Difference between revisions of "Part:BBa K1442034:Design"

Latest revision as of 14:55, 16 October 2014

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

RNA and secondary structure

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.

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.

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

@@ Line 1: / Line 1: @@
 __NOTOC__
 <partinfo>BBa_K1442034 short</partinfo>
@@ Line 6: / Line 5: @@
-===Design Notes===
+== Image of sequence ==
-N/A
+[[File:Promoters_Diagram.jpg]]
+== RNA and secondary structure ==
+[[File: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.
+[[File: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.
+[[File: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
-N/A
+“Crystal structure of a hepatitis delta virus ribozyme”, 1998, Adrian R. Ferré-D'Amaré, Kaihong Zhou and Jennifer A. Doudna
 ===References===