"
Team:Warwick/Modelling/Experiments
From 2014.igem.org
| Line 69: | Line 69: | ||
<h1>Experiments</h1> <br> | <h1>Experiments</h1> <br> | ||
<p>Besides modelling the overall system to gain a theoretical understanding as to how the system should behave, we modelled individual experiments so that we could help the biologists and directly compare results by fitting data. These were the experiments that we modelled:</p> | <p>Besides modelling the overall system to gain a theoretical understanding as to how the system should behave, we modelled individual experiments so that we could help the biologists and directly compare results by fitting data. These were the experiments that we modelled:</p> | ||
| - | <h3>IRES (Internal Ribosome Entry Site)</h3> | + | <h3 align="left">IRES (Internal Ribosome Entry Site) </h3> |
<p>We obtained two different IRES sequences whilst constructing our system, one for the NKRF IRES and one for the EMCV IRES. We knew that the EMCV had been shown to work in human cells (Huh-7 cells) [Lohmann 1999] but NKRF had been introduced into HeLa cells successfully <a href = "http://www.ncbi.nlm.nih.gov/pmc/articles/PMC85491/#!po=42.3077"> [1] </a> and be 92 times more efficient than EMCV in this cell line. We wanted to determine if the same held in Huh-7.5 cells which was our cell line of choice. We modelled the efficiency of the two IRESs and then fitted the data to our model to determine its accuracy.</p> | <p>We obtained two different IRES sequences whilst constructing our system, one for the NKRF IRES and one for the EMCV IRES. We knew that the EMCV had been shown to work in human cells (Huh-7 cells) [Lohmann 1999] but NKRF had been introduced into HeLa cells successfully <a href = "http://www.ncbi.nlm.nih.gov/pmc/articles/PMC85491/#!po=42.3077"> [1] </a> and be 92 times more efficient than EMCV in this cell line. We wanted to determine if the same held in Huh-7.5 cells which was our cell line of choice. We modelled the efficiency of the two IRESs and then fitted the data to our model to determine its accuracy.</p> | ||
<h3>3’ UTR</h3> | <h3>3’ UTR</h3> | ||
Revision as of 01:52, 17 October 2014
Experiments
Besides modelling the overall system to gain a theoretical understanding as to how the system should behave, we modelled individual experiments so that we could help the biologists and directly compare results by fitting data. These were the experiments that we modelled:
IRES (Internal Ribosome Entry Site)
We obtained two different IRES sequences whilst constructing our system, one for the NKRF IRES and one for the EMCV IRES. We knew that the EMCV had been shown to work in human cells (Huh-7 cells) [Lohmann 1999] but NKRF had been introduced into HeLa cells successfully [1] and be 92 times more efficient than EMCV in this cell line. We wanted to determine if the same held in Huh-7.5 cells which was our cell line of choice. We modelled the efficiency of the two IRESs and then fitted the data to our model to determine its accuracy.
3’ UTR
We found sequences for six different 3’ promoters, and similar to the IRES we wanted to determine which was most efficient. Our goal with the modelling was to determine which would be the best for our system, so that we could then incorporate it into our overall replicon.
Aptazyme
This functions as the kill switch for our system. We wanted to model the effectiveness of this switch, by using values obtained from the literature to obtain theoretical time scales as to how long it would take to stop replication of the system. We then conducted the experiment and fit the data to obtain a comparison as to how close the theoretical model and real values differ.
siRNA
This model was to illustrate the effects of siRNA as a method of gene silencing. We took to the literature and followed previously developed models from these papers, and then explored the dynamics of them using values closer to our systems to gauge a response as to how effective siRNA targeting is at silencing. In the testing this was specific to DPP-IV, however in practice we can take this and apply it to any siRNA targeting sequence. Therefore we made our model general to keep with our aim of reusability so that others in the future could take our model, and with a few tweaks be able to apply it to their systems.
