Team:Warwick/Parts

         <a href="/Team:Warwick"> <img class = "headerImage" style = "width: 12%;"  

<li> <a href = "/Team:Warwick/Parts/3promoter"> RNA PROMOTERS </a> </li>

<p> Below is an interactive schematic of our system in RNA (for simplicity, we have omitted in the drawing a T7 promoter and its terminator to transcribe the whole operon), hover your mouse over any part to see a brief description, and click to follow through to the Registry and see a more in depth description, and information regarding sequences and results. You can find the full list of parts we've submitted <a href="http://parts.igem.org/cgi/partsdb/pgroup.cgi?pgroup=iGEM2014&group=Warwick">here</a>. The parts labelled 'favourite' are our best parts. </p>

     <h2>5' RNA Promoter</h2>

     <p>This is derived from the 5' UTR of the HCV virus strain 1b isolate Con1. It contains the reverse complement of the RdRp initiation sequence. The secondary structure of this sequence acts as a binding site and initiates replication of the minus strand by RdRp. It also includes the first 16 amino acids of the first gene of HCV as this has been shown to increase the efficacy of binding of the RdRp to the 5' promoter. We define an RNA promoter as a sequence located at the 3' end of the transcript, consisting of a RdRp initiation sequence and a ribozyme that cleaves after the initiation sequence without leaving any scar. We foresee that such regulatory elements will allow constructing novel post-transcriptional circuits by adding operator sites in the promoters. Such operator sites could be for instance the MS2 hairpin or any other RNA sequence known to bind to a regulator. This is the RNA equivalent to a transcription factor.</p>

classical EMCV IRES used in many papers in our target cells,which has been shown to be compatible  

factor. We chose to test it also because during  

efficient; however, it had never been previously used in Huh7.</p>

site for the MS2 coat protein (formed  

downstream in the replicon), which  

represses translation, hence preventing  

exponential growth of the replicon. This "copy number control" also allows a switching off of the RdRp once a steady state is reached, which minimizes the possible interferences between RdRp and the ribosome.</p>

endogenous to mammalian cells,

hairpin.</p>

a protein which binds to the MS2 box - a hairpin  

efficiency and leaves no scar following the  

genome has one open reading frame  

     <h2>3' RNA Promoter</h2>

     <p>Each unique RNA dependent RNA polymerase (RdRP) initiates de novo replication of a RNA strand by interacting with RdRP-specific RNA sequences, henceforth called RdRP/RNA promoters. The promoter is "insulated" by using a ribozyme on its 3' end that self-cleaves without leaving a scar (and, therefore, producing the right 3' end for RdRp initiation).  The RdRP chosen for our project is taken from the Hepatitis C virus (HCV) and it recognizes a limited set of such initiation sequences. All of them possess a few common characteristics: an initiation cytidylate at the 3’ end, where the replication starts; and a stable secondary structure – single stranded tail and a stem of various length. For our project in addition to the indigenous to HCV RdRP promoters, we designed alternative RNA promoter sequences previously identified by Heinz et al. as templates for replication by the HCV RdRP. </p>

<a href="/Team:Warwick/Parts/3promoter" class="popper" data-popbox="pop1"><img src="https://static.igem.org/mediawiki/2014/7/7f/5%27_promoter-01-01.png" width="100%"></a>