Difference between revisions of "Part:BBa K3815011"
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This part was inserted in  L4440. The L4440 has two t7 promoters, and this ​parts are transcribed from both sides.
 
This part was inserted in  L4440. The L4440 has two t7 promoters, and this ​parts 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. aaaaaaa]]
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[[File:RNAgel2.png|300px|thumb|right|Fig1.1.L4440  2 vATPase-B  (7ug) 3 vATPase-B  (1ug )<br> 700bp band is target RNA]]
 
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<br>
 
We product ds RNA and purify this [2].<br>The 1L LB culture was started at 37C and  IPTG (final 0.2mM) was added when OD exceeded 0.4 to induce RNA production for 4 hours. <br>E. coli was disrupted and precipitated with NaCl, and RNA contained in the supernatant was purificated by phenol-chloroform treatment. <br>
 
We product ds RNA and purify this [2].<br>The 1L LB culture was started at 37C and  IPTG (final 0.2mM) was added when OD exceeded 0.4 to induce RNA production for 4 hours. <br>E. coli was disrupted and precipitated with NaCl, and RNA contained in the supernatant was purificated by phenol-chloroform treatment. <br>

Revision as of 09:25, 21 October 2021


Part of the V-ATPaseB gene of Frankliniella occidentalis to synthesize dsRNA for RNAi
This is a section of V-ATPaseB gene of Frankliniella occidentalis to synthesize dsRNA for RNAi. To product dsRNA this sequence inserted in 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 anneal.

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 ​parts are transcribed from both sides.

production and purification methods

Fig1.1.L4440 2 vATPase-B (7ug) 3 vATPase-B (1ug )
700bp band is target RNA


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

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

We could confirm RNA production.