Difference between revisions of "Part:BBa K1442103:Design"

 
 
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<partinfo>BBa_K1442103 short</partinfo>
 
<partinfo>BBa_K1442103 short</partinfo>
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===Design Notes===
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== Image of sequence ==
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...
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[[File:Promoters_Diagram.jpg]]
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== RNA and secondary structure ==
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[[File:5'.gif]]
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== Ribozyme ==
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The use of a ribozyme is necessitated due to the complicated binding process between the RNA template and the RdRP. In order to optimise the process and avoid any risk of unfavourable secondary structures or obstruction by unneeded nucleotides, a self-cleaving ribozyme was put after the RNA promoter. The rationale being that after transctiption (either in vitro for human cell tests or in vivo for e-coli tests), any added bases as a result of the T7 polymerase would be removed from the main template strand.
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[[File:Rib1.gif]]
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The chosen ribozyme taken from the Hepatitis Delta virus was investigated at length by the team of J. Doudna  and is reported to be the fastest naturally occurring self-cleaving protein. It also functions independently, without the need for adding chemical substances, and is resistant to denaturants. Its close genetic origin to the RdRP also contributes to a better working and more compatible system.
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The structure of the molecule and its active site in particular are shown below.
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[[File:Rib2.gif]]
  
  
 
===Source===
 
===Source===
 +
“Partial reconstitution of hepatitis C virus RNA polymerization by heterologous expression of NS5B polymerase and template RNA in bacterial cell”, 2005, Sangyoon Lee, Jong-Ho Lee, Young Hoon Kee, Mi Young Park, Heejoon Myung
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B. Heinz, C. Kao – “Template Requirements for RNA Synthesis by a Recombinant Hepatitis C Virus RNA-Dependent RNA Polymerase” - [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC113194/]
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“Hepatitis C virus type 1b complete genome, isolate Con1”- [http://www.ncbi.nlm.nih.gov/nuccore/AJ238799]
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“Replication of Subgenomic Hepatitis C Virus RNAs in a Hepatoma Cell Line”, 1999, V. Lohmann, F. Körner, J.-O. Koch, U. Herian, L. Theilmann, R. Bartenschlager
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 +
“Hepatitis C virus subtype 1b isolate 31 polyprotein gene, partial cds”-
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[http://www.ncbi.nlm.nih.gov/nuccore/KJ679443.1]
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“Sequences in the 5′ Nontranslated Region of Hepatitis C Virus Required for RNA Replication”, 2001, Peter Friebe, Volker Lohmann, Nicole Krieger, and Ralf Bartenschlager
  
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...
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“Crystal structure of a hepatitis delta virus ribozyme”, 1998, Adrian R. Ferré-D'Amaré, Kaihong Zhou and Jennifer A. Doudna
  
 
===References===
 
===References===

Latest revision as of 18:05, 16 October 2014

Reverse 5


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 142
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 397
    Illegal NgoMIV site found at 426
    Illegal AgeI site found at 236
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 69


Image of sequence

Promoters Diagram.jpg


RNA and secondary structure

5'.gif


Ribozyme

The use of a ribozyme is necessitated due to the complicated binding process between the RNA template and the RdRP. In order to optimise the process and avoid any risk of unfavourable secondary structures or obstruction by unneeded nucleotides, a self-cleaving ribozyme was put after the RNA promoter. The rationale being that after transctiption (either in vitro for human cell tests or in vivo for e-coli tests), any added bases as a result of the T7 polymerase would be removed from the main template strand.

Rib1.gif

The chosen ribozyme taken from the Hepatitis Delta virus was investigated at length by the team of J. Doudna and is reported to be the fastest naturally occurring self-cleaving protein. It also functions independently, without the need for adding chemical substances, and is resistant to denaturants. Its close genetic origin to the RdRP also contributes to a better working and more compatible system.

The structure of the molecule and its active site in particular are shown below.

Rib2.gif


Source

“Partial reconstitution of hepatitis C virus RNA polymerization by heterologous expression of NS5B polymerase and template RNA in bacterial cell”, 2005, Sangyoon Lee, Jong-Ho Lee, Young Hoon Kee, Mi Young Park, Heejoon Myung

B. Heinz, C. Kao – “Template Requirements for RNA Synthesis by a Recombinant Hepatitis C Virus RNA-Dependent RNA Polymerase” - [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC113194/]

“Hepatitis C virus type 1b complete genome, isolate Con1”- [http://www.ncbi.nlm.nih.gov/nuccore/AJ238799]

“Replication of Subgenomic Hepatitis C Virus RNAs in a Hepatoma Cell Line”, 1999, V. Lohmann, F. Körner, J.-O. Koch, U. Herian, L. Theilmann, R. Bartenschlager

“Hepatitis C virus subtype 1b isolate 31 polyprotein gene, partial cds”- [http://www.ncbi.nlm.nih.gov/nuccore/KJ679443.1]

“Sequences in the 5′ Nontranslated Region of Hepatitis C Virus Required for RNA Replication”, 2001, Peter Friebe, Volker Lohmann, Nicole Krieger, and Ralf Bartenschlager

“Crystal structure of a hepatitis delta virus ribozyme”, 1998, Adrian R. Ferré-D'Amaré, Kaihong Zhou and Jennifer A. Doudna

References