Difference between revisions of "Part:BBa K389004:Experience"

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[[Image:Bielefeld_Luc_qP.jpg|250px|center]] <div align="right">(1)</div>
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[[Image:Bielefeld_Luc_qP.jpg|300px|center]] <div align="right">(1)</div>
  
  
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plotted against the specific growth rate µ which was determined with equation (2)  
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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)  
  
  
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[[Image:Bielefeld_Luc_bildung_zu_wachstum.jpg|650px|thumb|center|'''Fig. 1A: Specific production rates q<sub>P</sub> of many different cultivations of <partinfo>K389015</partinfo> in ''Escherichia coli'' DB3.1, some induced with acetosyringone, some uninduced. Fig. 1B: Cultivation of <partinfo>K389307</partinfo> in ''E. coli'' TOP10.''']]
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[[Image:Bielefeld_Luc_bildung_zu_wachstum.jpg|750px|thumb|center|'''Fig. 1A: Specific production rates q<sub>P</sub> of many different cultivations of <partinfo>K389015</partinfo> in ''E. coli'' DB3.1, some induced with acetosyringone, some uninduced. Fig. 1B: Cultivation of <partinfo>K389307</partinfo> in ''E. coli'' TOP10.''']]
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====Kinetic of luciferin conversion====
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Look up the general luminescence measurement protocol [[Part:BBa_K389015:Experience#Measurement protocol | here]].
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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.
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[[Image:Bielefeld_Luc_kinetik.jpg|600px|thumb|center|'''Fig. 2: Kinetic of the luciferase reaction after adding luciferin to a cell lysate.''']]
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====Measurement protocol====
 
====Measurement protocol====

Revision as of 11:09, 26 October 2010

This experience page is provided so that any user may enter their experience using this part.
Please enter how you used this part and how it worked out.

Applications of BBa_K389004

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)


File:....
(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.


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


Measurement protocol

  • For the luciferase detection we used a [http://www.promega.com/tbs/tb281/tb281.pdf Promega Luciferase Assay System], containing a Cell Culture Lysis Reagent, Luciferase Assay Substrate and Luciferase Assay Buffer
  • Prepare reaction tubes with 10 µL of high salt buffer (1M K2HPO4, 20mM EDTA, pH 7.8)
  • Add 90 µL sample, mix and freeze at -80 °C
  • For the measurement thaw by placing the tubes in room temperature water
  • Mix and incubate the cells for 10 minutes at room temperature
  • Prepare the Luciferase Assay Reagent, by adding 10 mL of Luciferase Assay Buffer to the vial containing the Luciferase Assay Substrate
  • Fill each well of a white, flat bottom 96 well microtiter plate with 20 µL of cell lysate
  • For the detection of luciferase use a plate reading luminometer with injector for the Luciferase Assay Reagent and following settings (we used a Promega GloMax®-Multi Detection System with dual injector):
    • Injection volume of Luciferase Assay Reagent: 100 µL
    • Delay: 20 secs
    • Integration: 3 secs


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