Part:BBa_J23100:Experience
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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);
RPU measurement has the following advantages (under suitable conditions)
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:
Inducible systemsEvery experiment is performed on the following cultures:
For inducible systems several plots are reported. The first plot is a panel containing 4 subplots, numerated this way:
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
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