Difference between revisions of "Part:BBa K1442024"

Revision as of 12:12, 11 October 2014

NKRF IRES

This is an Internal Ribosome Entry Site (IRES) acting in mammalian cells. It allows translation initiation in the middle of an RNA strand which is required for the fuctioning of our replicon as there are multiple protein coding regions separated by structural nucleotide sequences interspersed throuhgout the RNA strand. We chose to investigate two IRES sequences to compare the relative efficiency and determine which was better for incorporation into our system. These were the Encephalomyocarditis virus (EMCV) and the IRES derived from the NF-kappaB repressor factor (NKRF) untranslated region.

<NKRF is a transcriptional repressor protein that is essential in the immune response and acts to mediate the translational efficiency of other cellular proteins. The IRES used in this experiment is dervied from the 3' untranslated region of the protein mRNA. Oumard and Hennecke 2000 indicates that this IRES has a 30 times higher translational efficiency than EMCV IRES and significantly higher than the poliovirus also, in HeLa cells. As our experiment is in Huh 7.5 cells we decided to test both the EMCV (already previously confirmed to work in Huh7.5) and NKRF which may or may not give us significantly better translational efficiency. </p>

Oumard et al (2000) showing that the flourescence when using an NKRF IRES before a firefly luciferase protein was significantly higher relative to both EMCV and poliovirus IRES and hence seemed optimal for our system where translational efficiency is key to maintaining the replicon in the cell.

The NKRF, like the EMCV IRES, forms a complex secondary structure

This shows the secondary conformation of this structural nucleotide sequence and acts to attract ribosomes to the site of the start codon of the internal protein and begin translation. Oumard and Hennecke 2000 and shows the sequence used in this experiment

Usage

This IRES was intended for used before the neomycin selectivity marker and the NS5B RdRp derived from the HCV strain 1b genome, as both of these proteins are integrated into the RNA strand with structural nucleotide sequences before and after.

This is a schematic of the entire replicon and indicating the positioning of the IRESes

Diagram generated by Genious.

part BBa_K1442109

This testing module was used to determine the efficacy of the IRESes by measuring the fluorescence in human HeLa and Huh7.5 cells using a tecan plate reader. The IRES was cloned between the AscI and BsrGI site following the T7 promoter and before the GFP. This was sequenced to ensure no mutations during the PCR amplification of the parts synthesised by IDT.

Sequence and Features