Difference between revisions of "Part:BBa K238013"
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after some experimental set ups following considerations need to be made:
 
after some experimental set ups following considerations need to be made:
 
* The promoter is weak (compared to standard I20260)
 
* The promoter is weak (compared to standard I20260)
−
* aside from light, temperature is a very important influence (if not a bigger one). for instance when working in low copy plasmids temperature will influence the promoter/repressor ratio. Cells are grown at 37°C will have too much promoter resulting in constitutive activity. When grown at 25°C the ratio is beter leading to a good repression of the promoter.
+
* aside from light, temperature is a very important influence (if not a bigger one). for instance when working in low copy plasmids temperature will influence the promoter/repressor ratio. Cells are grown at 37°C will have too much promoter resulting in constitutive activity. At 16°C, there will be more repressor and receptor but the low temperature will put the receptor in its active state thus inhibiting the repressor from doing its job. When grown at 25°C the ratio is beter and the receptor/represssor are still in there ground state leading to a good repression of the promoter.
  
  
 
{|
 
{|
−
| [[Image:weakness.png|thumb|365px|strength of our promoter in RPU]]
+
| [[Image:weakness.png|thumb|365px|Comparison of the blue light promotor (blue) to a standard promotor strength I20260 (orange). The blue light promotor shows at least half the strenght of the standard promotor in RPU, meaning it can be classified as a weak promotor]]
 
| [[Image:Light-dark.png|thumb|365px|Effect of blue light irradiance on promotor strenght. Cells grown in blue light irradiance (blue) show a stronger expression compared to cells grown in the dark (grey)]]
 
| [[Image:Light-dark.png|thumb|365px|Effect of blue light irradiance on promotor strenght. Cells grown in blue light irradiance (blue) show a stronger expression compared to cells grown in the dark (grey)]]
 
|}
 
|}
−
 
+
 
 +
our experimental set-up:
 +
*liquid cultures were ented (and reented in the early morning) and grown on two different temperatures: 37° and 25°. We noticed a more consistent response when the cells were still young.
 +
*from each of these growth conditions some of the cultures were put in 16° and the other at 25°. each of these groups were then again divided in a group that was lit with blue light and a group that wasn't.
 +
*measurements were done at following hours: 0h;0,5h;1,5h;3h;4,5h;6h;8h.
 +
*this was done for our ligation of blp with GFP in three different vector (pSB3K3, pBR322 and pSB1A2)
 +
*an example of a sample name is: 37/16/NB/LigA, grown at 37°, put in 16°, not lit and in vector pSB1A2 
 +
*note: enting of the cultures needs to be done in a dark room (red light) and it might be best to use medium that was put at 4°C overnight.
 +
 
 +
 
 +
this experiment was repeated some times and variations were done:
 +
* only growing cultures on 25°C and lighting them at room temperature. Always make sure your cultures can be shaken during the experiment, to keep the cells in suspension.
 +
 
 +
 
 +
== Experience ==
 +
 
 +
 
 +
{|width='80%' style='border:1px solid gray'
 +
|-
 +
|width='10%'|
 +
<partinfo>BBa_K238013</partinfo>
 +
<I>Glasgow iGEM 2011</I>
 +
|width='60%' valign='top'|
 +
'''Glasgow iGEM 2011'''
 +
 
 +
We coupled the bluf promoter to YFP and measured intensity after induction with white light. We found that expression showed a 2.4* increase in fold induction when compared with darkness as a control.
 +
 
 +
The promoter demonstrated some leaky expression with no light induction.
 +
 
 +
[[Image:Gla_Bluf plus YFP.jpg]]
 +
The image above demonstrates expression of YFP ligated to an RBS and the bluf promoter. The images are taken from two samples, one which was grown in darkness and the other grown with light present. The samples were shaken overnight at room temperature and the intensity of expression was measured. (See graph below)
 +
 
