Team:Northwestern/Project/Modeling

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| External IPTG || [[Image:Iex1.jpg|200px]] || [[Image:Iex2.jpg|200px]]
| External IPTG || [[Image:Iex1.jpg|200px]] || [[Image:Iex2.jpg|200px]]
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|-
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|  || '''Not Induced''' || '''Induced'''
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|-
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| Internal IPTG || [[Image:Iin1.jpg|200px]] || [[Image:Iin2.jpg|200px]]
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|-
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|  || '''Not Induced''' || '''Induced'''
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|-
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| IPTG bound Lac Repressor || [[Image:Ii1.jpg|200px]] || [[Image:Ii2.jpg|200px]]
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|-
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|  || '''Not Induced''' || '''Induced'''
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|-
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| Free Lac Repressor || [[Image:i1.jpg|200px]] || [[Image:i2.jpg|200px]]
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|-
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|  || '''Not Induced''' || '''Induced'''
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|-
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| Repressor Bound DNA || [[Image:Db1.jpg|200px]] || [[Image:Db2.jpg|200px]]
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|-
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|  || '''Not Induced''' || '''Induced'''
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| Free DNA || [[Image:Dunb1.jpg|200px]] || [[Image:Dunb2.jpg|200px]]
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|-
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|  || '''Not Induced''' || '''Induced'''
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| mRNA || [[Image:Re1.jpg|200px]] || [[Image:Re2.jpg|200px]]
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|-
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|  || '''Not Induced''' || '''Induced'''
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| Chitin Synthase 3 || [[Image:E1.jpg|200px]] || [[Image:E2.jpg|200px]]
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|-
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|  || '''Not Induced''' || '''Induced'''
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| N-Acetyl-Glucosamine || [[Image:S1.jpg|200px]] || [[Image:S2.jpg|200px]]
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|-
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|  || '''Not Induced''' || '''Induced'''
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|-
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| CHS3-NAG/Chitin Complex || [[Image:C1.jpg|200px]] || [[Image:C2.jpg|200px]]
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|-
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|  || '''Not Induced''' || '''Induced'''
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|-
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| Chitin || [[Image:P1.jpg|200px]] || [[Image:P2.jpg|200px]]
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|}

Revision as of 04:08, 27 October 2010


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Modeling Chassis Induction Chitin Apoptosis

Objective

The main purpose of modeling was to characterize the topography of the kinetics behind foreign protein/product production in recombinant E.Coli biofilm with a diffusing inducer, and the effect on the various species involved by primarily the following factors:


  • Time-derivative Spatial-Gradient Inducer Concentration
  • Repressor Concentration
  • Ribosome Binding Site (Rate of Transcription)


In terms of our iGEM project, this model was employed to explore the effect of IPTG concentration and diffusion, lacI concentration (determined by the combination part of constitutive promoter, ribosome binding site, lacI gene, double terminator, lac promoter/operon), and the ribosome binding site on the concentrations of all species involved - described in the following sections - and especially on Chitin Synthase and Chitin concentration and the corresponding rates.


Modeling

Overall Model

Using enzyme kinetics equations, we elected to mathematically simulate the following model:

SuperModeling.jpg


Variables

  • Iex: External Inducer, determined by diffusion through Fick's law (IPTG in our experiment)
  • Iin: Internal Inducer (IPTG)
  • Ii: Inducer bound to Repressor (IPTG bound to lacI)
  • i: Repressor (lacI)
  • Db: Repressor-bound DNA (lacI-bound DNA(CHS3) region in plasmid)
  • Dunb: transcribe-able or Repressor-unbound DNA (lacI-unbound DNA(CHS3))
  • Re: mRNA for Enzyme (CHS3 mRNA)
  • E: Enzyme (CHS3)
  • S: Substrate (N-Acetyl Glucosamine)
  • C: Enzyme Substrate Complex (CHS3-(N-Acetyl-Glucosamine)-Chitin or (NAG)n Complex)
  • P: Protein Product (Chitin or (NAG)n+1)


Equations

The differential of the variables were found as follows:

