IGEM:IMPERIAL/2009/M1/Modelling/oldpage
Background
Genetic circuit
In Module 1 (M1), LacI is constitutively produced. LacI represses the LacI repressible promoter which controls the production of PAH/cellulase.
In order to start off M1, we pipette in IPTG. This represses LacI production, which was originally repressing BBa_R0011. BBa_R0011 is the promoter responsible to kick start the production of PAH / cellulase. As the repression on the promoter is now lifted, the genes under the promoter can now be transcribed to produce the PAH/cellulase.
In conclusion, the addition of IPTG represses the production of functional LacI, and this will subsequently start protein production.
We must note that IPTG is toxic, and it has some significant effects on bacterial cell growth. These must be taken into account, and the Literature review on IPTG will highlight some key findings from papers and online sources.
The conclusion we can draw from these papers is that IPTG above certain amounts (1-2mM) are toxic to e-coli cells. See Literature review on IPTG for growth curves. These effects must be included in simulations, as amounts of IPTG will significantly play an effect in the production of the protein of interest.
Matthieu's FB:
- Constitutive expression of LacI is not part of module 1;talking about the genetic circuit
- Change title: You are not only talking about the circuit; you are describing how the whole module works
- Drawings please
Preliminary Model 1: Genetic circuit: Action of the LacI/IPTG repressive pathway
Protein production and enzyme kinetics
After IPTG/LacI induction, we reach to the stage of protein production:
The drugs we are producing are enzymes, and in order to detect them, they undergo a secondary set of reactions in our assays, so the assays tell us the amount of substrate and the activity of the enzyme, but not how much substrate we have produced.
To track the amounts of drug produced, we have to rely on Michaelis Menten assumptions. The problem with this is that it may not necessarily be the way enzymes behave. At the moment, we have included a very generic model, where the 2 assumptions:
- Total amounts of enzyme are constant throughout
- Rate of change of enzymatic complex is quasi-steady.
Furthermore, we don't know how cellulase and PAH (our two peptide drugs) bind to their enzyme complexes. The reactions may be more complicated, and they may undergo a secondary set of reactions. This data will be obtained from literature.
- Matthieu's FB: Create another page for enzymatic activity It will be much simpler for readers
I will only review protein production from now on
Preliminary Model 2: Enzyme kinetics
References
- Zahn K. Overexpression of an mRNA dependent on rare codons inhibits protein synthesis and cell growth. J Bacteriol. 1996 May;178(10):2926-33. DOI:10.1128/jb.178.10.2926-2933.1996 |
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Effect of Variable Isopropyl Beta-Thiogalactoside Concentrations on �-Galactosidase
Activity,Avinash M. Baktula and Genise A. Nolan, Department of Biology, Western Kentucky University
- Yan J, Zhao SF, Mao YF, and Luo YH. Effects of lactose as an inducer on expression of Helicobacter pylori rUreB and rHpaA, and Escherichia coli rLTKA63 and rLTB. World J Gastroenterol. 2004 Jun 15;10(12):1755-8. DOI:10.3748/wjg.v10.i12.1755 |
