Team:Aberdeen Scotland/parameters

From 2009.igem.org

University of Aberdeen iGEM 2009

Internal Dynamics Parameters

When modeling any process, it is essential to use parameters which reflect reality, otherwise the model will not give accurate predictions. Thus we spend a considerable time researching, analysing and estimating parameters so that our model will reflect real life behaviour as accuratly as possible.

Parameter Description Value Unit Reference
Rate of production of HSL from LuxI 0.45 1/s [1]
HSL Rate of diffusion of HSL in/out of the cell 0.4 1/s [1]
IPTG Rate of diffusion of IPTG in/out of the cell 0.014 1/s [3]
pproduction Number of plasmids 10 medium copy plasmid number; decided within the team
platch Number of plasmids 10 medium copy plasmid number; decided within the team
plysis Number of plasmids 10 medium copy plasmid number; decided within the team
panti-lysis Number of plasmids 10 medium copy plasmid number; decided within the team
pQS Number of plasmids 10 medium copy plasmid number; decided within the team
max_production Maximal production rate of lux box promoter 0.44 pops [1]
min_production Minimal production rate of lux box promoter 0.013 pops estimated
latch Maximal production rate of latch promoter 0.28 pops See Below
lysis Maximal production rate of lysis promoter 0.0426 pops See Below
anti-lysis Maximal production rate of anti-lysis promoter 0.0066 pops See Below
QS Maximal production rate of QS promoter 0.018 pops See Below
KLacI Dissociation constant for LacI to LacO 700 m See Explanation
KLacI-IPTG Dissociation constant for IPTG to LacI 1200 m See Explanation
KTetR Dissociation constant for TetR to TetO 7000 m See Explanation
KcI Dissociation constant for cI to operon 7000 m See Explanation
KP Dissociation constant for P to lux box 700 m See Explanation
nLacI Hill coefficient 2 [10]
ncI Hill coefficient 2 [11]
nTetR Hill coefficient 3 [1]
nP Hill coefficient 2 [1]
mRNA Degradation of mRNA 0.00288 1/s [2]
X Degradation of X 0.00000802 1/s [5]
Y Degradation of Y 0.00000802 1/s [5]
λ-CI Degradation of lambda CI 0.002888 1/s [5]
LacI Degradation of LacI 0.001155 1/s [8]
TetR Degradation of TetR 0.00288811 1/s tagged; half-life of 4 min
Holin Degradation of Holin 0.0002 1/s estmated; half-life of an hour
Endolysin Degradation of Endolysin 0.0002 1/s estimated; half-life of an hour
Antiholin Degradation of Antiholin 0.0002 1/s estimated; half-life of an hour
LuxI Degradation of LuxI 0.002888 1/s tagged; half-life of 4 min
LuxR Degradation of LuxR 0.0002 1/s [1]
HSL Degradation of HSL 0.00016667 1 [8]; value for AHL
Protein Translation rate of Protein 0.1 1/s [2]
P Rate of formation of the HSL-LuxI complex 0.00010 1/ms [1]
-P Rate of dissociation of the HSL-LuxI complex 0.003 1/s [1]



Calculating promoter strengths

A brief discription of how some of our promoter strengths were calculated follows:

The paper "Measuring the activity of BioBrick promoters using an in vivo reference standard" published in the Journal of Biological Engineering makes J23101 their reference standard, giving it a strength of 0.03 POPS.

We can directly compare the strengths of all the J series constititive promoters using other promoters in the above paper and the promoters in the standard registry of parts website. From this page we can calculate that if J23101 =0.03 pops then our original LuxI and LuxR promoter J23107 has a strength of ~0.018 POPS and our ammended model LuxR promoter has a strength of ~0.01 POPS. We also find that our constitutive Antiholin promoter has a strength of ~0.0066 POPS


LuxR promoter polymerase per second (PoPS) The following BioBrick F2620 defines the LuxR responsive promoter. This has been assessed as having 6.6 PoPS promoter activity, i.e. 6.6 RNA polymerisation events per second. However, this seemed unlikely; the most active promoter in E.coli has about 2 polymerisations per second. At the iGEM meeting in Edinburgh this year Dr Stansfield attended on behalf of the team were he met Barry Canton, the actual experimenter who deduced the 6.6 PoPS. When questioned he stated that he measured the PoPS value in PoPs per cell; the LuxR promoter/GFP construct was on a ‘15 copies per cell’ plasmid, thus there were 15 copies of the LuxR promoter. The actual PoPS = 6.6/15 = 0.44 PoPS i.e. consistent with known E.coli promoters.


An estimate for the strength of different E.coli promoters, including lambda P(L) is in the following paper:

Journal of Molecular Biology Volume 292, Issue 1, Activities of constitutive promoters in Escherichia coli1

S.-T Liang1, 2, M Bipatnath1, Y.-C Xu1, S.-L Chen2, P Dennis3

It states the PoPs values for several promoters as:

Pspc, 26 polymerase initiations per minute P(L), 17 polymerase initiations per minute Pbla 1.7 polymerase initiations per minute PRNAI, 17 polymerase initiations per minute


Using the P(L) strength of 17 polymerase initiations per minute we can easily calculate the PoPs value.



References

[1] Goryachev, A.B., D.J. Toh and T. Lee. “System analysis of a quorum sensing network: Design constraints imposed by the functional requirements, network topology and kinetic constant.” BioSystems 2006: 83, 178-187.

[2] Alon, Uri. “An Introduction to Systems Biology Design Principles of Biological Circiuts.” London: Chapman & Hall/CRC, 2007.

[3] Kepes, A., 1960, “Etudes cinetiques sur la galactoside-permease D'Escherichia coli. Biochim.” Biophys. Acta 40, 70-84.

[4] Canton, B. and Anna Labno. “Part: BBa_F2620.” BioBrick Registry. 13th August 2009. <http://partsregistry.org/Part:BBa_F2620>

[5] Andersen JB, Sternberg C, Poulsen LK, Bjorn SP, Givskov M, Molin S. “New Unstable Variants of Green Fluorescent Protein for Studies of Transient Gene Expression in Bacteria.” Appl Environ Microbiol. 1998 Jun; 64(6):2240-6.

[6] Elowitz MB, Leibler S.; “A synthetic oscillatory network of transcriptional regulators.” Nature 2000 Jan; 403(6767):335-8.;

[7] BCCS-Bristol 2008. “Modelling Parameters” iGEM wiki. 13th August 2009. <http://2008.igem.org/Team:BCCS-Bristol/Modeling-Parameters>

[8] Subhayu Basu; “A synthetic multicellular system for programmed pattern formation.” Nature April 2005: 434, 1130-1134

[9] KULeuven 2008. “Cell Death”iGEM wiki. 13th August 2009. <http://2008.igem.org/Team:KULeuven/Model/CellDeath>

[10] ETHZ 2007. “Engineering” iGem wiki. 13th August 2009. <http://2007.igem.org/ETHZ/Engineering>

[11] Bintu, Lacramioara, Terence Hwa. “Transcriptional regulation by the numbers: applications.” Current Opinion in Genetics & Development 2005, 15:125–135.