Part:BBa_K1864000

CSBV-RdRP

Our part is CSBV's RdRP,RdRP gene cloned into the L4440 vector,and transformed into E.coli strain HT115 to express dsRdRP.

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]

Design

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. After the enlage cultivation of HT115, we extracted dsRdRp from bacteria. And we added different concentrations into the honeybees' food. Meanwhile,We noticed that the induction of IPTG could improve the expression of dsRdRp. Therefore,we also tested the inductive effect of IPTG.

The control efficiency of dsRdRp in Chinese Scabrood Virus

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.

File:FAFU-CHIAN Parts information (1).png

IPTG inductive effect

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.

The inductive effect of different concentration IPTG

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

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.

The inductive effect of different time length

We tested dsRNA concentration with the induction time length from 0 to 9 hours. And we tested three group which involved concentration.

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