Other

Part:BBa_K1442034:Design

Designed by: Yin Ho Vong, Caroline de Cock, Waqar Yousaf, Iva Burova, Chelsey Tye   Group: iGEM14_Warwick   (2014-09-05)
Revision as of 14:55, 16 October 2014 by IvaBurova (Talk | contribs)

(diff) ← Older revision | Latest revision (diff) | Newer revision → (diff)

HCV 3


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 200
    Illegal PstI site found at 227
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 169
    Illegal PstI site found at 200
    Illegal PstI site found at 227
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 200
    Illegal PstI site found at 227
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 200
    Illegal PstI site found at 227
    Illegal NgoMIV site found at 241
    Illegal NgoMIV site found at 270
  • 1000
    COMPATIBLE WITH RFC[1000]


Image of sequence

Promoters Diagram.jpg


RNA and secondary structure

3'.jpg


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

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


“Genetic Analysis of Sequences in the 3′ Nontranslated Region of Hepatitis C Virus That Are Important for RNA Replication”, 2002, Peter Friebe and Ralf Bartenschlager


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

References