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Team:Imperial College London/Drylab/Protein Production
From 2009.igem.org
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| - | *[https://2009.igem.org/Team:Imperial_College_London/Drylab/ | + | =Introduction= |
| + | This module consists of producing the drug protein of interest. In order to describe the function of the module, two models have been developed. | ||
| + | |||
| + | ==Model for protein drug production:== | ||
| + | Based on the [https://2009.igem.org/Team:Imperial_College_London/M1/Genetic Genetic circuit], a LacI-IPTG inducible promoter is responsible for kickstarting the production of the drug. | ||
| + | * In the absence of IPTG, LacI represses the production of the drug (Cellulase or PAH) | ||
| + | * When IPTG is introduced, the LacI repressing pathway is “de-repressed”, and some output protein is produced. | ||
| + | [[Image:II09_NoIPTG_yesIPTG.jpg|350px|centre]] | ||
| + | |||
| + | <!-- [[Image:m1.fgc.JPG | 400px.jpg]]--> | ||
| + | |||
| + | ===Our goals=== | ||
| + | The modelling aims to provide an overview and better understanding of the M1 system’s function by: | ||
| + | *Characterizing the system. | ||
| + | *Modeling to account for several factors that may reduce/hinder the production of the protein drug such as: | ||
| + | **Lac promoter leakiness | ||
| + | **IPTG toxicity | ||
| + | **Stability of output protein | ||
| + | |||
| + | |||
| + | This module is an integral part of the design, as large-scale commercialization of the drug of interest depends on finding the optimal conditions for protein production. This model has also helped the Wetlab to plan some of the experiments. | ||
| + | |||
| + | More information on the model can be found in the [https://2009.igem.org/Team:Imperial_College_London/Drylab Drylab hub] | ||
| + | |||
| + | ==Model for drug protein enzyme kinetics== | ||
| + | |||
| + | The protein drug of interest is an enzyme. It will bind to specific substrates and increase the rate of their conversion into products. Therefore, by monitoring either the substrate concentration or the product concentration, we can indirectly see the activity of the enzyme. This is quantified by measuring by enzyme activity in the [https://2009.igem.org/Team:Imperial_College_London/Wetlab/Protocols/cellulase cellulase assay] and [https://2009.igem.org/Team:Imperial_College_London/Wetlab/Protocols/PAH PAH assay]. Enzyme activity is the rate of substrate utilisation /product formation per unit time. | ||
| + | |||
| + | ===Our Goals=== | ||
| + | |||
| + | This model aims to better understand the enzymatic action of the drug by: | ||
| + | |||
| + | * Characterizing its enzymatic activity | ||
| + | * Subsequently,by modeling the relationship between the quantity of protein being produced and the enzyme activity using a simple Michaelis-Menten enzyme kinetics model will enable us to take into account factors that could impair enzymatic activity | ||
| + | |||
| + | |||
| + | This model will allow an understanding of the activity of the enzymes and allow the WETLAB to have an idea of the magnitude of the enzymatic activity. More importantly, after the results from enzyme assays are obtained, the model should provide a means of relating the activity output data to the concentration of the enzyme. | ||
| + | |||
| + | More information on the model can be found in the [https://2009.igem.org/Team:Imperial_College_London/M1/Drylab Drylab hub] | ||
| + | |||
| + | |||
| + | <!-- | ||
| + | {{Imperial/Box1|Module 1: Protein production|Two models are required to explain the functionality of M1: | ||
| + | |||
| + | 1) Modelling protein production - Integral part of our design, responsible for manufacturing the drug of interest. | ||
| + | |||
| + | 2) Modelling enzyme kinetics - Since the drugs we are manufacturing (cellulase and PAH) are peptide drugs, which undergo a secondary set of enzymatic reactions in the assays, and we aim to relate their activity to the amount of drug produced by our system.}} | ||
| + | |||
| + | {{Imperial/Box2|Protein Production|M1 consists of the controlled production of a protein of interest. The construct for this module is shown below: | ||
| + | |||
| + | * In the absence of IPTG, LacI represses the production of the protein of interest (which is our drug, cellulase or PAH) | ||
| + | * When we add in IPTG, the LacI repressing pathway is “de-repressed”, and some output protein is produced.<br> | ||
| + | |}} | ||
| + | |||
| + | {{Imperial/Box1|The system||[[Image:II09_M1_OVERVIEW_1.jpg |550px]]}} | ||
| + | |||
| + | ===Goal of the modelling:=== | ||
| + | The modelling aims to provide an overview and better understanding of the M1 system’s function by: | ||
| + | |||
| + | *Characterizing the LacI-IPTG system that is responsible for the production of the drug of interest. | ||
| + | *Modeling and accounting for several factors that may reduce/hinder the production of our output protein of interest such as: | ||
| + | **Lac promoter leakiness | ||
| + | **IPTG toxicity | ||
| + | **Stability of output protein | ||
| + | |||
| + | The conclusions from the models should help people from the WETLAB to plan their experiments and take into account these considerations as possible limitations/factors to look out for. Note that this module is an integral part of our design, as large-scale commercialization of our product of interest depends on finding the optimal conditions for protein production, and we would preferably like to produce a tuneable output. Hence why we think that our simulations will be useful. | ||
| + | |||
