dsRdRp , the groups of medium concentration and high concentration are performing better.
Figure.2 Detection the virus-infected rate of offsprings of by RT-PCR. The infection rate is sharply decreased after feeding A.c.creana.