Team:Warwick/Project

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             <li> <a href = "/Team:Warwick"> HOME </a> </li>
             <li> <a href = "/Team:Warwick"> HOME </a> </li>
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             <li> <a href = "/Team:Warwick/Project"> PROJECT </a> </li>
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             <li> <a href = "/Team:Warwick/Project"> <span> PROJECT </span> </a> </li>
             <li> <a href = "/Team:Warwick/Team"> TEAM </a> </li>
             <li> <a href = "/Team:Warwick/Team"> TEAM </a> </li>
             <li> <a href = "/Team:Warwick/Parts"> PARTS </a> </li>
             <li> <a href = "/Team:Warwick/Parts"> PARTS </a> </li>
             <li> <a href = "/Team:Warwick/Modelling"> MODELLING </a> </li>
             <li> <a href = "/Team:Warwick/Modelling"> MODELLING </a> </li>
             <li> <a href = "/Team:Warwick/Notebook"> NOTEBOOK </a> </li>
             <li> <a href = "/Team:Warwick/Notebook"> NOTEBOOK </a> </li>
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             <li> <a href = "/Team:Warwick/Human"> HUMAN PRACTICES </a> </li>
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             <li> <a href = "/Team:Warwick/Human"> POLICY & PRACTICES </a> </li>
             <!--<li> <a href = "/Team:Warwick/Measurements"> MEASUREMENTS </a> </li>-->
             <!--<li> <a href = "/Team:Warwick/Measurements"> MEASUREMENTS </a> </li>-->
             <li> <a href = "/Team:Warwick/Interlab"> INTERLAB </a> </li>
             <li> <a href = "/Team:Warwick/Interlab"> INTERLAB </a> </li>
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             <li> <a href = "/Team:Warwick/Attributions"> SPONSORS </a> </li>
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             <li> <a href = "/Team:Warwick/Attributions"> ATTRIBUTIONS </a> </li>
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<li> <a href = "/Team:Warwick/Project/Motivation"> MOTIVATION </a> </li>
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<li> <a href = "/Team:Warwick/Project/Howitworks"> HOW IT WORKS </a> </li>
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<li> <a href = "/Team:Warwick/Project/Applications"> APPLICATIONS </a> </li>
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            <h1> PROJECT </h1> <br> <br>
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<h2>WELCOME TO RNA WORLD</h2>
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            <p>Disrupting the flow of biological information at the level of mRNA is a safer alternative to conventional gene therapy, wherein insertional mutagenesis can occur through integrating vectors. In addition, the ability to regulate the level of expression of a gene using such vectors proves difficult. Therefore, we aim to create a modular, self-replicating RNA system using Hepatitis C Virus (HCV) derived RNA dependent RNA polymerase (RdRp). This drives production of siRNA directed against the enzyme dipeptidyl peptidase-IV (DPP-IV) which is elevated in type 2 diabetes and is the target of major drug studies. The replicon contains control modules, exhibiting a negative feedback mechanism provided by: an MS2 domain linked to RdRp, thereby controlling RdRp translation and therefore controlling replication, and an aptazyme switch to regulate expression levels of our siRNA. Validation of our system and the testing of modules will be performed in human (Huh 7.5) and E. coli cells.
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<p> Welcome to the biological world which we have spent an entire summer creating and perfecting. We are introducing RNA to synthetic biology in a big way by directly contributing a number of key parts to iGEM. We have taken a risk, we have been told our project is unrealistic and should be scrapped, that a group of undergraduates are crazy for even thinking about doing something so complicated. At times, we doubted ourselves- there have been good times and bad times. Spurred on by a desire to make a difference to synthetic biology and to the world in which we live, we pressed forward - a new team to iGEM, not seasoned veterans. We take chances and risks to make a difference - whether success comes our way or failure, we embrace the moment, cherishing it. We hope that the synthetic biology community will appreciate our efforts, as RNA usage and popularity surges. DNA best serves as a template for information storage whereas RNA is multi-functional, catalytic and dynamic. Welcome to our world - the RNA world!<br>  
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<h2> RNA Dependent RNA Polymerase </h2>  
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<b>- Warwick iGEM Team 2014 </b>
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<p> RNA dependent RNA polymerase (RdRp) is an enzyme which catalyses the replication of RNA from an RNA template. An essential protein encoded within viruses that lack a DNA phase and replicate using negative sense RNA. The submitted RdRp part derives from the Hepatitis C virus con1 strain, RdRp is also referred to as non-structural protein 5B (NS5B). Part sequence was derived from Lohmann et al., 1997, with the authors achieving full expression and activity of RdRp in a baculovirus expression system. Membrane association of RdRp is essential for replication of HCV subgenomic regions, with the C-terminal tail containing 21 amino acids which confer high hydrophobicity and mediate insertion into the membrane (Moradpour et., 2004).
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The 21 amino acid residues have been removed in the part sequence, ensuring cytoplasmic RdRp activity, in line with previous analysis showing no significant loss of nucleotide polymerization activity (Vo et al., 2004). The C-terminal tail preceding the C-terminal hydrophobic insertion sequence interacts with structural elements including the β-hairpin loop of NS5b (Leveque et al., 2003). The β-hairpin loop inserts into the active site, believed to position the 3’ terminius of HCV viral RNA to initiate RNA synthesis (Hong et al., 2001). RdRp initiates RNA synthesis with nucleotide transfer activity found within the palm motif (Figure 1a & b),  with several amino acid residues implicated in nucleotide triphosphate contact (Bressanelli et al., 2002).  RdRp requires 5’ and 3’ untranslated regions (UTRs) found within the HCV genome to direct RNA synthesis.  The UTRs form ordered RNA structures and are evolutionary conserved.
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<h4> Aptazyme </h4>
 
