Difference between revisions of "Part:BBa K1442304:Design"

Latest revision as of 19:03, 16 October 2014

SLD3 RdRP Promoter 3

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal NgoMIV site found at 13
Illegal NgoMIV site found at 41
Illegal NgoMIV site found at 70
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI.rc site found at 34

Image of sequence

DNA

5’ GGGCTTGCATAGCAAGTCTGAGACC 3’

RNA and secondary structure

Features

Gel electrophoresis showing replication by HCV RdRP of a number of potential RNA promoters. The bands show the amount of replicated product and its approximate length, and quantify the tendency promoters to produce strands of wrong size. C2 (band 8) shows considerable efficiency in directing replication of the full template, although an amount of incorrect products were also present.

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

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

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/]

@@ Line 1: / Line 1: @@
 __NOTOC__
 <partinfo>BBa_K1442304 short</partinfo>
@@ Line 5: / Line 4: @@
 <partinfo>BBa_K1442304 SequenceAndFeatures</partinfo>
+== Image of sequence ==
-===Design Notes===
+[[File:Promoters_Diagram.jpg]]
-None
+== DNA ==
+’ GGGCTTGCATAGCAAGTCTGAGACC 3’
+== RNA and secondary structure ==
+[[File:SD3.png]]
+== Features ==
+[[File:1Paper.jpg]]
+Gel electrophoresis showing replication by HCV RdRP of a number of potential RNA promoters. The bands show the amount of replicated product and its approximate length, and quantify the tendency promoters to produce strands of wrong size. C2 (band 8) shows considerable efficiency in directing replication of the full template, although an amount of incorrect products were also present.
+== 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===
-http://www.ncbi.nlm.nih.gov/pmc/articles/PMC113194/
+“Crystal structure of a hepatitis delta virus ribozyme”, 1998, Adrian R. Ferré-D'Amaré, Kaihong Zhou and Jennifer A. Doudna
+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/]
 ===References===