Team:KULeuven/Modelling/Vanillin Production

From 2009.igem.org

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| Rate of locking of leaked RIBOLOCK.
| Rate of locking of leaked RIBOLOCK.
| [https://2009.igem.org/Team:KULeuven/Modelling/Vanillin_Production#References [5<html>]</html>]
| [https://2009.igem.org/Team:KULeuven/Modelling/Vanillin_Production#References [5<html>]</html>]
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! colspan="4" style="border-bottom: 1px solid #003E81;" | Sam8 catalysis
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| k<sub>cat</sub>
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| 0.015 s<sup>-1</sup>
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| [https://2009.igem.org/Team:KULeuven/Modelling/Vanillin_Production#References [8<html>]</html>]
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| K<sub>m</sub>
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| 15.5 microMolarity
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| [https://2009.igem.org/Team:KULeuven/Modelling/Vanillin_Production#References [8<html>]</html>]
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! colspan="4" style="border-bottom: 1px solid #003E81;" | Sam5 catalysis
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| k<sub>cat</sub>
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| 4.2385 s<sup>-1</sup>
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| [https://2009.igem.org/Team:KULeuven/Modelling/Vanillin_Production#References [8<html>]</html>]
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| K<sub>m</sub>
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| 432.0 microMolarity
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| [https://2009.igem.org/Team:KULeuven/Modelling/Vanillin_Production#References [8<html>]</html>]
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! colspan="4" style="border-bottom: 1px solid #003E81;" | COMT catalysis
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|-
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| k<sub>cat</sub>
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| 0.03319 s<sup>-1</sup>
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| [https://2009.igem.org/Team:KULeuven/Modelling/Vanillin_Production#References [8<html>]</html>]
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| K<sub>m</sub>
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| 43.0 microMolarity
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|
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| [https://2009.igem.org/Team:KULeuven/Modelling/Vanillin_Production#References [8<html>]</html>]
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! colspan="4" style="border-bottom: 1px solid #003E81;" | Fcs catalysis
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|-
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| k<sub>cat</sub>
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| 0.074068 s<sup>-1</sup>
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| [https://2009.igem.org/Team:KULeuven/Modelling/Vanillin_Production#References [8<html>]</html>]
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| K<sub>m</sub>
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| 43.0 microMolarity
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| [https://2009.igem.org/Team:KULeuven/Modelling/Vanillin_Production#References [8<html>]</html>]
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! colspan="4" style="border-bottom: 1px solid #003E81;" | Ech catalysis
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|-
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| k<sub>cat</sub>
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| 2.3 s<sup>-1</sup>
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| [https://2009.igem.org/Team:KULeuven/Modelling/Vanillin_Production#References [8<html>]</html>]
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| K<sub>m</sub>
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| 2.4 microMolarity
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| [https://2009.igem.org/Team:KULeuven/Modelling/Vanillin_Production#References [8<html>]</html>]
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Revision as of 12:06, 9 September 2009

Contents

Vanillin Production

overview

Vanillin production overview starting from Tyrosine.

The vanillin synthesis consists of a five step process starting from tyrosine, which is used quite regulary in the biosynthesis of vanillin in E. Coli.

The behaviour of this subsystem should be a production unit of vanillin which can be regulated by the instantaneous amount of RIBOKEY present in the cell.

E. coli controls its own tyrosine production which is an non essential amino acid. However,should this be insufficient, we can always add extra tyrosine. The production process will be tested in two steps. First, from tyrosine to ferulic acid; then from ferrulic acid to vanillin.

By locking both the transcription of the Sam 8 and COMT enzyme we prevent vanillin synthesis without the presence of RIBOKEY. Because we want the production of vanillin proportional to the amount of input key, it is essential that the regulated enzymes that catalyse the production of (intracellular) vanillin degrade as fast as possible. As proteins are normally very stable in the intracellular environment the SAM8 and COMT where labelled with a LVA-tag, which insures relatively fast half times. Due to the non zero half life time of the regulating enzymes, the production depends on the integrated amount of RIBOKEY over several half lives.

