Team:NYMU-Taipei/Project/pLac

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Contents

Methodology

It is an indirect method to quantitatively characterize the coding regions with specific functions (e.g. waste removal efficiency, tuner of oscillator) name QFCS (Quantitative Functional Calibration System). When we have an unknown coding region X, we can place this coding region after standard and calibrated Lac I promoter (pLac). The calibrated promoter has time-course standardized conversion between IPTG dosage and gene expression. Thus one can estimate the expression of unknown coding region X from calibration and compare with observed functional changes (e.g. the amount change of urea or phosphate to be removed, the period change of oscillators). This quantitative characterization procedure can precisely define the specification of functional genes between modeling and experimental verification.

QFC.png

  • Rx (Rate of unknown gene expression)
  • Rg (Rate of GFP expression in calibration system)
  • Rf (Rate of Functional change w.r.t. unknown functional gene X)


There are several technical issues should be concerned:

  • Rx =~ Rg (expression Rate of gene X can be estimated by GFP synthesis; However, it requires more detailed conversion, e.g. length of gene X, codon bias, etc.)
  • inconsistent growth between (pLac + GFP) and (pLac + gene X)
  • Non-linearity between Rx and Rf

Calibration of pLac (R0010)

pLac + E0240 under 0 and 50uM IPTG

PLac 0 vs. 50uM IPTG.png

pLac + E0240 under 50, 500 and 1,000uM IPTG

PLac 50,500,1000uM IPTG.png

References

  • [http://science.nhmccd.edu/biol/operon.html gene regulation and operon]
  • [http://rothlab.ucdavis.edu/protocols/beta-galactosidase-3.html b-Galactosidase Activity Assay]
  • [http://www.bio.davidson.edu/Courses/Molbio/MolStudents/spring2003/Causey/pET.html The pET Expression System]
  • [http://www.escience.ws/b572/L11/L11.htm The lac operon]
  • [http://www.springerlink.com/content/8texkfla66v0hcxc/ The Effect of the lacY Gene on the Induction of IPTG Inducible Promoters, Studied in Escherichia coli and Pseudomonas fluorescens, Current Microbiology 1997]
  • [http://www.pnas.org/content/104/14/6043.abstract Combinatorial transcriptional control of the lactose operon of Escherichia coli, PNAS 2007 (Thomas Kuhlman and et. al)]
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