Team:Bielefeld-Germany/Results/Characterization/K389004

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Contents

Characterization of BBa_K389004

On this page the experiments and results that lead to the BBa_K389004 characterization data presented on our characterization page are shown in detail.

Accumulation of luciferase

The production of the reporter gene luciferase by Escherichia coli is dependent from the specific growth rate µ (fig. 1A, eq. (2)) and the growth phase (fig. 1B). Fig. 1A shows the specific production rate qP which was determined with equation (1)


Bielefeld Luc qP.jpg
(1)


with the amount of product P, the cell count X and the relative luminescence units RLU plotted against the specific growth rate µ which was determined with equation (2)


Bielefeld Specific growth rate.jpg
.
(2)


With decreasing growth rate the luciferase production slows down and with stopping growth the luciferase is degraded. Especially in the stationary growth phase the luciferase degradation is high (fig. 1B).


Fig. 1A: Specific production rates qP of many different cultivations of BBa_K389015 in E. coli DB3.1, some induced with acetosyringone, some uninduced. Fig. 1B: Cultivation of BBa_K389307 in E. coli TOP10.


Kinetic of luciferin conversion

Look up the general luminescence measurement protocol here.

To determine the ideal time of RLU measurement after adding luciferin to the cell lysate a kinetic of this reaction was recorded with the luminometer (fig. 2). A maximum is seen between 20 - 40 s. So a measurement with a delay of at least 20 s from the time luciferin is added to the cell lysate is recommended. In addition, the time between adding the luciferin to the cell lysate and the measurement should not be more than 40 s.


Fig. 2: Kinetic of the luciferase reaction after adding luciferin to a cell lysate.


Sensitivity

To determine the sensitivity of the reporter gene luciferase the limit of detection (LOD) and the limit of quantification (LOQ) are calculated. For this calculation the noise (standard derivation sR) and the arithmetic mean (xR) of the reference measurement, so cells without luciferase gene, are collected. Afterwards the LOD is calculated with equation (3) and the LOQ with equation (4):


Bielefeld LOD.jpg
(3)


Bielefeld LOQ.jpg
(4)


The LOD is 162 RLU and the LOQ is 306 RLU. This equals 0.3 % and 0.7 %, respectively, of the maximal RLU measured with a luciferase gene under the control of the weak constitutive promoter BBa_J23103 in pSB1A2 (measured with part BBa_K389302 in pSB1A2). So the luciferase is a very sensitive reporter gene.


mRFP vs. luciferase as reporter gene

In the following table a short comparison between luciferase and mRFP as reporter genes for promoter activity is shown. A (+) means high and a (-) means low. Click on a (+) or (-) in the table to see the adequate results.

Table 1: Comparison between luciferase and mRFP as reporter genes.
Parameter Luciferase mRFP
Sensitivity (+) (-)
Stability (-) (+)
Response time (+) (-)
Degradation (+) (-)
Reproducibility (-) (+)
Growth dependent (+) (+)
Costs / work (+) (-)
Sample volume (-) (+)
Online measuring (-) (+)


References

http://www.promega.com/tbs/tb281/tb281.pdf