Biological model

Biologie vanillin synthesis.png

Sequences used to test the vanillin production, the pathway from ferulic acid to vanillin in E. Coli is better investigated than the first. [7]

Biologie vanillin synthesis short.png
Biologie vanillin synthesis shortII.png

Mathematical model

Vanillin production.png
Parameter values (Vanillin Production)
Name Value Comments Reference
Degradation Rates
dmRNA 2.3105E-3 s-1 [1]
dSam8 2.8881E-4 s-1 Fast degradation through to LVA tag [2]
dSam5 1.9254E-5 s-1 [3]
dCOMT 2.8881E-4 s-1 Fast degradation through to LVA tag [2]
dFcs 1.9254E-5 s-1 [3]
dEch 1.9254E-5 s-1 [3]
dVanillin 8.6643E-6 s-1 Equivalent degradation dilution rate of vanillin [4]
Transcription Rates
ktranscription 0.00848 s-1 estimate [6]
ktranslation 0.167 s-1 estimate [6]
Key Lock Parameters
kunlock 0.00237 s-1 Rate of unlocking the RIBOLOCK through key [5]
klock 0.00416 s-1 Rate of locking of unlocked RIBOLOCK-Key complex. [5]
kopen 7.5 s-1 Rate of unlocking RIBOLOCK when no key is present (LEAK). [5]
kclose 500 s-1 Rate of locking of leaked RIBOLOCK. [5]
Sam8 catalysis
kcat 0.015 s-1 [8]
Km 15.5 microMolarity [8]
Sam5 catalysis
kcat 4.2385 s-1 [8]
Km 432.0 microMolarity [8]
COMT catalysis
kcat 0.03319 s-1 [8]
Km 43.0 microMolarity [8]
Fcs catalysis
kcat 0.074068 s-1 [8]
Km 43.0 microMolarity [8]
Ech catalysis
kcat 2.3 s-1 [8]
Km 2.4 microMolarity [8]

Simulation

Because COMT and SAM8 are regulated by the amount of mRNA key its worthy to investigate the change of COMT and SAM8 production in function of the input amount of mRNA key. Because Sam8 and COMT are located behind the same lock they are both translated at the same rate, therefore only the concentration of COMT is shown, the translation rate of both RNA strands is considered equal.

The time scale on which COMT and Sam8 reaches equilibrium conditions is about 2 magnitudes faster than the time scale on which vanillin reaches equilibrium. Therefore only the steady state levels of COMT and Sam8 are relevant for further investigation regulatory role of both enzymes in the production of vanillin.

Because we expect only to encounter small amount of free RIBOKEY (because of the fast decay times of untranslated RNA), the steady state levels of COMT and Sam8 are relatively linearly controlled by the RIBOKEY.

Concentration of COMT in function of time and input key
.

Due to the complicated non linear (Michaelis-Menten kinetics) pathway from tyrosine to vanillin, the steady state level of vanillin well not be linearly depend on the concentration of RIBOKEY, also it takes a relatively large amount of time (hours) to reach steady state conditions.

The following figure shows that the steady state level of vanillin concentration is (non linearly) regulated by the amount of mRNA input key.

Molecules of Vanillin in function of time and input key

References

[1] J.A. Bernstein et al., “Global analysis of mRNA decay and abundance in Escherichia coli at single-gene resolution using two-color fluorescent DNA microarrays,” Proceedings of the National Academy of Sciences of the United States of America, vol. 99, Jul. 2002, pp. 9697–9702

[2] J.B. Andersen et al., “New Unstable Variants of Green Fluorescent Protein for Studies of Transient Gene Expression in Bacteria,” Applied and Environmental Microbiology, vol. 64, Jun. 1998, pp. 2240–2246

[3] K. Nath et al., “Protein degradation in Escherichia Coli,” The Journal of Biological Chemistry, vol. 246, Nov. 1971, pp. 6956-6967

[4] https://2009.igem.org/Team:KULeuven/Modeling/Vanillin_Receptor

[5] https://2008.igem.org/Team:KULeuven/Model/Filter

[6] S.L. Gotta et al., “rRNA Transcription Rate in Escherichia Coli,” Journal of Bacteriology, vol. 173, Oct. 1991, pp. 6647-6649

[7] E.G. LEE et al., “Directing Vanillin Production From Ferulic Acid by Increased Acetyl-CoA Consumption in Recombinant Escherichia coli,” Biotechnology and Bioengineering, Jul. 2008

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