Team:UNIPV-Pavia/Project/Results

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UNIPV TEAM 2011

Results

Contents


NB: unless differently specified, all tests were performed in E. coli MGZ1 in M9 supplemented medium at 37°C. For the cloning of the parts, E. coli TOP10 was used.

Parts assembly

Characterization of basic modules

Characterization of promoters pTet and pLux

Characterization of enzymes AiiA and LuxI

Characterization of the efficiency of RBSs from the community collection

Identification of bacterial growth parameters

The bacterial growth curve has been modelled as a logistic curve and is represented by the following equation:

dN/dt=N*μ*(Nmax-N)/Nmax
N(0)=n0

where μ represents the growth rate of the cells (E. coli MGZ1 in M9 supplemented medium) and Nmax represents the maximum number of cells in the well. For a detailed description of the parameters, see modelling section. For details on parameters identification, see identification section. The growth curves in all the performed experiments are measured in O.D.600. Since the N species in the model is expressed in cell number, a conversion factor has been estimated. The conversion factor KO.D.toC.F.U. has been estimated as follows.

    • Two cultures C1 and C2 (MGZ1 cells) were grown in M9 medium till saturation (ON liquid culture, 37°C, 220 rpm).
    • Next morning, both C1 and C2 were diluted with the following dilution factors
      • 1:1
      • 1:10
      • 1:100
      • 1:1000
      in fresh M9 medium. These cultures were grown for further 1 hour at 37°C, 220 rpm.
    • After 1 hour, O.D.600 was measured using TECAN microplate reader (don't forget to measure a M9 sample for blanking!)
      NB: from now on, cultures must be placed in ice to stop cell growth.
    • At the same time, proper dilution of the cultures were plated on LB agar plates.
    • Plates were grown overnight and next morning C.F.U. were counted.
Culture O.D.600 TECAN O.D.600 Spectrophotometer C.F.U. Dilution Factor (10^) N
C1 0,367950002 0,758004416 990 5 990000000
C1 0,044649998 0,091982322 141 6 1410000000
C1 0,004799999 0,009888356 136 5 136000000
C1 0,000549998 0,001133037 20 6 200000000
C2 0,54840003 1,129744917 165 4 16500000
C2 0,058200002 0,119896339 23 5 23000000
C2 0,008700002 0,017922652 251 3 2510000
C2 0,000100002 0,000206011 24 4 2400000

Estimation of the spontaneous degradation of HSL in M9 medium and in cultures at different pH values

Characterization of BBa_T9002 biosensor

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