  • dIin = kIin*Iex - kIex*Iin + kIir*Ii - kIif*Iin*i
  • dIi = kIif*Iin*i - kIir*Ii
  • di = kIir*Ii - kIif*Iin*i + kDbr*Db - kDbf*i*Dunb
  • dDb = kDbf*i*Dunb - kDbr*Db
  • dDunb = kDbr*Db - kDbf*i*Dunb
  • dRe = ktscribe*Dunb - kRdeg*Re
  • dE = ktslate*Re - kEdeg*E + kCr*CC + kP*CC - kCf*E*Si
  • dSi = ksin*So - kCf*E*Si + kCr*CC
  • dCC = kCf*E*Si - kCr*CC - kP*CC
  • dP = kP*CC - kPdeg*P


IPTG Diffusion

First, Fick's Law of Diffusion was modeled through MATLAB. The diffusion constant used was 220um^2/s.[4]

Loopydoops.gif

IPTG was sprayed at the top of the colony, which then diffuses as according to Fick's law.

The spatially different local IPTG concentration will then differentially induce downstream processes.

This distinction was necessary in our project in order to establish a Chitin layer on the top of the biofilm.

Semi-Empirical Variable/Constant Determination

Status: Under Development

The initial plan was to use lacI-constitutive expression / lac-operon (CP-LacpI) part with Green Fluorescent Protein to acquire empirical data.

By testing various combinations of CP/LacpI, RBS, and IPTG concentrations, the acquisition of a broad range of expression level (GFP fluorescence) over time could be acquired through a plate reader.

This data would be used to determine many of the rate constants as well as initial concentration values, thus generating a more accurate semi-empirical kinetics model.

However, at the time of the wiki-freeze, data acquisition is incomplete.

Current Model

Status: Under Development

Not Induced Induced
External IPTG Iex1.jpg Iex2.jpg
Not Induced Induced
Internal IPTG Iin1.jpg Iin2.jpg
Not Induced Induced
IPTG bound Lac Repressor Ii1.jpg Ii2.jpg
Not Induced Induced
Free Lac Repressor I1.jpg I2.jpg
Not Induced Induced
Repressor Bound DNA Db1.jpg Db2.jpg
Not Induced Induced
Free DNA Dunb1.jpg Dunb2.jpg
Not Induced Induced
mRNA Re1.jpg Re2.jpg
Not Induced Induced
Chitin Synthase 3 E1.jpg E2.jpg
Not Induced Induced
N-Acetyl-Glucosamine 200px S2.jpg
Not Induced Induced
CHS3-NAG/Chitin Complex C1.jpg C2.jpg
Not Induced Induced
Chitin P1.jpg P2.jpg

Future Concerns

blahblahblah

References

1. A novel structured kinetic modeling approach for the analysis of plasmid instability in recombinant bacterial cultures

William E. Bentley, Dhinakar S. Kompala Article first published online: 18 FEB 2004 DOI: 10.1002/bit.260330108 http://onlinelibrary.wiley.com/doi/10.1002/bit.260330108/pdf


2. Mathematical modeling of induced foreign protein production by recombinant bacteria

Jongdae Lee, W. Fred Ramirez Article first published online: 19 FEB 2004 DOI: 10.1002/bit.260390608 http://onlinelibrary.wiley.com/doi/10.1002/bit.260390608/pdf


3. Pool Levels of UDP N-Acetylglucosamine and UDP NAcetylglucosamine-Enolpyruvate in Escherichia coli and Correlation with Peptidoglycan Synthesis

DOMINIQUE MENGIN-LECREULX, BERNARD FLOURET, AND JEAN VAN HEIJENOORT* E.R. 245 du C.N.R.S., Institut de Biochimie, Universit' Paris-Sud, Orsay, 91405, France Received 9 February 1983/Accepted 15 March 1983 http://www.ncbi.nlm.nih.gov/pmc/articles/PMC217602/pdf/jbacter00247-0262.pdf


4. Diffusion in Biofilms

Philip S. Stewart Center for Biofilm Engineering and Department of Chemical Engineering, Montana State University–Bozeman, Bozeman, Montana, 59717-3980 http://www.ncbi.nlm.nih.gov/pmc/articles/PMC148055/pdf/0965.pdf


5. Regulation of the Synthesis of the Lactose Repressor

PATRICIA L. EDELMANN' AND GORDON EDLIN Department of Genetics, University of California, Davis, California 95616 Received for publication 21 March 1974 http://www.ncbi.nlm.nih.gov/pmc/articles/PMC245824/pdf/jbacter00335-0105.pdf


MATLAB file provided upon request.

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