Team:Imperial College London/Drylab/Protein production/Analysis
From 2009.igem.org
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- | <font face='Calibri' size='3'><b>Assumptions</b></font><br> | + | <font face='Calibri' size='3'><b>Assumptions</b></font><br><br> |
*<b>LacI assumptions:</b> | *<b>LacI assumptions:</b> | ||
**The lac operon represses by constitutively synthesizing the LacI gene product [ref1]. | **The lac operon represses by constitutively synthesizing the LacI gene product [ref1]. | ||
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- | <font face='Calibri' size='3'><b>Model Predictions</b> </font> | + | <font face='Calibri' size='3'><b>Model Predictions</b> </font><br><br> |
Taking these assumptions into account, we can predict what the overall Qualitative behavior of the system will be. | Taking these assumptions into account, we can predict what the overall Qualitative behavior of the system will be. | ||
*In the absence of IPTG, the output amount of our protein of interest will depend on the steady state of LacI protein. | *In the absence of IPTG, the output amount of our protein of interest will depend on the steady state of LacI protein. | ||
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<font face='Calibri' size='3'><b>The actual model...</b></font> | <font face='Calibri' size='3'><b>The actual model...</b></font> | ||
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[https://2009.igem.org/Team:Imperial_College_London/Drylab/M1/Protein_production/Analysis/Detailed Click here for details] | [https://2009.igem.org/Team:Imperial_College_London/Drylab/M1/Protein_production/Analysis/Detailed Click here for details] | ||
{{Imperial/09/TemplateBottom}} | {{Imperial/09/TemplateBottom}} |
Revision as of 14:56, 18 September 2009
- Overview
- The model
- Simulations
Assumptions
- LacI assumptions:
- The lac operon represses by constitutively synthesizing the LacI gene product [ref1].
- Here we have assumed that all the other complicated dynamical pathways are at dynamic equilibrium [ref2], and we are only considering the LacI gene.
- If these effects were included, the dynamics of the entire system will be more complicated, and the outcome of the model will be different.
- In a culture grown overnight, levels of LacI expression will have reached steady state. We can assume that this is the case before we add in IPTG.
- IPTG assumptions:
- LacI and IPTG undergo a secondary set of reactions when IPTG is added in. We assume that 1 molecule of IPTG binds to 1 molecule of LacI, creating an intermediate reaction complex:
- We also assume that the concentration of IPTG we put in is below the threshold of toxicity that could significantly start killing our cells. ([http://openwetware.org/wiki/IGEM:IMPERIAL/2009/M1/Modelling/Analysis/literatureIPTG Literature review on IPTG])
- In this model we have neglected the degradation rate of IPTG and also of the [IPTG-LacI] complex.
Model Predictions
Taking these assumptions into account, we can predict what the overall Qualitative behavior of the system will be.
- In the absence of IPTG, the output amount of our protein of interest will depend on the steady state of LacI protein.
- If the levels of LacI at steady state are low, we get a relatively high basal production of protein of interest, as the steady state is dependent on the lac promoter strength and its degradation. A weak promoter does not repress sufficiently the production of protein of interest.
- If the levels of LacI at steady state are high, we are repressing more the production of protein of interest, so we will get a lower initial rate of protein production as the Lac promoter is stronger (higher PoPs output)
- If we take into account leakiness of the Lac promoter, we will see a higher basal amount of protein of interest initially than in the non-leaky case.
- When IPTG is added in, it will undergo a secondary reaction with LacI, thus, de-repressing the pathway, so we will see a bump in production of the protein of interest.
- These effects are transient. LacI has a constitutive equilibrium, so after sometime, it will return to its original state and repress production of protein of interest once again.
- The more IPTG we add in, the more LacI we will remove initially from the system, so the more output protein produced for a given period of time.
The actual model... Click here for details