 +
Scale = 10 microns.
 +
 
 +
Fluorescence filter : 515-565nm
 +
 
 +
[[Image:Bluf graph.jpg]]
 +
 
 +
The graph clearly shows that expression has been significantly induced by white light for expression with the bluf domain. When averaged out, the total fold induction on this graph shows an increase in YFP fluorescence by 2.4 times, however, it was noted that the promoter does exhibit some leaky expression.
 +
 
 +
The construct used to test fluorescence is available here (https://parts.igem.org/wiki/index.php?title=Part:BBa_K660601)
 +
 
 +
|};
  
 
For more info: 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 and development23: 522-534 (2009)  
 
For more info: 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 and development23: 522-534 (2009)  

Latest revision as of 01:09, 30 September 2011

Exact promoter of the blue light receptor

This is a promoter which normally lies before the ycgZ gene, a part of an operon consisting of several genes used by E.coli for regulating e.g. biofilm production. It is regulated by the ycgF/E system. This system consists of a receptor, ycgF, which is responsive to blue light. When blue light strikes, the receptor changes conformation and dimerizes. This allows it to bind to the ycgE repressor through its EAL-domain, releasing the repressor from the promoter region. The inverted repeats shown here are expected to represent the binding site for ycgE. YcgE binding results in transcription repression.

after some experimental set ups following considerations need to be made:

  • The promoter is weak (compared to standard I20260)
  • aside from light, temperature is a very important influence (if not a bigger one). for instance when working in low copy plasmids temperature will influence the promoter/repressor ratio. Cells are grown at 37°C will have too much promoter resulting in constitutive activity. At 16°C, there will be more repressor and receptor but the low temperature will put the receptor in its active state thus inhibiting the repressor from doing its job. When grown at 25°C the ratio is beter and the receptor/represssor are still in there ground state leading to a good repression of the promoter.


Comparison of the blue light promotor (blue) to a standard promotor strength I20260 (orange). The blue light promotor shows at least half the strenght of the standard promotor in RPU, meaning it can be classified as a weak promotor
Effect of blue light irradiance on promotor strenght. Cells grown in blue light irradiance (blue) show a stronger expression compared to cells grown in the dark (grey)

our experimental set-up:

  • liquid cultures were ented (and reented in the early morning) and grown on two different temperatures: 37° and 25°. We noticed a more consistent response when the cells were still young.
  • from each of these growth conditions some of the cultures were put in 16° and the other at 25°. each of these groups were then again divided in a group that was lit with blue light and a group that wasn't.
  • measurements were done at following hours: 0h;0,5h;1,5h;3h;4,5h;6h;8h.
  • this was done for our ligation of blp with GFP in three different vector (pSB3K3, pBR322 and pSB1A2)
  • an example of a sample name is: 37/16/NB/LigA, grown at 37°, put in 16°, not lit and in vector pSB1A2
  • note: enting of the cultures needs to be done in a dark room (red light) and it might be best to use medium that was put at 4°C overnight.


this experiment was repeated some times and variations were done:

  • only growing cultures on 25°C and lighting them at room temperature. Always make sure your cultures can be shaken during the experiment, to keep the cells in suspension.


Experience

BBa_K238013 Glasgow iGEM 2011

Glasgow iGEM 2011

We coupled the bluf promoter to YFP and measured intensity after induction with white light. We found that expression showed a 2.4* increase in fold induction when compared with darkness as a control.

The promoter demonstrated some leaky expression with no light induction.

Gla Bluf plus YFP.jpg The image above demonstrates expression of YFP ligated to an RBS and the bluf promoter. The images are taken from two samples, one which was grown in darkness and the other grown with light present. The samples were shaken overnight at room temperature and the intensity of expression was measured. (See graph below)

Scale = 10 microns.

Fluorescence filter : 515-565nm

Bluf graph.jpg

The graph clearly shows that expression has been significantly induced by white light for expression with the bluf domain. When averaged out, the total fold induction on this graph shows an increase in YFP fluorescence by 2.4 times, however, it was noted that the promoter does exhibit some leaky expression.

The construct used to test fluorescence is available here (https://parts.igem.org/wiki/index.php?title=Part:BBa_K660601)

;

For more info: 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 and development23: 522-534 (2009)


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]