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An aptazyme is an RNA based switch  that operates by ribozyme-mediated cleavage of RNA. This original sequence for this part derives from Hartig et al., 2002 and requires the addition of theophylline to induce hammerhead ribozyme activation and cleavage, as depicted in Figure 1a. The part sequence is modified to contain a stop codon at the end, as an RBS is present. 
 
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<h6> EMCV IRES </h6>
 
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This internal ribosme entry site (IRES) is derived from the encephalomyocarditis virus (EMCV) -  it is a noncoding RNA fragment that is able to initiate high levels of cap-independent translation in mammalian and cell free extracts. This part derives from bases 273-845bp as found in Pamenberg et al., 2006.  Due to the extensive and dynamic secondary structure produced (Figure 1a), optimum activity is retained with the presence of  273-845. Viral bases 400-836 also retain activity, although this is partial (Pamenberg et al., 2006) . The IRES AUG at the 5’ end of the cistron is critical to ensure 40S ribosomal subunit recruitment  (Caruso et al.,;  Pongnonec et al., 2006) and has been retained. 
 
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<h7> NKRF IRES </h7>
 
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This NKRF IRES is found within the mammalian X chromosome and is  from the long 5’ untranslated region of the NF-kB repressing factor, a multifunctional cytokine interferon-β. This forms a secondary structure, shown below with sequence, which directs ribosomes to the downstream start codon by a cap-dependent mechanism. Following experimentation this was shown to have a 30-fold higher than picornaviral IRESs (Oumard, 2000). This was compared to the EMCV IRES and the poliovirus IRES in HeLa cells, murine embryonic stem cells and embryonic fibroblasts, using the firefly luciferase. The level of fluorescence seen was 92-fold higher than EMCV and 130-fold more active than the poliovirus IRES. NKRF acts in a distance independent manner and has a very high efficiency of translation initiation. 
 
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<h7> NKRF IRES </h7>
 
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This NKRF IRES is found within the mammalian X chromosome and is  from the long 5’ untranslated region of the NF-kB repressing factor, a multifunctional cytokine interferon-β. This forms a secondary structure, shown below with sequence, which directs ribosomes to the downstream start codon by a cap-dependent mechanism. Following experimentation this was shown to have a 30-fold higher than picornaviral IRESs (Oumard, 2000). This was compared to the EMCV IRES and the poliovirus IRES in HeLa cells, murine embryonic stem cells and embryonic fibroblasts, using the firefly luciferase. The level of fluorescence seen was 92-fold higher than EMCV and 130-fold more active than the poliovirus IRES. NKRF acts in a distance independent manner and has a very high efficiency of translation initiation. 
 
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<h9> MS2 Coat Protein <h9>
 
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<p> This MS2 bacteriophage coat protein part derives from Fussenegger et al., 2012. The MS2 coat protein binds a specific stem-loop structure, referred to as the MS2 box. This acts as a silencing mechanism of RNA through translational repression (Ni et al., 1995). The full sequence found in Fussenegger et al., 2012 has been retained, as previous analysis has indicated alteration of MS2 coat protein reduces cooperative protein-RNA binding (Uhlenbeck et al., 1995). MS2 is frequently used in biochemical purification of RNA-protein complexes and is combined with GFP to detect RNA in living cells. </p>
 
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<h11> MS2 Hairpin </h11>
 
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This non-coding part forms an operator hairpin to facilitate binding by the bacteriophage MS2 Coat protein. Naturally, this mechanism is used to repress the translation of the viral replicase (Uhlenbeck et al., 2002). The protein-RNA interaction is depicted in Figure 1. The MS2 hairpin represents an ideal part that can be used for translational control
 
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Latest revision as of 03:40, 18 October 2014

WELCOME TO RNA WORLD

Welcome to the biological world which we have spent an entire summer creating and perfecting. We are introducing RNA to synthetic biology in a big way by directly contributing a number of key parts to iGEM. We have taken a risk, we have been told our project is unrealistic and should be scrapped, that a group of undergraduates are crazy for even thinking about doing something so complicated. At times, we doubted ourselves- there have been good times and bad times. Spurred on by a desire to make a difference to synthetic biology and to the world in which we live, we pressed forward - a new team to iGEM, not seasoned veterans. We take chances and risks to make a difference - whether success comes our way or failure, we embrace the moment, cherishing it. We hope that the synthetic biology community will appreciate our efforts, as RNA usage and popularity surges. DNA best serves as a template for information storage whereas RNA is multi-functional, catalytic and dynamic. Welcome to our world - the RNA world!
- Warwick iGEM Team 2014



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