Difference between revisions of "Part:BBa K3815011"
 
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<partinfo>BBa_K3815011 short</partinfo>
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<partinfo>BBa_K3815011 short</partinfo><br>
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ATPaseB gene of Frankliniella occidentalis to synthesize dsRNA for RNAi. To product dsRNA this sequence inserted in L4440 plasmid, and transformed into HT115(DE3).
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This is a section of the V-ATPaseB gene of Frankliniella occidentalis to synthesize dsRNA for RNAi. To product dsRNA, this sequence is inserted in the L4440 plasmid, and transformed into HT115(DE3).
 
<h3><font size="3"></font> purpose </h3>
 
<h3><font size="3"></font> purpose </h3>
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We focused on feeding RNAi to kill these insects, based on previous studies showing that oral ingestion of dsRNA can induce RNAi-induced gene knockdown in Frankliniella occidentalis[1].
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vATPase-B is a gene that encodes a protein that is part of a subunit of vATPase, a type of ATP-dependent proton pump that regulates the pH of various cell organelles. Since this gene is essential for thrips, knockdown of vATPase-B results in the death of the thrips Frankliniella occidentalis.[1]
 
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<!-- Add more about the biology of this part here
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===Usage and Biology===
 
===Usage and Biology===
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RNAi<br>
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RNAi (RNA interference) is a process in which externally introduced dsRNA suppresses the expression of genes that have complementary sequences to the dsRNA.<br><br>
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L4440<br>
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L4440 is a plasmid vector having two convergent T7 promoters adjacent to the multi-cloning site. By inserting a portion of the target gene sequence into the multi-cloning site of this plasmid, the target sequence is transcribed from both sides, and dsRNA can be obtained when both parts are annealed.<br><br>
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HT115(DE3)<br>
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HT115 (DE3) is an RNase III-deficient E. coli strain that has been modified to express T7 RNA polymerase from an IPTG-inducible promoter. It lacks dsRNA-specific RNase III, which allows it to produce high levels of specific dsRNA. These attributes allow HT115 (DE3) to be a promising strain for the preparation of large amounts of viral dsRNA in vivo.<br><br>
  
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<h3><font size="3"></font>Sequence and features </h3>
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<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K3815011 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K3815011 SequenceAndFeatures</partinfo>
  
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<h3><font size="3"> cloning </font> </h3>
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This part was inserted in  L4440. The L4440 has two t7 promoters, and this ​part are transcribed from both sides.
 
<h3><font size="3"> production and purification methods </font> </h3>
 
<h3><font size="3"> production and purification methods </font> </h3>
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[[File:RNAgel2.png|300px|thumb|right|Fig1.1.L4440  2 vATPase-B  (total 7ug nucleotic acid were loaded) 3 vATPase-B  (total 1ug nucleotic acid were loaded)<br>  band of 700bp may be target RNA]]
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<br>
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We produced dsRNA and purify this [2].<br>The 1L LB culture was started at 37°C and  IPTG (final 0.2mM) was added when OD exceeded 0.4 to induce RNA production for 4 hours. <be><i>E. coli</i> was disrupted and precipitated with NaCl, and RNA contained in the supernatant was purified by phenol-chloroform treatment. <br>
  
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Electrophoresis in acrylamide gel was performed to confirm whether the target RNA was produced.
  
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We could confirm RNA production.
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<h3><font size="3"> Assay against Frankliniella occidentalis </font> </h3>
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Specifically, 200 μl of a 200 ng/μl dsRNA solution targeting vATPase-B (Milli-Q was used as a negative control) was pipetted in a PCR tube and the petiole of a green bean primary leaf was immersed. After 2 hours, three leaf fragments of 1 cm × 2 cm were cut from the soaked bean leaves and placed in three petri dishes with about 10 thrips. The mortality and leaf damage were checked after 24, 48, 72, and 96 hours.<br>
  
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RNA purification was followed this protocol[2].
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[[File:RNAi2.png|500px|thumb|center|Fig.2 RNAi assay]]
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[[File:RNAgel.png|200px|thumb|right|Fig1. ethylene pathway]]
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1. Add 1 ml of ampicillin per 1 L of autoclaved LB. <br>
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2.Spread 3ml of LB on a plate, suspend the colonies, and return to LB. <br>
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3.Start incubation at 37℃.(180rpm)<br>
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4.After incubation, when the OD reaches 0.4, add 200ul of 1M IPTG and induce RNA production with 0.2mM IPTG.<br>
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5.After the addition of IPTG, the culture was continued at 37c for 4h, and then the precipitates were collected.<br>
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6.The collected precipitates were suspended in 5ml 70%Etoh in PBS.<br>
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7.4c 5 min incubate<br>
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8.Centrifuge at 10000 rpm for 10 min <br>
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9.Drain off supernatant<br>
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10.Suspend the precipitates in 3ml 150mM NaCl<br>
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11.4c 1h incubate<br>
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12.10min 10000 rpm centrifuge<br>
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13.Collect supernatant (also freeze precipitate at -20c)<br>
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14.Add 8ml of 100% EtOH to supernatant<br>
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15.-20c overnight<br>
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16.centrifuge at 4C 10000rpm for 30min.<br>
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17.Discard the supernatant.<br>
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18.Wash the precipitates with 1ml of 85% EtOh.<br>
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19.Centrifuge at 4c 10000rpm for 3min.<br>
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20.Discard the supernatant<br>
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21.4c 10000rpm 1min centrifugation<br>
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22.Discard the supernatant.<br>
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23.Elute the precipitates into 100ul of DW.<br>
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24.The amount of RNA estimated by PAGE electropharased .<br>
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In particular, there was a large difference in the mean mortality rate between dsRNA-treated and negative control thrips on day 3: 8.3% and 31.3%, respectively. This result confirms the insecticidal effect of dsRNA indirectly feeding to the western flower thrips.
 
