Difference between revisions of "Part:BBa K1442023"

Latest revision as of 21:43, 21 October 2019

EMCV 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.

IRES' were first discovered in EMCV and polioviruses and the EMCV has been the favoured IRES for use in replicon systems such as Lohmann (1999) and Lee (2005).

IRESes are commonly used by viruses to ensure translation is active even during times when host translation is inhibited. This is ideal for our system as in order to maintain the RNA in the cell for the maximum length of time the translation of the RdRp should be constant to balance the instability and subsequent degradation of the RNA strands.

The EMCV IRES functions via its secondary structure, a complex series of hairpin loops and double stranded sections including a bifarcation loop, which attracts ribosomes to the vicinity of the start codon of the protein coding region of the RNA.

The secondary structure of the EMCV IRES predicted using Vienna RNAfold calculating minimum free energy of nucleotide pairing

This is detailed in the Bochkov and Palmenberg (September 2006) paper on translational efficiency of EMCV IRES and details the minimum and preferred regions included for functional translational action.

We used the extended preferred region referenced in this image, in order to optimize the efficacy of this subunit of the replicon.

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.

Model

The way in which the IRES should work was modelled using the following equation:

R=positive strand RNA

t=transfection time

and G=GFP levels

Experimentation

We compared efficiency of EMCV and NKRF in both HeLa and Huh 7.5 cells. EMCV and HeLa have been previously investigated in HeLa cells however have not been directly compared in Huh 7.5 cells. We compared each of these at the peak of fluorescence for most of the cells, 1800 seconds after loading. Due to the nature of the measurement equipment we were unable to supply CO2 to the cells during measurement and the cells gradually died during the 25 hour measurement period explaining the exponential decline in fluorescence.

This shows an exponential decline as the cells die due to lack of CO2 supply and shows the same overall trend in all the cells with maximal fluorescence occurring at approximately 1800 seconds

Method

We transfected the cells with 0.1μg of the IRES or control RNA, transcribed in vitro using Invitrogen RNA transcription kit, into each well of cells in a 96-well plate. The cells were plated at a concentration of 0.15x10⁵ determined using a haemocytometer. The cells were transfected using lipofectamine delivery in which lipid nanoparticles details of which can be found at Biontex K2 transfection system. Flourescence of the media and cells themselves were also taken into account. Each IRES had 10 biological repeats in each cell while the control had 5. Technical repeats were very difficult to achieve as it required splitting of the cells in each well as both HeLa and Huh 7.5 are attached cells, therefore the likelihood of spillage and contamination between the wells was too high to warrant the added accuracy of technical repeats.

Results

The fluorescence of each type of transfected cell was taken at 1800 seconds and averaged across the groups while fluorescence was adjusted for media and cell alone fluorescence and consolidated in this table.

We measured fluorescence using a tecan Magellan plate reader at wavelength 465nm - 530nm (that corresponding to GFP). Each IRES had ten replicates in each cell and hence error bars had an accuracy of 0.1. The fluorescence of media and cells themselves were accounted for in each case and the negative control used was the human optimised forwards GFP part:BBa_K1442106 without IRES included.

HeLa cell experimentation

In HeLa cells the NKRF IRES have previously been directly compared with firefly luciferase association in [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC85491/#__ffn_sectitle Oumard et al 2000]

This clearly indicates that the NKRF IRES is significantly more efficient at initiating translation of GFP following this IRES conferring 2.4 times greater intensity than EMCV. Strangely the negative control showed higher fluorescence than the EMCV IRES also. This may have been due to increased growth rate of the cells or slower depletion of the growth media hence increasing overall fluorescence. Due to time restraints and limited number of wells in the plate we did not have as many replicates with this control.It may also be due to inefficient transfection of the cells with EMCV DNA strand.

Huh 7.5 experimentation

This is, to our knowledge, the first investigation of NKRF relative efficiency in Huh 7.5 cells. The results indicated that the NKRF IRES would be more efficient for use in our replicon but is conferred a less significantly increased relative fluorescence intensity than in HeLa cells as seen in the bar chart below.

This bar chart indicates that the results of the IRES efficiency in Huh cells is not significantly different as the standard error bars overlap somewhat. The negative control, as in the HeLa cells, indicates that NKRF does work efficiently and significantly in HeLa cells.

These results indicate that overall the NKRF transfection was more efficient and would be more appropriate and reliable for use in our system.<p> [http://2019.igem.org/Team:AFCM-Egypt# Team:AFCM-Egypt 2019] have characterized this part In order to reduce CAR exhaustion and prevent undesired expression,, the introduction of an internal ribosomal entry site (IRES) to lower tonic signaling and ligand-independent phosphorylation of the CAR-CD3ζ chain which has been shown to improve CAR-T cell expansion as compared to CAR-T cells not containing the IRES element. Thus, we have added IRES to the construction of our TRUCK CAR and we also characterized it in terms of enhancement to expression of our TRUCK CAR HDR insertion in engineered T-Cells For results of functional characterization please refer to our parts Page EGYPT-AFCM 2019 Parts as This real time qPCR shows the quantitative increase in expression of the CAR insertion in schistosomiasis associated bladder cancer patient T-cells engineered with IRES in comparison to relatively lower expression in engineered T cells without IRES.

Sequences and Features