Regulatory

Part:BBa_J23100:Experience

Designed by: John Anderson   Group: iGEM06_Berkeley   (2006-08-04)
Revision as of 10:47, 25 October 2009 by Lor18 (Talk | contribs)

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Applications of BBa_J23100

Characterization experiment on BBa_J23100, BBa_J23101, BBa_J23118 - UNIPV-Pavia Team (Test performed by L. Pasotti, S. Zucca, E. Del Fabbro)

Description

These three promoters are from the Anderson Promoter Collection, which is a library of constitutive sigma70 bacterial promoters. The strength of each promoter of the library has already been estimated in saturation growth phase cultures in LB, but here we provide the characterization of BBa_J23100 and BBa_J23118 in standard units (RPUs) in LB medium, in order to add experience and data for these BioBricks. BBa_J23101 is the reference standard promoter, so it has RPU=1 for definition.

The data shown below are referred to BBa_K173000, BBa_K173001 and BBa_K173002 that are the measurement parts of BBa_J23100, BBa_J23101 and BBa_J23118 respectively.

Characterization

Compatibility: E. coli TOP10 in pSB1A2

Part LB
Doubling time [minutes] RPU
BBa_J23100
(in BBa_J61002 plasmid)		 36 not computed
BBa_J23101
(in BBa_J61002 plasmid)		 37 not computed
BBa_J23118
(in BBa_J61002 plasmid)		 36 not computed
BBa_K173000 36 2.04 [1.99 ; 2.08]
BBa_K173001 36 1 (reference standard)
BBa_K173002 35 0.69 [0.64 ; 0.73]

[[Image:PV_Jxx.png|700px|thumb|No part name specified with partinfo tag. or BBa_B0033, which are symmply RBSs do not have expression systems for reporter genes);
  • ABS is the blanked absorbance (O.D.600) of the culture, computed as described in "Growth curves" section.

RPU measurement has the following advantages (under suitable conditions)

  • it is proportional to PoPS (Polymerase Per Second), a very important parameter that expresses the transcription rate of a promoter;
  • it uses a reference standard and so measurements can be compared between different laboratories.

The hypotheses on which RPU theory is based can be found in Kelly J. et al., 2008, as well as all the mathematical steps. From our point of view, the main hypotheses that have to be satisfied are the following:

  • the reporter protein must have a half life higher than the experiment duration (we use GFPmut3, BBa_E0240, which has an estimated half life of at least 24 hours, and the experiments duration is always less than 7 hours);
  • strain, plasmid copy number, antibiotic, growth medium, growth conditions, protein generator assembled downstream of the promoter must be the same in the promoter of interest and in J23101 reference standard.
  • steady state must be valid, so (dF/dt)/ABS (proportional to the GFP synthesis rate per cell) must be constant.
Inducible systems

Every experiment is performed on the following cultures:

  • the culture of interest (system studied expressing GFP)
  • the benchmarck used to evaluate R.P.U. (BBa_K173001 measurement part, that is BBa_J23101 with BBa_E0240 downstream)
  • a negative control (generally, BBa_B0033 RBS)

For inducible systems several plots are reported. The first plot is a panel containing 4 subplots, numerated this way:

(1) (2)
(3)

Plot (1) contains growth curves of the cultures, after blank value has been removed. Every curve is calculated averaging on three replicates of the same culture and subtracting the blank for each time sample. Blank is calculated averaging the replicates of blank wells.

Plot (2) shows the logarithm of absorbance in exponential phase of bacterial growth, determined by a visual inspection of log-plots. These values are used to evaluate doubling time and R.P.U..

Plot (3) contains (dGFP/dt)/O.D., the value named S_cell in Kelly J. et al., 2008 procedure for RPU evaluation.

In these plots are reported black veritcal lines that define the range of values used to evaluate RPU. It is important to underline, as explained in next paragraph, that RPU are calculated on cultures at the same O.D. level, not at the same time.

The second graphic shows S_cell VS O.D.. This plot allows the conparison of S_cell values between different cultures, that are supposed to reach the same level of growth not at the same time, but at the same O.D. value.

The third graphic shows the induction curve. The RPU value is calculated on S_cell values corresponding to O.D. values in exponential phase (typically, from 0.05 to 0.16). The curve is obtained averaging in time S_cell values corresponding to exponential phase.

Error bars rapresent the minimum and maximum value of R.P.U. belonging to the range of O.D. in exponential phase.


Materials
  • Long term glycerol stocks were stored at -80°C with a final glycerol concentration of 20%
  • Antibiotics were: Ampicillin (Amp) 100 ug/ml. It was stored at -20°C in 1000x stocks. Amp was dissolved in water.
  • LB medium was prepare with: 1% NaCl, 1% bactotryptone, 0.5% yeast extract. The medium was not buffered with NaOH.

User Reviews

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iGEM Groningen 2009

We used a number of the constitutive promoter family members for testing our biobricks. The constitutive promoters show the expected level of fluorescence when transformed into E. coli TOP10 cells. Placing parts behind the promoters turned out to be relatively straight forward. We used this part in combination with several biobricks for building our constructs e.g. BBa_I750016 was placed behind the promoters.

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