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== Reference==
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1 Andongma, A.A., Greig, C., Dyson, P.J., Flynn, N., and Whitten, M.M.A. (2020). Optimization of dietary RNA interference delivery to western flower thrips Frankliniella occidentalis and onion thrips Thrips tabaci. Arch. Insect Biochem. Physiol. 103, e21645.
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2 Papić, L., Rivas, J., Toledo, S., and Romero, J. (2018). Double-stranded RNA production and the kinetics of recombinant Escherichia coli HT115 in fed-batch culture. Biotechnol Rep (Amst) 20, e00292.
  
 
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Latest revision as of 02:22, 22 October 2021


Part of the V-ATPaseB gene of Frankliniella occidentalis to synthesize dsRNA for RNAi
This is a section of the V-ATPaseB gene of Frankliniella occidentalis to synthesize dsRNA for RNAi. To product dsRNA, this sequence is inserted in the L4440 plasmid, and transformed into HT115(DE3).

purpose

vATPase-B is a gene that encodes a protein that is part of a subunit of vATPase, a type of ATP-dependent proton pump that regulates the pH of various cell organelles. Since this gene is essential for thrips, knockdown of vATPase-B results in the death of the thrips Frankliniella occidentalis.[1]

Usage and Biology

RNAi
RNAi (RNA interference) is a process in which externally introduced dsRNA suppresses the expression of genes that have complementary sequences to the dsRNA.

L4440
L4440 is a plasmid vector having two convergent T7 promoters adjacent to the multi-cloning site. By inserting a portion of the target gene sequence into the multi-cloning site of this plasmid, the target sequence is transcribed from both sides, and dsRNA can be obtained when both parts are annealed.

HT115(DE3)
HT115 (DE3) is an RNase III-deficient E. coli strain that has been modified to express T7 RNA polymerase from an IPTG-inducible promoter. It lacks dsRNA-specific RNase III, which allows it to produce high levels of specific dsRNA. These attributes allow HT115 (DE3) to be a promising strain for the preparation of large amounts of viral dsRNA in vivo.

Sequence and features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 452
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 320

cloning

This part was inserted in L4440. The L4440 has two t7 promoters, and this ​part are transcribed from both sides.

production and purification methods

Fig1.1.L4440 2 vATPase-B (total 7ug nucleotic acid were loaded) 3 vATPase-B (total 1ug nucleotic acid were loaded)
band of 700bp may be target RNA


We produced dsRNA and purify this [2].
The 1L LB culture was started at 37°C and IPTG (final 0.2mM) was added when OD exceeded 0.4 to induce RNA production for 4 hours. <be>E. coli was disrupted and precipitated with NaCl, and RNA contained in the supernatant was purified by phenol-chloroform treatment.

Electrophoresis in acrylamide gel was performed to confirm whether the target RNA was produced.

We could confirm RNA production.

Assay against Frankliniella occidentalis

Specifically, 200 μl of a 200 ng/μl dsRNA solution targeting vATPase-B (Milli-Q was used as a negative control) was pipetted in a PCR tube and the petiole of a green bean primary leaf was immersed. After 2 hours, three leaf fragments of 1 cm × 2 cm were cut from the soaked bean leaves and placed in three petri dishes with about 10 thrips. The mortality and leaf damage were checked after 24, 48, 72, and 96 hours.

Fig.2 RNAi assay

In particular, there was a large difference in the mean mortality rate between dsRNA-treated and negative control thrips on day 3: 8.3% and 31.3%, respectively. This result confirms the insecticidal effect of dsRNA indirectly feeding to the western flower thrips.


Reference

1 Andongma, A.A., Greig, C., Dyson, P.J., Flynn, N., and Whitten, M.M.A. (2020). Optimization of dietary RNA interference delivery to western flower thrips Frankliniella occidentalis and onion thrips Thrips tabaci. Arch. Insect Biochem. Physiol. 103, e21645.

2 Papić, L., Rivas, J., Toledo, S., and Romero, J. (2018). Double-stranded RNA production and the kinetics of recombinant Escherichia coli HT115 in fed-batch culture. Biotechnol Rep (Amst) 20, e00292.