| + | Note that the pros of this type of modeling are that, although it provides us a general understanding of the system, there are several factors that we are still not accounting for. Biological processes are stochastic and there is no limit to how much “realism” we can include in our simulations. These are preliminary models, and the compromise level of detail is sufficient to provide us with the understanding we need, but the conclusion is that there is always room for improvement. | ||
| + | |||
| + | == Enzymatic reactions == | ||
| + | |||
| + | After IPTG is added, the Lac promoter is induced. Our protein of interest, either PAH or cellulase, is synthesized and builds up within the bacteria. In the small intestines, the capsule will be degraded and the protein of interest will released. The protein of interest will now show enzyme activity and act upon its substrate within the small intestines. | ||
| + | |||
| + | The protein of interest is an enzyme. It will bind to specific substrates and increase the rate of their conversion into products. Therefore, by monitering either the substrate concentration or the product concentration, we can indirectly see the activity of the enzyme. This is quantitated by enzyme activity, which is the rate of substrate utilisation / product formation per unit time. | ||
| + | |||
| + | The raw data to calculate enzyme activity comes from the enzyme assays conducted in the wet lab. To track the quantity of protein of interest being produced, we rely on the [http://en.wikipedia.org/wiki/Michaelis%E2%80%93Menten_kinetics Michaelis-Menten] plot to relate enzymatic activity to concentration of protein present. | ||
| + | |||
| + | === Goal of the modelling === | ||
| + | |||
| + | This modelling aims to better understand the enzymatic action of our protein of interest:<br> | ||
| + | |||
| + | * Characterise the enzymatic activity of our protein of interest | ||
| + | * Subsequently, model the relationship between the quantity of protein being produced and the enzyme activity using a simple Michaelis-Menten enzyme kinetics model, taking into account factors that could impair enzymatic activity | ||
| + | |||
| + | This model is useful in more accurately predicting the effect of our protein of interest as these are not just simple proteins, but also have enzymatic activity in themselves. <br> | ||
| + | |||
| + | This model will allow an understanding of the activity of the enzymes and allow the WETLAB to have an idea of the magnitude of the activity of the enzymes. More importantly, after the results from enzyme assays are obtained, the model should provide a means of relating the activity output data to the concentration of the enzyme. <br> | ||
| + | |||
| + | Details of the model are found [https://2009.igem.org/Team:Imperial_College_London/M1/Drylab HERE] | ||
| + | |||
| + | --> | ||
| + | |||
| + | |||
| + | |||
*[https://2009.igem.org/Team:Imperial_College_London/Drylab/M1/Protein_production/Analysis The Model]<br> | *[https://2009.igem.org/Team:Imperial_College_London/Drylab/M1/Protein_production/Analysis The Model]<br> | ||
*[https://2009.igem.org/Team:Imperial_College_London/Drylab/M1/Protein_production/Simulations Simulations]<br> | *[https://2009.igem.org/Team:Imperial_College_London/Drylab/M1/Protein_production/Simulations Simulations]<br> | ||
{{Imperial/09/TemplateBottom}} | {{Imperial/09/TemplateBottom}} | ||
Revision as of 12:18, 17 September 2009
Contents |
Introduction
This module consists of producing the drug protein of interest. In order to describe the function of the module, two models have been developed.
Model for protein drug production:
Based on the Genetic circuit, a LacI-IPTG inducible promoter is responsible for kickstarting the production of the drug.
- In the absence of IPTG, LacI represses the production of the drug (Cellulase or PAH)
- When IPTG is introduced, the LacI repressing pathway is “de-repressed”, and some output protein is produced.
Our goals
The modelling aims to provide an overview and better understanding of the M1 system’s function by:
- Characterizing the system.
- Modeling to account for several factors that may reduce/hinder the production of the protein drug such as:
- Lac promoter leakiness
- IPTG toxicity
- Stability of output protein
This module is an integral part of the design, as large-scale commercialization of the drug of interest depends on finding the optimal conditions for protein production. This model has also helped the Wetlab to plan some of the experiments.
More information on the model can be found in the Drylab hub
Model for drug protein enzyme kinetics
The protein drug of interest is an enzyme. It will bind to specific substrates and increase the rate of their conversion into products. Therefore, by monitoring either the substrate concentration or the product concentration, we can indirectly see the activity of the enzyme. This is quantified by measuring by enzyme activity in the cellulase assay and PAH assay. Enzyme activity is the rate of substrate utilisation /product formation per unit time.
Our Goals
This model aims to better understand the enzymatic action of the drug by:
- Characterizing its enzymatic activity
- Subsequently,by modeling the relationship between the quantity of protein being produced and the enzyme activity using a simple Michaelis-Menten enzyme kinetics model will enable us to take into account factors that could impair enzymatic activity
This model will allow an understanding of the activity of the enzymes and allow the WETLAB to have an idea of the magnitude of the enzymatic activity. More importantly, after the results from enzyme assays are obtained, the model should provide a means of relating the activity output data to the concentration of the enzyme.
More information on the model can be found in the Drylab hub
