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After much deliberation over many project ideas, either expanding on previous projects or alternative substrates for existing parts, we decided we wanted to open up a whole new world of opportunities for Synthetic Biology. Developing the basics for a new realm in the field of RNA. Using a combination of experiments in Escherichia coli (E.coli) and human cells, both HeLa and Huh7.5 we attempted to turn on the lights to RNA world experimentation.

Until now RNA has been used sparingly in iGEM with teams tiptoeing around the idea with little advancement, we wanted to set the groundwork for future teams to have the option of classical Synthetic Biology i.e. DNA projects or new projects in RNA world. We feel this is a hugely exciting new area for research to begin as we were initially struggling with originality of our project due to the exponential increase in iGEM teams and projects done previously and underway. RNA is a fascinating alternative for projects. We decided the fundamentals were: an RNA repressor, promoter, ribosome binding site (RBS), kill switch, a replication system and demonstrating the potentials with our own part. These were combined into a self-replicating RNA strand or “Replicon”. These demonstrate a use of all the elements together however the potential permutations and adaptations of these parts are endless.

Deciding on how to utilise our system we had a huge number of potential experiments but decided we would focus our efforts on current and important world health problems. These were narrowed down to; type II diabetes mellitus and, on further research into current events, Ebola. Type II diabetes is a pandemic of epic proportions, on the rise in all corners of the globe of all race and age, in part due to the increase trend in obesity and glucose consumption.

America	England
Comparison by race	Worldwide

This costs the healthcare systems in all countries billions of dollars already and many are living undiagnosed that could double this figure. Almost every individual in America and Europe will have a friend or family member affected with this disease and causes heartache to thousands more following deaths of sufferers.

Treatments for Type II diabetes range from the simple; lose weight and consume less sugar to the expensive; gene therapy and dipeptidylpeptidase IV (DPP-IV) inhibitors, and the painful; amputations. DPP-IV, due to its cellular, genomic origin seemed like an appropriate target to tackle using our system. DPP-IV inhibitors are administered in stage two of treatment directly after lifestyle changes and are used to slow the degradation of incretins such as glucose like peptides which is accelerated by DPP-IV. Incretins act to increase the duration of

Gene therapy has been long sought after in biology. Its applications are extremely wide ranging, as by manipulating the genome a cell, you can in theory completely manipulate a cell to make it do whatever you want. This is essentially the same goal as synthetic biology, however the key difference is that gene therapy focuses on modifying the genes of cells in animals that have already grown beyond embryonic stage, rather than individual cells on their own. There are many problems with current gene therapy, including it being dangerous, being extremely difficult to perform, its possibility of modifying the DNA of cells in ways that can't be predicted, or the gene therapy not being permanent. Our project is to try and solve some of these problems by using a type of RNA called a 'replicon'.

What is a replicon?

A replicon is RNA that acts to replicate itself on its own using only the ribozymes of the cell. RNA usually degrades very quickly in cells, but a replicon should last permanently, because it should replicate faster than it can be degraded. Several viruses use replicons as their method of manipulating cells. Our idea is to take parts from the genome of hepatitis C (HCV) and modify it so that instead of doing damage to the body, it 'silences' harmful genes. To do this, we want to add an siRNA sequence to a replicon sequence. siRNA (small interfering RNA) is RNA that contains part of a complimentary nucleotide sequence to a particular RNA sequence. The human cell breaks this sequence down, and then continues to break down complimentary RNA sequences to this, including the sequence we want to target.

What are the advantages of this over conventional gene therapy?

Nowhere in this process is the actual DNA of the cell modified, so this removes the danger of DNA of the cell being modified in a way that is unwanted. This also improves upon conventional gene silencing, which involves siRNA only, as the replicon represents a permanent source of siRNA, greatly increasing the efficiency of the gene silencing. Our proposed method of delivery is to use a viral vector, technology which unfortunately doesn't exist yet, but could be developed within the next few decades. This method would allow for gene silencing in multicellular organisms, the holy grail of gene therapy.

PROJECT

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.

RNA Dependent RNA Polymerase

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). 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.

Aptazyme

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.

EMCV IRES

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.

NKRF IRES

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.

NKRF IRES

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.

MS2 Coat Protein

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.

MS2 Hairpin

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