Difference between revisions of "Part:BBa K568002"

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===Functional Parameters===
 
===Functional Parameters===
 
<partinfo>BBa_K568002 parameters</partinfo>
 
<partinfo>BBa_K568002 parameters</partinfo>
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Induction ideally at 465 nm
 
Induction ideally at 465 nm
  
  
−
The part works, however no complete on/off reaction was observed. Compared to a sample in the dark, cells under blue light (450 nm) expressed about 1.7 times as much beta-galactosidase. This was tested in a Miller Assay.
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'''The part works, however no complete on/off reaction was observed. This Assay was conducted in a high copy plasmid pSB1A2. For better repressor/activator ratio a low copy vector should be used to avoid constitutive on promoter activity.'''  Compared to a sample in the dark, cells under blue light (450 nm) expressed about 1.7 times as much beta-galactosidase. As described for part <html><a href="https://parts.igem.org/Part:BBa_K238013">BBa_K238013</a></html>, expression is delayed and rises strongly after about four hours. This was tested in a Miller Assay.
  
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There is a '''temperature dependency''', the blue light promotor works better at lower temperatures compared to 37 °C.
  
−
[[Image:Miller Assay Diagramm prozentual 12.9.2011.jpg|left|thumb|1200px|Results of the Miller Assay of BBa_K568002. Compared to the sample grown in room light and in the dark, blue light (450 nm) yielded a higher expression of beta-galactosidase. As the absolute values differed strongly, values were calculated relative to the sample "dark" which was set to 100 %.]]
+
 
 +
[[Image:Miller Assay Diagramm prozentual 12.9.2011.jpg|center|thumb|800px|Experiment 1: At RT. Tracking of beta-galactosidase expression over the course of five hours. As the Miller values at various points of time differed greatly, they were normalized to the "dark" sample of each point of time, which was set to 100 %.]]
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<html><div style="clear:both;color:white">.</div></html>
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[[Image:Miller Assay of blue light promoter + lacZ 4 h.jpg|center|thumb|800px|Experiment 2: Two growth temperatures: 37 °C and RT. Only values at 4 hours after splitting of the cultures and start of lighting in different conditions shown. Not shown: diagrams of experiment at other times, which exhibit the same tendency.]]
 +
<html><div style="clear:both;color:white">.</div></html>
  
 
===Experimental Testing===
 
===Experimental Testing===
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two cultures in LB medium:  
 
two cultures in LB medium:  
  
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blue light - lacZ in DH5alpha with three incubation conditions: lighted in room light, dark and blue light (450 nm)
+
blue light promoter - lacZ in DH5alpha with three incubation conditions: lighted in room light, dark and blue light (450 nm)
  
 
cpH8 in DH5alpha as a control under room light
 
cpH8 in DH5alpha as a control under room light
  
−
conditions of bacterial growth: 23,5 °C, shaken, both cultures grown in the dark prior to the experiment, two hours before the experiments: cultures were subdivided and lit as described
+
conditions of bacterial growth:  
 +
depending on the experiment, cells were grown at 23,5 °C (= room temperature, RT), or for comparisons at 37 °C and RT, in all cases shaken at 200 rpm, both cultures grown in the dark prior to the experiment, two hours before the experiments: cultures were subdivided and lit as described
  
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volume of bacterial suspension used: 50 µl (or dilutions with LB medium)
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incubation at 37 °C
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+
−
incubation time for Miller Assay: 5 min (37 °C)
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- incubation at 37 °C
 
- incubation at 37 °C
  
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- stop reaction with 50 μl of 1 M Na2CO3 = STOP after exactely 5 min  
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- stop reaction with 50 μl of 1 M Na2CO3 = STOP   
  
 
- measurement of Abs (420nm) and Abs (550nm)
 
- measurement of Abs (420nm) and Abs (550nm)
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[[Image:TU_Munich_2011_Equation_Miller_Assay.jpg]]
 
[[Image:TU_Munich_2011_Equation_Miller_Assay.jpg]]
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 +
===reference===
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 +
temperature dependency:
 +
 +
Natalia Tschowri, Susan Busse and Regine Hengge, "The BLUF-EAL protein YcgF acts as a direct anti-repressor in a blue-light response of Escherichia coli" <i>Genes Dev. 2009 23: 522-534</i>

Latest revision as of 10:40, 14 October 2011

blue light promoter lacZ reporter part

blue light promotor + lacZ

This part is based on the blue light sensor (BBa_K238013) with an additional lacZ Gene. Part (BBa_B0034) was used as RBS.


Usage and Biology

The part provides the possibility to induce lacZ transcription via blue light irradiation.

Blue light leads to dimerisation of YcgF and binding of the repressor YcgE. The formation of the YcgE-YcgF complex leads to the unbinding of YcgE from the YcgZ promoter which activates the transcription of lacZ.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Functional Parameters

n/ablue light promoter lacZ reporter part


Induction ideally at 465 nm


The part works, however no complete on/off reaction was observed. This Assay was conducted in a high copy plasmid pSB1A2. For better repressor/activator ratio a low copy vector should be used to avoid constitutive on promoter activity. Compared to a sample in the dark, cells under blue light (450 nm) expressed about 1.7 times as much beta-galactosidase. As described for part BBa_K238013, expression is delayed and rises strongly after about four hours. This was tested in a Miller Assay.

There is a temperature dependency, the blue light promotor works better at lower temperatures compared to 37 °C.


Experiment 1: At RT. Tracking of beta-galactosidase expression over the course of five hours. As the Miller values at various points of time differed greatly, they were normalized to the "dark" sample of each point of time, which was set to 100 %.

.

Experiment 2: Two growth temperatures: 37 °C and RT. Only values at 4 hours after splitting of the cultures and start of lighting in different conditions shown. Not shown: diagrams of experiment at other times, which exhibit the same tendency.

.

Experimental Testing

two cultures in LB medium:

blue light promoter - lacZ in DH5alpha with three incubation conditions: lighted in room light, dark and blue light (450 nm)

cpH8 in DH5alpha as a control under room light

conditions of bacterial growth: depending on the experiment, cells were grown at 23,5 °C (= room temperature, RT), or for comparisons at 37 °C and RT, in all cases shaken at 200 rpm, both cultures grown in the dark prior to the experiment, two hours before the experiments: cultures were subdivided and lit as described

incubation at 37 °C


Start of Assay: - measurement of Abs(600nm)

- new eppi with 0,5 ml of Zbuffer + 20 μl of freshly prepared 0,1% SDS+ 40 μl of Chloroform (under fume hood) in 2 ml tube

- 50 μl (as a dilution 25 µl + 25 µl LB medium or 10 μl + 40 μl LB medium) of the samples are taken and transferred to the Zbuffer

- mix the solution by vortexing for 10 s (all samples with equal vortexing time)

- transferring of 100 μl supernatant to 96 well plate (for photometer)

- initiation of assay with 20 μl of ONPG (4mg/ml) = START

- incubation at 37 °C

- stop reaction with 50 μl of 1 M Na2CO3 = STOP

- measurement of Abs (420nm) and Abs (550nm)


for this diagram:

  • calculation of Miller Units
  • mean of triplicate measurement
  • adjusted to 100 %
  • corrected for outliers

TU Munich 2011 Equation Miller Assay.jpg

reference

temperature dependency:

Natalia Tschowri, Susan Busse and Regine Hengge, "The BLUF-EAL protein YcgF acts as a direct anti-repressor in a blue-light response of Escherichia coli" Genes Dev. 2009 23: 522-534